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W P Lafuse

Publications and source records attributed to W P Lafuse.

At least 19 recordsLinked to original sources
PubMed2005

Modulation of human dUTPase using small interfering RNA.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is responsible for maintaining low intracellular levels of dUTP, thus preventing the incorporation of dUTP into DNA. A 21 bp double-stranded RNA molecule (siRNAdUT3) targeted against motif 3 of human dUTPase resulted in a time- and dose-dependent decrease in dUTPase activity in transfected cells. dUTPase activity was reduced approximately 95+/-5% in all cell lines tested 48 h after transfection with 2 microg siRNAdUT3 and it was maintained at this decreased level for at least 72 h. Down-regulation of dUTPase resulted in a significant increase in intracellular dUTP and a decreased proliferation of the transfected cells. Therefore, we conclude that dUTPase activity/expression can be down-regulated using siRNA specifically targeted to dUTPase mRNA and that this approach can be used to elucidate the role of dUTPase in DNA metabolism, as well as, to determine whether dUTPase is a valid target for drug development.

Cell Line, Tumor↗
PubMed2001

NFkappaB and Sp1 elements are necessary for maximal transcription of toll-like receptor 2 induced by Mycobacterium avium.

We have previously reported that Toll-like receptor (TLR) 2 mRNA was induced after infection with Mycobacterium avium. To investigate the molecular basis of TLR2 expression in macrophages, we cloned and analyzed the murine putative 5'-proximal promoter. Transient transfection of a 326-bp region from nucleotides -294-+32 relative to the first transcription start site was sufficient to induce maximal luciferase activity at the basal level and after infection with M. avium in J774A.1 cells. Sequence analysis showed that the region lacked a TATA box but contained two typical stimulating factor (Sp) 1 sites, two NF-kappaB sites, one IFN-regulatory factor site and one AP-1 site. Site-directed mutagenesis revealed that the NF-kappaB and Sp1 sites but not the IFN-regulatory factor site or the AP-1 site contributed to the basal level and the induction of luciferase activity during M. avium infection. Binding of Sp1/Sp3 and NF-kappaB (p50/p65) was confirmed by EMSA. Further studies showed that three copies of Sp1 elements or NF-kappaB elements are not sufficient to confer M. avium induction on a heterologous promoter. By contrast, overexpression of NF-kappaB p65 caused a strong increase in transcription from an intact TLR2 promoter, whereas it caused only a partial increase in promoter activity when cotransfected with the TLR2 promoter with one of the Sp1 sites mutated. Sp1 and NF-kappaB were the minimum mammalian transcription factors required for effective TLR2 transcriptional activity when transfected into Drosophila Schneider cells. Together, these data provide genetic and biochemical evidence for NF-kappaB as well as Sp1 in regulating TLR2 transcription.

Animals↗
PubMed2001

Infection with Mycobacterium avium differentially regulates the expression of iron transport protein mRNA in murine peritoneal macrophages.

Iron is an important element for the growth of microorganisms as well as in the defense of the host by serving as a catalyst for the generation of free radicals via the Fenton/Haber-Weiss reactions. The iron transporter natural resistance-associated macrophage protein 1 (Nramp1) confers resistance to the growth of a variety of intracellular pathogens including Mycobacterium avium. Recently several other proteins that are involved in iron transport, including the highly homologous iron transporter Nramp2 and the transferrin receptor-associated protein HFE (hereditary hemochromatosis protein), have been described. The relationship of these proteins to host defense and to the growth of intracellular pathogens is not known. Here, we report that infection with M. avium differentially regulates mRNA expression of the proteins associated with iron transport in murine peritoneal macrophages. Both Nramp1 and Nramp2 mRNA levels increase following infection, while the expression of transferrin receptor mRNA decreases. The level of expression of HFE mRNA remains unchanged. The difference in the expression of the mRNA of these proteins following infection or cytokine stimulation suggests that they may play an important role in host defense by maintaining a delicate balance between iron availability for host defense and at the same time limiting iron availability for microbial growth.

Animals↗
PubMed2001

Iron transport into mycobacterium avium-containing phagosomes from an Nramp1(Gly169)-transfected RAW264.7 macrophage cell line.

