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C H Barton

Publications and source records attributed to C H Barton.

At least 37 records · Page 2Linked to original sources
PubMed1997

Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis.

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poorly characterized in vitro, with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

Animals↗
PubMed1996

The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNos activity in a murine macrophage cell line.

Human immunodeficiency virus type 1 (HIV-1) infection is frequently associated with concurrent infection by opportunistic pathogens, against which production of nitric oxide by host macrophages provides a first line of defence. We have investigated whether regulatory HIV-1 proteins, such as Tat, can modulate the activity of the inducible nitric oxide synthase (iNos) gene when expressed in stable transfectant lines of RAW264.7 cells. A bioassay for Tat, based on transactivation of an HIV-1 LTR-CAT reporter gene, allowed selection of Tat-expressing cells. Parental and Tat-expressing macrophages accumulated identical levels of nitrite following lipopolysaccharide (LPS) stimulation. Interferon gamma (IFN-gamma) stimulation however, resulted in reduced levels of nitrite accumulation as a direct consequence of Tat expression. Conditioned media from Tat-expressing cells reduced the level of nitrite accumulation in parental cells following IFN-gamma stimulation but not stimulation with LPS. These results implicate HIV-1 Tat as a modulator of the IFN-gamma-specific signal transduction pathways leading to iNos expression.

Animals↗
PubMed1995

Opposing effects of interferon-gamma on iNOS and interleukin-10 expression in lipopolysaccharide- and mycobacterial lipoarabinomannan-stimulated macrophages.

Previous studies have demonstrated that, like bacterial lipopolysaccharide (LPS), arabinofuranosyl-terminated lipoarabinomannan (AraLAM) from an attenuated strain of Mycobacterium induces potent early gene (c-fos, KC, JE and TNF-alpha) responses in murine macrophages, whereas extensively alpha-Manp capped LAM (ManLAM) from virulent M. tuberculosis do not. In this study we have extended analysis of the influence of mycobacterial LAM on macrophage function by demonstrating that AraLAM (but not ManLAM), like bacterial LPS, is a potent stimulator of inducible nitric oxide synthase (iNOS) expression independent of the autocrine activity of co-stimulated tumour necrosis factor-alpha (TNF-alpha) release. The inability of ManLAM to induce iNOS expression was not due to induction of the 'deactivating' cytokine interleukin-10 (IL-10). Indeed, like LPS, AraLAM was also a potent inducer of IL-10 expression. However, analysis of AraLAM- or LPS-induced responses in the presence of interferon-gamma (IFN-gamma) showed that, whereas IFN-gamma acts as a potent co-stimulus for iNOS, it completely inhibits the IL-10 response. Hence, the presence of IFN-gamma early in infection will have an important immunomodulatory role in determining the macrophage response. These results have important implications for the pathogenesis of virulent and avirulent mycobacteria in vivo.

Amino Acid Oxidoreductases↗
PubMed1995

Genomic organization and sequence of the human NRAMP gene: identification and mapping of a promoter region polymorphism.

BACKGROUND: Murine Nramp is a candidate for the macrophage resistance gene Ity/Lsh/Bcg. Sequence analysis of human NRAMP was undertaken to determine its role in man. MATERIALS AND METHODS: A yeast artificial chromosome carrying NRAMP was subcloned and positive clones sequenced. The transcriptional start site was mapped using 5' RACE PCR. Polymorphic variants were amplified by PCR. Linkage analysis was used to map NRAMP. RESULTS: NRAMP spans 12kb and has 15 exons encoding a 550 amino acid protein showing 85% identity (92% similarity) with Nramp. Two conserved PKC sites occur in exon 2 encoding the Pro/Ser rich SH3 binding domain, and in exon 3. Striking sequence similarities (57 and 53%) were observed with yeast mitochondrial proteins, SMF1 and SMF2, especially within putative functional domains: exon 6 encoding the second transmembrane spanning domain, site of the murine susceptibility mutation; and exon 11 encoding a conserved transport motif. No mutations comparable to the murine susceptibility mutation were found. The transcriptional initiation site mapped 148 bp 5' of the translational initiation codon. 440bp of 5' flanking sequence contained putative promoter region elements: 6 interferon-gamma response elements, 3 W-elements, 3 NF kappa B binding sites and 1 AP-1 site. Nine purine-rich GGAA core motifs for the myeloid-specific PU.1 transcription factor were identified, two combining with imperfect AP1-like sites to create PEA3 motifs. TATA, GC and CCAAT boxes were absent. A possible enhancer element containing the Z-DNA forming dinucleotide repeat t(gt),ac(gt),ac(gt),g was polymorphic (4 alleles; n = 4,9,10,11), and was used to map NRAMP to 2q35. CONCLUSIONS: This analysis provides important resources to study the role of NRAMP in human disease.

