Biomedical subjects

K Zahedi

Publications and source records attributed to K Zahedi.

At least 19 recordsLinked to original sources

PubMed2000

Role of STAT3 and C/EBP in cytokine-dependent expression of the mouse serum amyloid P-component (SAP) and C-reactive protein (CRP) genes.

Inflammation is accompanied by a rapid increase in blood levels of acute phase proteins synthesized by hepatocytes in response to cytokines. Although C-reactive protein (CRP) levels increase dramatically in most mammals, the major acute phase protein in the mouse is the homologous pentraxin, serum amyloid P-component (SAP), whereas CRP is a minor acute phase reactant. The molecular basis for the pronounced difference in SAP and CRP gene expression in the mouse is unknown. Transfection of ++/Li mouse hepatoma cells with CAT-reporter constructs containing the 5'-flanking region of the mouse CRP gene indicated that transcription was stimulated by either IL-6, or IL-6 plus IL-1, when > or =360 bp of the 5'-proximal DNA was present. Examination of the 5'-flanking region of the mouse SAP gene revealed that the region between -433 and -397 from the transcription start site responded to IL-1 and IL-6 by binding both STAT3 and C/EBPbeta. This responsive region consisted of two adjacent C/EBPbeta consensus sites that overlap with two STAT3 consensus sites and was found to bind C/EBPbeta at an upstream site of -427 to -409 and STAT3 at a downstream site of -415 to -397. By contrast, the 360 bp promoter of the CRP gene was bound only by STAT3 at consensus sites at -93, -142, -173, and -287 from the start site; however, a single consensus site for C/EBP at -75 was not recognized. STAT3 appears to be necessary for both mouse SAP and CRP gene transcription since overexpression of an inactive, deletion mutant of STAT3 inhibited transcription of both genes. The results indicate that both STAT3 and C/EBPbeta participate in mouse SAP gene expression, whereas only STAT3 is involved in mouse CRP gene expression. The findings for mouse SAP gene expression are consistent with the reported interaction between these two transcription factors for human CRP gene transcription.

Animals↗

PubMed1999

The promoter of the C1 inhibitor gene contains a polypurine.polypyrimidine segment that enhances transcriptional activity.

The C1 inhibitor (C1INH) promoter is unusual in two respects: 1) It contains no TATA sequence, but instead contains a TdT-like initiator element (Inr) at nucleotides -3 to +5; 2) it contains a polypurine.polypyrimidine tract between nucleotides -17 and -45. Disruption of the Inr by the introduction of point mutations reduced promoter activity by 40%. A TATA element inserted at nucleotide -30 in the wild-type promoter and in promoter constructs containing the mutated Inr led to a 2-fold increase in basal promoter activity. Previous studies suggested that the potential hinged DNA-forming polypurine.polypyrimidine tract might be important in the regulation of C1INH promoter activity. The present studies indicate that this region is capable of such intramolecular triple helix formation. Disruption of the polypurine.polypyrimidine sequence by substitution of 5 of the 23 cytosine residues with adenine prevented triple helix formation. Site-directed mutagenesis experiments demonstrate that the regulation of promoter activity is independent of hinged DNA-forming capacity but requires an intact AC box (ACCCTNNNNNACCCT) or the overlapping PuF binding site (GGGTGGG). The C1INH gene also contains a number of potential regulatory elements, including an Sp-1 and an hepatocyte nuclear factor-1 binding site and a CAAT box. The role of these elements in regulation of the C1INH promoter was examined. Elimination of the hepatocyte nuclear factor-1 site at nucleotides -94 to -81 by truncation reduced the activity of the promoter by approximately 50%. Similarly, site-directed mutations that disrupt this site reduce promoter activity by 70%.

5' Untranslated Regions↗

PubMed1998

Regulation of C1 inhibitor synthesis.

The primary biologic roles of C1 inhibitor (C1-INH) are the regulation of activation of the classical complement pathway and of the contact system of kinin formation. Heterozygosity for deficiency or dysfunction of C1-INH results in hereditary angioedema (HAE). This deficiency results in loss of homeostasis with unregulated complement and contact system activation. Due to the consequent C1-INH consumption, plasma levels of C1-INH in patients with HAE are decreased below 50% of normal. In addition, diminished synthesis contributes to the lowered levels in some patients. The hepatocyte is the primary source of C1-INH, although a number of other cell types, including peripheral blood monocytes, microglial cells, fibroblasts, endothelial cells, the placenta, and megakaryocytes also synthesize and secrete the protein both in vivo and in vitro. Interferon-gamma and alpha (IFN), colony stimulating factor-1, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) all induce C1-INH synthesis in a variety of cell types. The IFN-response elements in the 5'-flanking region and in the first intron have been partially characterized, as have several of the promoter elements that direct basal transcription of the gene. However, although androgen therapy, in vivo, results in an increase in C1-INH plasma levels, a direct effect of androgens on C1-INH synthesis has not been convincingly demonstrated. Although the C1-INH gene contains a potential glucocorticoid/androgen response element, this element does not appear to respond to androgen. Continued analysis of the transcriptional regulation of the C1-INH gene may lead to new approaches to therapy of HAE.

