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A S Whitehead

Publications and source records attributed to A S Whitehead.

At least 19 recordsLinked to original sources
PubMed1992

Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoprotein.

A novel serum amyloid A protein (SAA) has been identified as a normal apolipoprotein component of non-acute phase high density lipoprotein. This novel SAA has been designated "constitutive" SAA (C-SAA) to distinguish it from "acute phase" SAA (A-SAA). C-SAA was partially sequenced, and immunochemical analyses indicated that it constitutes a distinct subclass of apolipoproteins within the SAA superfamily. A C-SAA cDNA clone was isolated from a human liver library and sequenced. The clone predicts a pre-C-SAA molecule of 130 residues from which an 18-residue leader peptide is cleaved. The 112-residue mature molecule is 8 residues longer than human A-SAA; the size difference is due to the presence of an octapeptide between positions 70 and 77 that is not found in the corresponding region of human A-SAA. Paradoxically, octapeptides of similar composition are found at similar positions in the A-SAAs of a number of other species. The C-SAA octapeptide specifies the first two residues of a NSS tripeptide, the only potential N-linked glycosylation site in the molecule. Studies indicate that approximately 50% of these sites are glycosylated, thereby giving rise to two size classes, 14 and 19 kDa, of C-SAA in vivo. Human acute phase liver contains little C-SAA mRNA relative to the levels of A-SAA mRNA, and the treatment of PLC/PRF/5 hepatoma cells with monocyte-conditioned medium does not induce C-SAA mRNA concentrations to detectable levels, in contrast to the massive induction of A-SAA mRNA observed. C-SAA is therefore not a major acute phase reactant.

Amino Acid Sequence↗
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

Nucleotide sequence and expression of the human gene encoding apolipoprotein H (beta 2-glycoprotein I).

Human apolipoprotein H (ApoH), also called beta 2-glycoprotein I, is a 50-kDa serum glycoprotein whose function is not clearly defined. We have cloned and sequenced ApoH cDNAs both from human liver and from a human hepatoma cell line (HepG2). Both cDNA sequences predict a protein 345 amino acids (aa) in length. This sequence includes a 19-aa hydrophobic, N-terminal signal sequence which is not present in the mature protein [Lozier et al., Proc. Natl. Acad. Sci. USA 81 (1984) 3640-3644]. It differs from this previously reported aa sequence at two positions, both of which strengthen the conservation among the four short consensus repeats within the ApoH molecule. COS-1 cells transiently transfected with the ApoH cDNA in a eukaryotic expression vector produced a single species of ApoH mRNA and secreted in the ApoH protein. The level of ApoH mRNA expressed by HepG2 cells is downregulated by incubation with inflammatory mediators, implying that ApoH is a negative acute-phase protein.

Acute-Phase Reaction↗
PubMed1991

Heterogeneous modulation of acute-phase-reactant mRNA levels by interleukin-1 beta and interleukin-6 in the human hepatoma cell line PLC/PRF/5.

The acute-phase response to tissue injury and inflammation is accompanied by a dramatic increase in the hepatic synthesis of plasma proteins known as acute-phase reactants (APRs). This response is mediated by cytokines produced in part by activated macrophages at the site of inflammation; glucocorticoids have also been implicated as playing a regulatory role. The effects of the cytokines interleukin (IL)-1 beta and -6, alone or in combination, and in the absence or presence of the synthetic glucocorticoid dexamethasone, on the levels of APR mRNAs in the human hepatoma cell line PLC/PRF/5 were analysed. The accumulation of APR mRNAs [the complement components C3, factor B and Cl inhibitor; the major APRs C-reactive protein (CRP) and serum amyloid A protein and the CRP analogue serum amyloid P protein] was determined in dose-response and time-course studies. The APRs differed from each other in their responses to IL-1 beta alone, IL-6 alone, and IL-1 beta plus IL-6. Dexamethasone enhanced the cytokine-driven induction of a subset of APR mRNAs. These studies detail the heterogeneity of the 'in vitro' acute-phase response to defined mediators.

Acute-Phase Proteins↗
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

Dog serum amyloid A protein. Identification of multiple isoforms defined by cDNA and protein analyses.

Five distinct serum amyloid A (SAA) cDNA clones have been isolated from a library constructed using hepatic mRNA isolated from an individual beagle dog with canine pain syndrome. This implies the existence of at least three SAA genes in the dog genome. One clone predicts a truncated "amyloid A-like" SAA molecule and offers a possible alternative mechanism for the pathogenesis of secondary amyloidosis. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified by each of the dog cDNA clones. The existence of this peptide in all acute phase dog SAA proteins was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of acute phase high density lipoprotein and provides supporting evidence for gene conversion as a mechanism for maintaining the homogeneity of the SAA gene family within a species. Analysis of hepatic RNA following induction of an acute phase response shows a dramatic increase in SAA mRNA concentration; the SAA transcripts show a transient increase in size early in inflammation due to an increase in polyadenylation.

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

Structure of the human sialophorin (CD43) gene. Identification of features atypical of genes encoding integral membrane proteins.