Alleles↗
PubMed1995

Nramp transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on oxidative burst and nitric oxide pathways.

BACKGROUND: The Ity/Lsh/Bcg gene on mouse chromosome 1 regulates priming/activation of macrophages for antimicrobial and tumouricidal activity. A candidate gene expressed in macrophages has been identified by positional cloning and full-length sequence analysis, and encodes the Natural resistance-associated macrophage protein (Nramp). In this study, we have tested the hypothesis that the Nramp gene corresponds to Ity/Lsh/Bcg. MATERIALS AND METHODS: In vitro transfection was used to introduce the resistant allele into the macrophage cell line RAW 264.7 derived from the recessive susceptible BALB/c mouse strain. Expression of the transgene was monitored on the background of the endogenous susceptible allele by allele-specific oligonucleotide hybridization. RESULTS: Expression of the transgene correlated with three Lshr-associated lipopolysaccharide/interferon-gamma-regulated macrophage activation phenotypes: respiratory burst, nitrite release, and uptake of L-arginine. Endogenous and stimulated L-arginine fluxes were inhibitable with the radical scavengers nordihydroguaiaretic acid and butylated hydroxyanisole. The mitochondrial electron transport inhibitors, rotenone and thenoyltrifluoroacetone, inhibited respiratory burst, and rotenone suppressed L-arginine flux, implying that mitochondrial-derived oxygen radicals are important mediators in Nramp-regulated signal transduction pathways. CONCLUSIONS: These data provide the first direct evidence that Nramp is the product of the Ity/Lsh/Bcg gene, and are consistent with the hypothesis that the many pleiotropic effects of this gene on macrophage activation may all derive from the requirement for mitochondrial generation of oxygen radicals for intracellular signaling.

Animals↗
PubMed1994

Genetic and physical mapping of 2q35 in the region of the NRAMP and IL8R genes: identification of a polymorphic repeat in exon 2 of NRAMP.

Recent interest has focused on the region of conserved synteny between mouse chromosome 1 and human 2q33-q37, particularly over the region encoding the murine macrophage resistance gene Ity/Lsh/Bcg (candidate Nramp) and members of the Il8r interleukin-8 (IL8) receptor gene cluster. In this paper, identification of a restriction fragment length polymorphism in the IL8RB gene in 35 pedigrees previously typed for markers in the 2q33-q37 interval provided evidence (lod scores > 3) for linkage between IL8RB and the 2q34-q35 markers FN1, TNP1, VIL1, and DES. Physical mapping, using yeast artificial chromosomes isolated with VIL1, confirmed that IL8RA, IL8RB, and the IL8RB pseudogene map within the NRAMP-VIL1 interval, with the physical distance (155 kb) from 5' LSH to 3' VIL1 representing approximately 3-fold that observed in the mouse. Partial sequencing of NRAMP confirmed the presence of the N-terminal proline/serine-rich putative SH3 binding domain in exon 2 of the human gene. Further analysis of Brazilian leprosy and visceral leishmaniasis pedigrees identified a rare second allele varying in a 9-nucleotide repeat motif of the exon 2 sequence but segregating independently of the disease phenotype.

Animals↗
PubMed1994

NH2-terminal sequence of macrophage-expressed natural resistance-associated macrophage protein (Nramp) encodes a proline/serine-rich putative Src homology 3-binding domain.

The Lsh/Ity/Bcg locus on mouse chromosome 1 regulates macrophage (m phi) priming/activation for antimicrobial activity against intracellular pathogens. A candidate Bcg gene, designated natural resistance-associated m phi protein (Nramp), recently isolated from a pre-B cell cDNA library encodes a polytopic integral membrane protein with structural features common to prokaryotic and eukaryotic transporters. In the present study, an activated m phi cDNA library yielded new Nramp clones that differ in the 5' region from the published pre-B cell-derived clone sequence, resulting in addition of 64 amino acids at the NH2 terminus of the predicted protein. This new domain is rich in proline, serine, and basic amino acids, and includes three protein kinase C phosphorylation sites and a putative Src homology 3 binding domain. RNAs containing this domain are the only form found in the m phi. Hence, the protein encoded by this RNA is the candidate molecule mediating natural resistance to intra-m phi pathogens.