Nramp1 is an important determinant of innate resistance of macrophages to the growth of intracellular microorganisms. We previously showed that Nramp1 functions to transport iron from the cytoplasm into phagosomes of Mycobacterium avium-infected macrophages. The purpose of this investigation was to further characterize the factors that regulate Nramp1-mediated iron transport into phagosomes. Treatment of Nramp1(Gly169) macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly. We found that macrophage-activating cytokines, including TNF-alpha, IFN-gamma, IL-1alpha, and GM-CSF, when added prior to M. avium, increased the transport of iron into the phagosome. This increase in iron transport was not a result of an increased amount of Nramp1 protein in the phagosome nor to new protein synthesis. Treatment of Nramp1(Gly169)-transfected macrophages with inhibitors of protein kinase C (PKC) diminished the import of iron into the phagosomes. Iron import was inhibited by an anti-Nramp1 antibody against the putative fourth outer-loop region of Nramp1 but not by an anti-Nramp1 antibody against the carboxy terminus. The significance of these results on the orientation of Nramp1 in the phagosome membrane and on the transport of iron is discussed.

Animals↗
PubMed2000

Regulation of toll-like receptor 2 expression by macrophages following Mycobacterium avium infection.

Recent studies have implicated Toll-like receptors (TLR), especially TLR2 and TLR4, as sentinel receptors that signal the interaction of macrophages with bacterial pathogens via a NF-kappaB-mediated pathway. The regulation of TLR gene expression, however, has not been intensively studied. Here, we report that TLR2 mRNA was induced following infection of murine macrophages with Mycobacterium avium. The changes in TLR2 mRNA correlated with an increase in TLR2 surface expression. Infection with M. avium resulted in a concomitant decrease in TLR4 mRNA. The effect of M. avium infection on TLR2 mRNA appeared to be mediated, in part, by TLR2 because the induction of the mRNA was partially blocked by preincubation of the macrophages with an anti-human TLR2 Ab. In contrast, the effect of LPS stimulation was mediated via TLR4 because infection of macrophages from LPS(d) mice, which do not express active TLR4, resulted in an increase in TLR2 mRNA, while treatment of macrophages from these mice with LPS failed to induce TLR2 mRNA. Several cytokines, including TNF-alpha, IL-1alpha, and GM-CSF, but not IFN-gamma, induced TLR2 mRNA. M. avium infection resulted in the induction of TLR2 mRNA by macrophages from both TNFRI knockout and NF-kappaB p50 knockout mice.

Animals↗
PubMed2000

Regulation of Nramp1 mRNA stability by oxidants and protein kinase C in RAW264.7 macrophages expressing Nramp1(Gly169).

The murine Nramp1 (natural-resistance-associated macrophage protein) locus confers innate resistance against intracellular macrophage pathogens. The gene encodes a transporter molecule, which is rapidly recruited to the phagosome. Nramp1 functions as an iron transporter by transporting iron into the phagosome. Within the phagosome iron mediates anti-microbial killing by hydroxyl radical formation through the iron-catalysed Fenton/Haber-Weiss reaction. In addition to its effects on the growth of intracellular pathogens, Nramp1 exerts a wide range of pleiotropic effects in activated macrophages. One of these pleiotropic effects is the increased stability of a variety of mRNA species, including Nramp1 mRNA. In the present study, the stability of Nramp1 mRNA in Mycobacterium avium infected RAW264. 7 macrophages expressing either the Nramp1(Gly169) resistant allele or the Nramp1(Asp169) susceptible allele was examined. Nramp1 mRNA stability was greater in Nramp1(Gly169) macrophages than in Nramp1(Asp169) macrophages. The increase in Nramp1 mRNA stability in resistant macrophages was inhibited by antioxidants and protein kinase C (PKC) inhibitors, suggesting that Nramp1 mRNA stability is regulated by an oxidant-generated signalling pathway that requires PKC activity. This was corroborated by treating Nramp1(Asp169) macrophages with menadione, which generates reactive oxygen species within cells. Menadione increased Nramp1 mRNA stability to the level observed in resistant macrophages; this increase was also inhibited by a PKC inhibitor. Further, PKC activity was found to be greater in M. avium-infected Nramp1(Gly169) macrophages than in infected Nramp1(Asp169) macrophages and inhibited by treatment with an antioxidant.

Animals↗
PubMed1999

Beta2-adrenergic receptor stimulation inhibits nitric oxide generation by Mycobacterium avium infected macrophages.