A human sialophorin (CD43) specific genomic clone was isolated, and a 6.5 kb fragment containing the 4.6 kb sialophorin gene was sequenced. The promoter region contains no TATA or CAAT boxes, but is highly enriched in G and C nucleotides and contains short repeat sequences similar to those found in the promoters of 'housekeeping' genes. S1-nuclease protection and primer-extension experiments established that the sialophorin gene has two major transcription initiation sites. There is a single intron of 378 bp that interrupts the sequence specifying the mRNA 5' untranslated region. The gene is therefore unusual in that the discrete extracellular, transmembrane and intracellular regions of the protein, including repeat sequences in the extracellular region, are not encoded by separate exons. Utilization of alternative polyadenylation signals was previously shown to generate two sialophorin mRNAs of 1.9 and 4.3 kb, which differ in the length of their 3' untranslated regions. Sequence analysis of the gene establishes that a single polyadenylation signal 2301 bp downstream of the first major transcription initiation site and five overlapping polyadenylation signals beginning a further 2290 bp downstream define the 3' termini of the 1.9 and 4.3 kb mRNA species respectively. The gene contains potential Z-DNA structures, Aly sequences, and elements that may be involved in regulating mRNA stability.

Amino Acid Sequence↗
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↗
PubMed1990

IL-1 and IL-6 mediate increased production and synthesis by hepatocytes of acute-phase reactant mouse serum amyloid P-component (SAP).

Primary mouse hepatocytes exposed to the inflammatory cytokines IL-1 and IL-6 in vitro displayed an increase in the production of the major acute-phase reactant, serum amyloid P-component (SAP). Antiserum to recombinant human IL-6 selectively neutralized the SAP-inducing activity secreted by human diploid fibroblasts. Purified mouse interferon-beta (IFN-beta), but not IFN-alpha, also induced SAP production. Addition of 0.05 ng/ml of recombinant mouse IL-1 alpha induced a 10-fold increase in SAP production, whereas recombinant human and recombinant mouse IL-6 displayed optimal SAP-inducing activity of four-fold and seven-fold at 10 ng/ml and 1 unit/ml/2 x 10(5) mouse hepatocytes, respectively. The SAP-inducing activity was neutralized by antibodies to each of the recombinant cytokines. The kinetics of the SAP response in vitro was similar for all of the cytokines. Addition of a mixture of IL-1 and IL-6 to the hepatocytes resulted in SAP production that was not synergistic, but additive, over a range of concentrations for each cytokine. The increase in SAP production mediated by the cytokines was in part the result of an increase in the level of SAP mRNA. Metabolic incorporation of [35S]methionine into mouse SAP occurred in response to both IL-1 and IL-6. Therefore, mouse SAP should be classified among the subset of acute-phase proteins that can be induced by the direct action of either IL-1 or IL-6 on hepatocytes.

Animals↗
PubMed1989

Acute phase induction of mouse serum amyloid P component. Correlation with other parameters of inflammation.

Hepatic mRNA levels of the mouse major acute phase proteins serum amyloid P component (SAP) and serum amyloid A component (SAA) were monitored at timed intervals after i.p. injection of thioglycollate or s.c. injection of azocasein. Both mRNA increased dramatically in response to either inflammatory stimulus. The increase in SAA mRNA levels accompanied an abrupt change in mRNA size from 650 to 750 bases. Peak SAA mRNA concentrations were observed 18 h after either stimulus; by 72 h concentrations had returned to preinflammatory levels. Peak SAP mRNA concentrations were observed 8 h after thioglycollate and 12 to 18 h after azocasein injection; by 36 h concentrations were close to preinflammatory levels. All mRNA species studied (SAP, SAA and the complement components C3, C5 and factor B) were induced more rapidly by the thioglycollate stimulus and reached higher peak concentrations. SAP mRNA levels were correlated with other parameters of inflammation: infiltration of peritoneal exudate cells (PEC) into the peritoneum after thioglycollate injection, and serum concentrations of SAP after azocasein injection. Serum SAP concentrations rose 20-fold in response to the latter stimulus by 36 h, i.e., 18 to 24 h after the peak SAP mRNA levels. The highest numbers of PEC were present 24 h after the thioglycollate stimulus, i.e. 16 h after the maximum SAP mRNA concentration, indicating the continuation of an active local inflammation many hours after one aspect of the systemic response has ceased.

Animals↗
PubMed1989

Characterization of the gene encoding mouse serum amyloid P component. Comparison with genes encoding other pentraxins.

A CBA/J-strain mouse serum amyloid P component (SAP) genomic clone was isolated and analysed. The clone contains the entire SAP gene and specifies a primary transcript of 1065 nucleotide residues. This comprises a first exon of 206 nucleotide residues containing the mRNA 5'-untranslated region and sequence encoding the pre-SAP leader peptide and the first two amino acid residues of mature SAP separated by a single 110-base intron from a 749-nucleotide-residue second exon containing sequence encoding the bulk of the mature SAP and specifying the mRNA 3'-untranslated region. The overall organization is similar to that of the human SAP gene, and the coding region and intron sequences are highly conserved. The SAP RNA cap site was defined by primer extension analysis of polyadenylated acute-phase liver RNA. The 5'-region of the mouse SAP gene contains modified CAAT and TATA promoter elements preceded by a putative hepatocyte-nuclear-factor-1-recognition site; these structures are in a region that is highly homologous to the corresponding region of the human SAP gene. Comparisons of the mouse SAP gene structure and derived amino acid sequence with those of other mammalian pentraxins were made.

Acute-Phase Proteins↗
PubMed1989

Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome.

Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here we describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residue C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed.

Amino Acid Sequence↗