Amino Acid Sequence↗
PubMed1994

Genetic regulation of leishmanial and mycobacterial infections: the Lsh/Ity/Bcg gene story continues.

A common basis to genetic regulation of leishmanial and mycobacterial infections is provided by the action of the murine Lsh/Ity/Bcg gene in controlling the priming/activation of macrophages for antimicrobial activity. This relies on the TNF-alpha-dependent sustained expression of the inducible nitric oxide synthase (iNOS) gene responsible for the generation of large amounts of toxic nitric oxide (NO). The Lsh/Ity/Bcg gene has many pleiotropic effects, including differential expression of the early response gene KC following stimulation of macrophages with bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM). The major signal transduction pathway involved in KC induction requires the generation of low levels of NO via constitutive nitric oxide synthase (cNOS) activity, leading to activation of guanylate cyclase and the cGMP-dependent kinase pathway. NO therefore appears to provide a common link between the early influence of Lsh in regulating the expression of genes which mediate many pleiotropic effects, and the later production of NO as the final effector mechanism for kill. The recently cloned candidate for Lsh/Ity/Bcg, designated Nramp for Natural resistance associated macrophage protein, encodes a polytopic integral membrane protein that has structural features common to prokaryotic and eukaryotic transporters and includes a conserved binding-protein-dependent transport motif which may be involved in interaction with peripheral ATP-binding subunits. The N-terminal sequence also carries a proline/serine rich putative SH3 binding domain, consistent with a role for tyrosine kinases in regulating Nramp function. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The inducible form of nitric oxide synthase (NOS2) isolated from murine macrophages maps near the nude mutation on mouse chromosome 11.

Nitric oxide synthase has been shown to mediate streptozocin-induced diabetes and to act as an antimicrobial agent in murine macrophages. Using a cDNA probe for the inducible form of nitric oxide synthase (Nos2) isolated from murine macrophages we have determined that the gene maps within 1 cM of the nude mutation on mouse Chromosome 11. The position of Nos2 was also mapped relative to the markers 115, Evi2, Cchlbl (previously unmapped), and Gfap. This map location is discussed relative to map locations for disease susceptibility loci involved in mediating cutaneous leishmaniasis (ScII) and autoimmune type-I diabetes (Idd4).

Animals↗
PubMed1994

Plasma levels and urinary excretion of fibrinolytic and protease inhibitory proteins in nephrotic syndrome.

Nephrotic syndrome is associated with numerous blood coagulation abnormalities and a marked propensity to thromboembolism. The present study was undertaken to examine the status of the fibrinolytic system in this hypercoagulable state. We measured the antigen concentrations or activities of plasminogen, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI), alpha 2-antiplasmin, alpha 1-antitrypsin, and alpha 2-macroglobulin as well as total antiplasmin activity and D-dimer concentration in the plasma of 39 patients with nephrotic syndrome and 32 normal controls subjects. In addition, antigen concentrations of plasminogen, alpha 2-antiplasmin, alpha 1-antitrypsin, and alpha 2-macroglobulin were measured in the urine of the study populations. The nephrotic group showed marked elevations of plasma t-PA, plasminogen, alpha 2-macroglobulin, and D-dimer and a significant reduction of plasma alpha 2-antiplasmin and alpha 1-antitrypsin as compared with the normal control group. Plasma alpha 2-macroglobulin was directly related to 24-hour urinary protein excretion and inversely related to serum albumin concentration. None of the proteins measured were detectable in the urine of normal controls. However, substantial amounts of plasminogen, alpha 2-antiplasmin, and alpha 1-antitrypsin and small amounts of alpha 2-macroglobulin were recovered in the urine of patients with nephrotic syndrome. Despite the lack of clinically demonstrable thrombosis, plasma D-dimer was markedly elevated in the nephrotic group, suggesting concurrent activation of coagulation and fibrinolytic pathways. In addition, the study revealed multiple abnormalities of the plasma fibrinolytic proteins and documented their urinary excretion in patients with nephrotic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Macrophage activation: lipoarabinomannan from avirulent and virulent strains of Mycobacterium tuberculosis differentially induces the early genes c-fos, KC, JE, and tumor necrosis factor-alpha.