Acute-Phase Reaction↗

PubMed1997

Characterization of the IFN-gamma-responsive element in the 5' flanking region of the C1 inhibitor gene.

Treatment of a variety of cell lines with IFN-gamma leads to enhanced synthesis and secretion of C1 inhibitor (C1inh). The induction of C1inh synthesis by IFN-gamma is primarily regulated at the transcriptional level, and is controlled by elements in the 5' flanking region and the first intron of the C1inh gene. Hep3B cells transfected with reporter constructs containing truncated segments between -738 and -81 of the 5' flanking region and stimulated with IFN-gamma expressed increased levels of chloramphenicol acetyl transferase. These data as well as the data obtained from studies using constructs with mutated IFN-gamma-activated sequence (GAS) indicate that the most proximal GAS element (GAS 4) that mapped to nucleotides -126 to -118 is responsible for this IFN-gamma responsiveness. Electrophoretic mobility shift assays using GAS 4 yielded a single band that appeared within 5 min after stimulation with IFN-gamma. In competition experiments, both GAS 4 and consensus GAS probes, but not a mutated GAS probe, competed for the GAS binding protein present in Hep3B and U-937 cell extracts. The identity of the GAS binding protein was confirmed using anti-STAT-1alpha Abs in supershift assays. The results indicate that STAT-1alpha binds to GAS 4, which is the primary element in the 5' flanking region responsible for IFN-gamma induction of the C1inh gene.

Carcinoma, Hepatocellular↗

PubMed1997

Characterization of the binding of serum amyloid P to laminin.

Serum amyloid P (SAP) is a member of the pentraxin family. These are evolutionarily conserved proteins made up of five noncovalently bound identical subunits that are arranged in a flat pentameric disc. Although a variety of activities have been attributed to SAP and other pentraxins, their biological functions remain unclear. In humans SAP is a constitutive serum protein that is synthesized by hepatocytes. It is encoded by a single copy gene on chromosome 1. SAP is a component of all amyloid plaques and is also a normal component of a number of basement membranes including the glomerular basement membrane. The association and distribution of SAP within the glomerular basement membrane are altered or completely disrupted in a number of nephritides (e.g. Alport's Syndrome, type II membranoproliferative glomerulonephritis, and membranous glomerulonephritis). In the present study the binding of SAP to laminin was characterized. SAP binds to human laminin and merosin as well as mouse and rat laminins. The binding of SAP to mouse laminin is saturable and calcium-dependent. The Kd of this interaction is 2. 74 x 10(-7) M, with a SAP/laminin molar ratio of 1:7.1. Competition binding assays indicate that the binding of SAP to laminin is inhibited by both SAP and its analog, C-reactive protein, as well as phosphatidylethanolamine. In turbidity assays SAP enhanced the polymerization of laminin in a concentration-dependent manner. However, SAP did not alter the ability of laminin to serve as a cell adhesion substrate. Previous observations indicating that SAP binds to extracellular matrix components such as type IV collagen, proteoglycans, and fibronectin in concert with the data presented here suggest that SAP may play an important role in determining the structure of those basement membranes with which it is associated.

Animals↗

PubMed1996

Characterization of the binding of serum amyloid P to type IV collagen.

Serum amyloid P (SAP), a member of the evolutionarily conserved pentraxin family, is a normal component of a number of basement membranes, including glomerular and alveolar. In vitro SAP binds to a variety of proteins including fibronectin, proteoglycans, and the collagen-like region of the complement component C1q. In these studies, binding of SAP to type IV collagen, a major component of basement membrane, was examined. Purified SAP binds to human and mouse type IV collagen but not type I, II, or III collagens. Binding of SAP to type IV collagen is dependent on the presence of Ca2+. This binding is saturable with a Kd approximately 1.2 x 10(-7) M based on solid phase binding and 4 x 10(-8) M based on the IC50 value from fluid phase binding data. Binding of SAP to type IV collagen was inhibited by both SAP and C-reactive protein (CRP). However, a 5-fold molar excess of CRP as compared with SAP was required to inhibit the SAP binding by 50%. Binding of SAP to type IV collagen was inhibited by both collagen IV and C1q but not by phosphatidylethanolamine or bovine serum albumin. The inhibition data indicate that SAP may bind to the triple helical region of type IV collagen via a site distinct from its galactan binding site. The most likely site of SAP involved in its interaction with type IV collagen may be the region spanning amino acid residues 108-120, which shows a great deal of sequence homology (60% strict identity) with the CRP region implicated in its binding to the collagen-like region of the C1q molecule.

Animals↗

PubMed1994

Transcriptional regulation of the C1 inhibitor gene by gamma-interferon.

Treatment of the hepatoma cell line, Hep3B, with gamma-interferon (IFN) enhanced expression of C1 inhibitor (C1INH) mRNA, primarily due to an enhanced transcription rate. Hep3B cells transfected with reporter constructs containing various regions of the C1INH gene between positions -1182 and +587, and stimulated with gamma-IFN, expressed increased levels of chloramphenicol acetyltransferase in the presence of the first intron and as few as 12 bases of the 5'-flanking region. However, a 66% reduction in the inducibility of the constructs was observed when the upstream region between -582 and -252 was eliminated. Successive deletions mapped the first intron IFN-responsive elements to a region between +368 and +410. The data indicate that both the upstream and the first intron sequences can independently enhance induction of C1INH gene expression. Examination of the immediate upstream sequence of the C1INH gene reveals the absence of a TATA box. The promoter of the C1INH gene was mapped to a region within 81 bases of the upstream sequence and the first exon. Further examination indicated two regions that were potentially important for promoter activity as follows: 1) a G-C-rich region from -81 to -49, and 2) an initiator element at -3 to +5. The results indicate that the upstream sequences including -81 to -49 and the H-DNA region between -48 and -17 are not necessary for promotor activity. The initiator element from -3 to +5 is sufficient and necessary for promoter function.

Base Sequence↗

PubMed1993

Regulation of mouse serum amyloid P gene expression by cytokines in vitro.

Inflammation is accompanied by an increase in the plasma levels of a number of proteins collectively known as acute-phase reactants (APRs). Serum amyloid P component (SAP) is a major mouse APR: hepatic SAP mRNA and plasma SAP protein concentrations increase by up to 20-fold in mice undergoing an inflammatory response. In-vitro studies, using primary hepatocyte cultures, have previously shown that SAP mRNA and protein levels increase in response to stimulation with a variety of cytokines such as monocyte-conditioned medium (MCM), interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta). In this report, we have examined a mouse hepatoma cell line in which SAP gene regulation closely resembles that of primary hepatocytes. Accumulation of SAP mRNA in the +/+ Li mouse hepatoma cell line after stimulation with MCM, IL-1, IL-6 and the combination of IL-1 and IL-6 was demonstrated. This increase in the cellular content of SAP mRNA did not require new protein synthesis and was at least partially due to an increase in the transcription rate of the SAP gene.

Animals↗

PubMed1993

C1 inhibitor.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1993

Structure, expression, and evolution of guinea pig serum amyloid P component and C-reactive protein.

The structure and expression of the pentraxins, serum amyloid P component (SAP), and C-reactive protein (CRP), have been investigated in the guinea pig. Northern blot analysis of hepatic RNA from animals in which acute inflammation had been induced by intraperitoneal injection of thioglycollate established that neither SAP or CRP is a major acute phase reactant in the guinea pig. Genomic clones of SAP and CRP were isolated and sequenced, and the gene and the derived protein sequences were compared with other mammalian homologues. Both genes have organizations typical of the pentraxin genes of other species, but some differences were defined in the regions that potentially determine the capacity of the pentraxin gene to be induced during acute inflammation. Nucleotide substitutions in coding regions have occurred at similar rates in the two pentraxin genes. Nonsynonymous substitution rates indicate that SAP and CRP are subject to similar, relatively low levels of constraint; at the amino acid sequence level the rate of evolution is approximately two replacements per site per 10(9) years. An estimate of the phylogenetic relationship among the pentraxin genes suggests that SAP and CRP arose as the result of a gene duplication event that occurred very early in mammalian evolution, but subsequent to the divergence of the reptilian ancestors of the mammalian and avian lineages. This raises doubts about the identity of proteins from fish, which have been previously characterized as CRP and SAP.