Lipoarabinomannan (LAM) is a major cell-wall associated glycolipid produced by Mycobacterium tuberculosis and Mycobacterium leprae. Previous work demonstrated that LAM from avirulent (H37Ra) and virulent (Erdman) strains of M. tuberculosis differ in structure at their non-reducing termini. In this study the effects of the H37Ra and Erdman LAM on the activation of murine bone marrow-derived macrophages has been investigated. Their abilities to elicit immediate early gene responses at mRNA (c-fos, JE, KC) and protein (TNF-alpha secretion) levels, and nitrite production, was examined. H37Ra LAM, but not Erdman LAM, elicited TNF-alpha secretion at 1000 ng/ml. Neither stimulated production of reactive nitrogen intermediates (RNI). Addition of 25 U/ml IFN-gamma enhanced TNF-alpha secretion in response to H37Ra LAM, reducing the threshold level of LAM required to 10 to 100 ng/ml. In contrast, Erdman LAM at concentrations up to 1000 ng/ml could not induce macrophage TNF-alpha secretion even in the presence of 25 U/ml IFN-gamma. H37Ra LAM also synergized with IFN-gamma to stimulate enhanced production of RNI, whereas IFN-gamma and Erdman LAM did not elicit RNI production. Examination of events before TNF-alpha and RNI production revealed that H37Ra LAM, like LPS, was able to induce increased levels of mRNA expression for c-fos, KC, and JE, with similar kinetics but reduced potency compared with LPS. Erdman LAM in concentrations up to 2500 ng/ml was unable to stimulate c-fos, KC, or JE expression. IFN-gamma at 25 U/ml was itself a potent stimulus of JE expression, and synergized with 1000 ng/ml H37Ra, and to a lesser extent, Erdman LAM for the induction of JE. In contrast, IFN-gamma inhibited H37Ra LAM stimulation of KC expression. The phenomenon of avoiding the stimulation of macrophage immediate early gene expression may be an important determinant of mycobacterial virulence.

Animals↗
PubMed1992

Plasma concentration and urinary excretion of erythropoietin in adult nephrotic syndrome.

PURPOSE: Nephrotic syndrome (NS) is associated with a significant alteration of protein metabolism. While lowering the plasma concentrations of certain proteins, the disease often raises the level of certain other proteins. The current study was undertaken to determine the effect of NS on erythropoietin (EPO) metabolism. PATIENTS AND METHODS: We measured the EPO concentration in plasma and urine of 26 patients with NS by an immunologic assay using a rabbit antiserum against recombinant human EPO. The results were compared with those obtained in a group of 12 normal control subjects. RESULTS: Despite a significant reduction in the hemoglobin concentration in the NS group compared with the control group (125 +/- 25 g/L versus 148 +/- 11 g/L, p less than 0.05), the plasma EPO concentration in the NS group was not significantly different from that seen in the control group (6.2 +/- 4.5 mU/mL versus 6.7 +/- 2.4 mU/mL, p = NS). No significant correlations were found between plasma EPO and hemoglobin concentration, serum creatinine, serum albumin, or urinary albumin excretion rate. Moreover, comparison of the NS patients with serum creatinine concentrations less than or equal to 1.5 mg/dL (133 mumol/L) with those exhibiting creatinine concentrations exceeding 1.5 mg/dL did not reveal a significant difference in mean plasma EPO concentration. Significant amounts of EPO were found in the urine of the patients with NS, while none was detected in the urine of the control subjects. CONCLUSION: We conclude that plasma EPO is inappropriately low in patients with NS. This is due, at least in part, to the urinary/renal losses of this protein and can potentially contribute to anemia in NS patients or compound the problem in those with concurrent renal insufficiency and diminished EPO production.

Adolescent↗
PubMed1992

Mapping Creb-1 to chromosome 1 in the mouse.

The gene CREB1 encoding the cyclic AMP response element DNA binding protein was previously assigned to human 2q32.3-q34. In this study, a panel of 207 backcross mice made between C57BL/10ScSn (=B10) females and (B10 x B10.L-Lsh)F1 males were used to map Creb-1 with respect to Cryg and Lsh/Vil on mouse chromosome 1. A reverse-transcribed, polymerase chain reaction-amplified cDNA probe covering bp 39 to 554 of the human sequence identified restriction fragment length polymorphisms with 7/18 restriction endonucleases used to digest whole genomic mouse DNA from the parental strains. BglII and DraI RFLPs for Creb-1 were scored on a subpanel of 16/207 known recombinants between Cryg and Lsh/Vil, yielding 2/16 recombinants between Cryg and Creb-1 and 14/16 recombinants between Creb-1 and Lsh/Vil. The 16/207 recombinants observed between Lsh/Vil and Cryg provide an estimated recombination frequency of 0.077 +/- 0.019, equivalent to a map distance of 7.7 +/- 1.9 cM. This is in good agreement with previously published map distances. The number of recombinants observed between Creb-1 and the other markers place Creb-1 approximately 1 cM distal to Cryg and 7 cM proximal to Lsh/Vil.