Acute-Phase Reaction↗

PubMed1993

Structure and regulation of the C1 inhibitor gene.

C1 esterase inhibitor is a M(r) approximately 105,000 glycoprotein and the sole regulation of the activities of C1r and C1s. As such, it plays an extremely important role in the regulation of the classical complement pathway. Hereditary angioedema (HAE) is the clinical manifestation of C1INH deficiency. Two types of HAE have been described. Type I HAE is characterized by low antigenic and functional levels of C1INH, while Type II HAE is characterized by normal or increased antigenic levels of C1INH with low levels of functionally active protein. C1INH is encoded by a single gene on chromosome 11. The C1INH gene consists of 8 exons and 7 introns and is approximately 1.7 x 10(4) base pairs in length. Expression of C1INH in vivo is enhanced by androgens. In vitro studies indicate that C1INH mRNA and protein levels are increased by up to 20 fold after stimulation with interferon-gamma (gamma-IFN) and to a lesser extent in response to alpha-interferon (alpha-IFN), tumor necrosis factor-alpha (TNF-alpha), Interleukin 6 (IL-6) and monocyte colony stimulating factor (M-CSF). In this chapter, we will discuss the structure of the C1INH gene and mechanisms of its regulation as well as some of the elements which may contribute to its transcriptional regulation.

Angioedema↗

PubMed1992

C5 deficiency in A/J mice is not associated with resistance to the development of secondary amyloidosis.

The aim of the study was to determine whether C5 deficiency in the mouse is associated with resistance to the development of secondary amyloidosis. Chronic inflammation was induced in the F2 progeny, derived from matings between amyloid-susceptible and amyloid-resistance mice, by daily injections of azocasein for thirty days. Using a restriction fragment length polymorphism generated by digestion of genomic DNA with the restriction enzyme HindIII, C5 sufficient and deficient DNA can be clearly differentiated. Eight mice were found to be C5 sufficient, 32 were heterozygotes and 14 were found to be C5 deficient. Grading of the splenic amyloid load from negative to 4+ was performed after staining tissue squashes with Congo red and viewing them under a polarizing microscope. Seventeen mice were noted to have negative to trace, 18 had moderate (1+ - 2+) and 19 had heavy (3+ - 4+) amyloid deposition. There was no correlation between splenic amyloid load and C5 deficiency. Based on these results it is clear that C5 deficiency and resistance to secondary amyloidosis are not associated.

Amyloid↗

PubMed1991

Molecular characterization of the mouse mannose-binding proteins. The mannose-binding protein A but not C is an acute phase reactant.

Mannose-binding proteins play a role in first line host defense against a variety of pathogens. We report the molecular cloning of two mouse mannose-binding proteins designated A and C based on their close identity with their rat homologues. The deduced amino acid sequence of the mouse mannose-binding proteins, as with rat and the human forms, have an NH2 terminus that is rich in cysteine that stabilizes a collagen alpha helix followed by a carboxyl- terminal carbohydrate binding domain. We further show that the mouse mannose-binding protein A mRNA, as with the human, is induced like the acute phase reactant serum amyloid P protein, yet the expression of mouse mannose-binding protein C mRNA is not regulated above its low baseline level. The expression of both mannose-binding proteins A and C mRNA is restricted to the liver under basal and stress conditions.

Acute-Phase Proteins↗

PubMed1991

Mouse IL-1 receptor antagonist protein. Molecular characterization, gene mapping, and expression of mRNA in vitro and in vivo.

The IL-1R antagonist protein (IL-1RN/IL-1rn) is a member of the IL-1 family of inflammatory mediators. We have isolated and analyzed a mouse IL-1rn cDNA clone and established that the derived mouse IL-1rn protein sequence is highly homologous to the human counterpart molecule. Mouse IL-1rn mRNA may be induced in P388D1 monocytic cells with PMA and in mouse liver in vivo by development of an experimental inflammation via s.c. injection of azocasein. This latter observation implies the existence of an autocrine hepatic negative feedback loop that down-regulates the acute phase response and is itself induced at the same time as the major acute-phase proteins. The mouse Il 1rn gene was mapped to the proximal region of chromosome 2 between the centromere and Spna2; the other known members of the mouse IL-1 gene family, Il 1a and Il 1b, both map to the same chromosome, although not in close linkage.

Amino Acid Sequence↗

PubMed1991

Haplotypic polymorphisms of the TNFB gene.