Animals↗
PubMed1991

Genetic regulation of macrophage priming/activation: the Lsh gene story.

This paper describes functional and genetic studies on the macrophage resistance gene Lsh/Ity/Bcg first described almost two decades ago. Working in vitro with resident peritoneal, liver (Kupffer cells) and bone marrow derived macrophages from congenic B10 (LshS) and B10.L-LshR mice it has been possible to demonstrate that the final effector mechanism for the gene in regulating antileishmanial activity involves production of reactive nitrogen rather than reactive oxygen intermediates. This in turn is dependent upon priming/activation of macrophages for enhanced TNF-alpha release which acts back on the macrophage in an autocrine manner to increase nitric oxide production. The precise point at which Lsh acts to control macrophage priming/activation has not been identified, but studies of early response gene expression show differences in KC mRNA levels at 2 h after LPS stimulation, and in c-fos mRNA as early as 20 min after stimulation with PMA plus ionophore, in peritoneal macrophages from congenic LshS and LshR mice. Data available suggest that both negative and positive signals may be involved in macrophage priming/activation, with LshS macrophages down-regulating their capacity for continued response to the autocrine loop. Work in progress will examine the role of TPA and cAMP response element-binding proteins in regulating gene expression in Lsh congenic mice. A major new initiative has also commenced to clone the Lsh gene by reverse genetics using yeast artificial chromosomes to walk towards Lsh from the closet proximal and distal markers on mouse chromosome 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Factor XIII and its substrates, fibronectin, fibrinogen, and alpha 2-antiplasmin, in plasma and urine of patients with nephrosis.

Plasma and urine concentrations of factor XIII and its circulating substrates (fibronectin, fibrinogen, and alpha 2-antiplasmin) were measured in a group of 36 patients with nephrotic syndrome. The results were compared with those obtained in a group of 32 normal volunteers (control group) and 12 patients with end-stage renal disease (ESRD). A mild but significant reduction in plasma level and an abnormal urinary excretion of alpha 2-antiplasmin was found in the nephrotic group. Plasma concentrations of factor XIII, fibronectin, and fibrinogen were significantly elevated in patients with nephrosis. In contrast, patients with ESRD showed no significant difference in the plasma concentrations of either factor XIII, fibronectin, or alpha 2-antiplasmin and only a modest elevation of fibrinogen when compared with normal controls. No significant correlation was found between serum creatinine concentration and plasma levels of factor XIII and its circulating substrates in the nephrotic group. No measurable quantities of factor XIII and only small quantities of fibronectin were found in the urine of patients with nephrosis. Elevation of plasma factor XIII, fibronectin, and fibrinogen concentrations in the nephrotic group is considered to be the result of a combination of increased synthesis and possibly contracted intravascular distribution of these macromolecular proteins in the face of their negligible urinary losses. The presence of the observed abnormalities in the nephrotic group and their absence in the non-nephrotic ESRD group tends to exclude renal failure as a cause of these abnormalities. Although the clinical significance of these abnormalities is uncertain, they can potentially contribute to the thrombophilic diathesis and platelet hyperaggregability in nephrotic syndrome.

Adolescent↗
PubMed1990

Characterization of the human N-CAM promoter.

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5' exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

Animals↗
PubMed1990

Enhanced myogenesis in NCAM-transfected mouse myoblasts.

The fusion of mononucleate precursor myoblasts to form the multinucleated skeletal muscle fibre is proceeded by a series of complex cell-cell interactions but the cell-surface molecules involved in these events have not been characterized. During myogenesis in vivo and in vitro, expression of the neural cell adhesion molecule (NCAM) undergoes an isoform transition that precisely correlates with terminal myoblast differentiation and myotube formation. Altered processing of RNA results in the replacement of the transmembrane NCAM (relative molecular mass, 145,000 (145K) in proliferating myoblasts by a predominant 125K NCAM form linked to glycosyl phosphatidylinositol in myotubes. We now report that mouse myoblasts transfected to constitutively express the human muscle-specific 125K glycosylphosphatidylinositol-linked NCAM isoform more readily fuse to form myotubes. This suggests that NCAM plays a part in myoblast fusion and that the isoform switch may promote this function.

Animals↗