The TNFB genes from two major histocompatibility complex (MHC) ancestral haplotypes have been compared. The genes carried by the ancestral haplotypes 8.1 (A1,B8,BfS,C4AQ0, C4B1,DR3) and 57.1 (A1, B57. BfS, C4A6, C4B1, DR7) were cloned and sequenced to determine the degree of polymorphism. In this report we show that the respective TNF genes are allelic and have unique nucleotide sequences. The data demonstrate the presence of three nucleotide differences between the TNFB alleles of 8.1 and 57.1. Two of the differences occur in untranslated regions of the gene but the third nucleotide change results in an amino acid difference in the mature TNFB protein. These polymorphisms may have implications with respect to differential regulation in disease- and nondisease-associated haplotypes.

Alleles↗

PubMed1991

Major acute-phase reactant synthesis during chronic inflammation in amyloid-susceptible and -resistant mouse strains.

Hepatic levels of mRNA specific for total serum amyloid A (SAA), the SAA1 and SAA2 isotypes, serum amyloid P (SAP), C-reactive protein (CRP), and fibronectin, as well as the plasma concentrations of SAA and SAP were examined in amyloid-resistant (A/J) and amyloid-susceptible (CBA/J) mice during azocasein-induced chronic inflammation. In both strains hepatic SAA and SAP mRNA levels and plasma SAA and SAP protein concentrations increased dramatically during the early stages of inflammation; this was followed by a decrease to concentrations that were maintained at levels considerably higher than background. The ratios of SAA1 and SAA2 mRNA and plasma protein were 1:1 throughout. This indicated that there was no preferential accumulation of mRNA specifying a particular isotype and no preferential synthesis or clearance of a particular isotype during chronic inflammation and the early stages of amyloidogenesis in either strain. Similarly, hepatic SAP mRNA levels in both strains increased dramatically during the early stages of inflammation and were subsequently maintained at elevated levels. Plasma SAP concentrations increased rapidly during the first three days of the study in both A/J and CBA/J mice; however, during the later stages of inflammation, A/J plasma SAP levels decreased to a steady-state concentration that was approximately half that observed in CBA/J mice. Our results identify differences in the hepatic mRNA and plasma protein levels of the major mouse acute-phase reactants (APR) in the amyloid-resistant A/J and amyloid-susceptible CBA/J mouse strains. These findings are consistent with circulating inflammatory APR concentrations contributing, together with other factors, to the onset and pathogenesis of secondary amyloidosis.

Acute-Phase Proteins↗

PubMed1991

Amyloid resistance in A/J mice is not determined by genetic variants at, or close to, the serum amyloid P component locus.

An experimental model to assess the utility of variants of the serum amyloid P component (SAP) gene in predicting resistance to amyloidosis resulting from chronic inflammation was developed. F2 mice were generated from amyloid resistant (A/J) and amyloid susceptible (C57BL/6J) progenitor strains. Mice were injected daily with azocasein for 30 days, a treatment protocol determined to produce little amyloid deposition in the A/J progenitor strain and substantial amyloid deposition in the C57BL/6J progenitor strain. In a blind study the F2 spleens were subjectively scored for amyloid deposition and DNA was isolated from F2 livers and subjected to Southern blot analysis to determine the inheritance of progenitor strain specific SAP restriction fragment length polymorphisms (RFLPs). No correlation between relative amyloid resistance and SAP genotype was observed, indicating that SAP RFLPs have no value in predicting predisposition to amyloid disease in the mouse model studied.

Amyloidosis↗

PubMed1990

Mouse C-reactive protein. Generation of cDNA clones, structural analysis, and induction of mRNA during inflammation.

A full-length C-reactive protein (CRP) cDNA clone has been isolated from a CBA/J-strain-mouse acute-phase liver library. The 1614-nucleotide cDNA specifies mRNA 5' and 3' untranslated regions of 81 and 858 bases respectively that flank 675 bases encoding mouse pre-CRP. The derived amino acid sequence predicts a 19-residue leader peptide followed by a 206-residue mature mouse CRP that shows considerable sequence identity with both human and rabbit CRP. Northern-blot analysis of mouse liver CRP mRNA concentrations after inflammatory stimuli and comparison with hepatic induction of mRNA for the major mouse acute-phase protein serum amyloid P component established that CRP, a major acute-phase reactant in human and rabbit, is a minor acute-phase reactant in mouse. The size and organization of the mouse CRP mRNA 5' and 3' untranslated regions are significantly different from those of human and rabbit CRP mRNA and may have implications for its anomalous minimal induction during acute inflammation.

Amino Acid Sequence↗