Catecholamine regulation of nitric oxide (NO) production by IFNgamma-primed macrophages infected with Mycobacterium avium was investigated. Epinephrine treatment of IFNgamma-primed macrophages at the time of M. avium infection inhibited the anti-mycobacterial activity of the cells. The anti-mycobacterial activity of macrophages correlated with NO production. Using specific adrenergic receptor agonists, the abrogation of mycobacterial killing and decreased NO production by catecholamines was shown to be mediated via the beta2-adrenergic receptor. Elevation of intracellular cAMP levels mimicked the catecholamine-mediated inhibition of NO in both M. avium infected and LPS stimulated macrophages. Specific inhibitors of both adenylate cyclase and protein kinase A prevented the beta2-adrenoceptor-mediated inhibition of nitric oxide production. Beta2-adrenoreceptor stimulation at the time of M. avium infection of IFNgamma-primed macrophages also inhibited expression of iNOS mRNA. These observations show that catecholamine hormones can affect the outcome of macrophage-pathogen interactions and suggest that one result of sympathetic nervous system activation is the suppression of the capacity of macrophages to produce anti-microbial effector molecules.

Animals↗
PubMed1999

Mycobacterium avium infection of mouse macrophages inhibits IFN-gamma Janus kinase-STAT signaling and gene induction by down-regulation of the IFN-gamma receptor.

Macrophage activation is required to control the growth of intracellular pathogens. Recent data indicate that macrophages become functionally deactivated during mycobacterial infection. We studied macrophage deactivation by examining the expression of a panel of IFN-gamma-inducible genes and activation of Janus Kinase (JAK)-STAT pathway in Mycobacterium avium-infected macrophages. Reduced expression of IFN-gamma-inducible genes-MHC class II gene E beta; MHC class II transactivator; IFN regulatory factor-1; and Mg21, a gene coding for a GTP-binding protein-was observed in M. avium-infected macrophages. Decreased tyrosine phosphorylation and DNA binding activity of STAT1 in M. avium-infected macrophages stimulated with IFN-gamma was observed. Tyrosine phosphorylation of JAK1, JAK2, and IFN-gamma R alpha was also reduced in infected cells. Northern and Western blot analyses showed that a down-regulation of IFN-gamma R alpha- and beta-chain mRNA and protein occurred in M. avium-infected macrophages. The down-regulation of IFN-gamma R and inhibition of STAT1 activation were time dependent and required 4 h of infection for down-regulation of the IFN-gamma R and 8 h for STAT1 inhibition. These findings suggest that M. avium infection inhibits induction of IFN-gamma-inducible genes in mouse macrophages by down-regulating IFN-gamma R, resulting in reduced phosphorylation of IFN-gamma R alpha, JAK1, JAK2, and STAT1.

Animals↗
PubMed1999

Synergistic interaction of catecholamine hormones and Mycobacterium avium results in the induction of interleukin-10 mRNA expression by murine peritoneal macrophages.

The results of this investigation provides evidence that catecholamine hormones interact with macrophages that are infected with Mycobacterium avium resulting in the induction of IL-10 mRNA and protein. The effect of catecholamine hormones was prevented by treating the cells with the beta-adrenergic receptor antagonist propranolol but not by alpha-adrenergic antagonist phentolamine. The effect of catecholamine stimulation was mimicked by the addition of beta-2 adrenergic agonists and by the addition of cAMP to the infected macrophage cultures. These observations suggest that sympathetic nervous system activation together with microbial infection results in a synergistic interaction that could result in the control of inflammatory processes.

Adrenergic alpha-Agonists↗
PubMed1999

Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169.

The transport of iron by RAW264.7 macrophage cell lines transfected with either Nramp1Gly169 (resistant) or Nramp1ASp169 (susceptible) alleles was assessed. We found no difference between resistant and susceptible cells in the rate of Fe import or export when Fe transport was measured in intact cells. In contrast, the rate of Fe import by latex-bead phagosomes isolated from resistant cells was more than double the rate by latex-bead phagosomes from susceptible cells. Similarly, phagosomes isolated from resistant cells that had been pre-labeled with 55Fe-citrate before phagocytosis contained up to four times as much Fe as the corresponding phagosomes from susceptible cells. Phagocytosis of Mycobacterium avium was accompanied by an increase in the production of hydroxyl radicals by Nramp1cGly169-transfected macrophages but not by macrophages transfected with the susceptible allele. These results are consistent with the hypothesis that Nramp1 functions to transport Fe into the bacterium-containing phagosome where it serves as a catalyst for the Haber-Weiss reaction, which accounts for the increased capacity of these cells to limit mycobacterial growth.

Animals↗
PubMed1999

The mouse p52 subunit of the transcription/DNA repair factor TFIIH is located in the class III region of the H2 complex: cloning and sequence polymorphism.

Loci controlling susceptibility to a number of diseases, including cortisone-induced cleft palate, experimental allergic orchitis, and chemically-induced transplacental lung tumors have been mapped to a 27 kilobase (kb) region within the class III region of the mouse major histocompatibility complex (H2). This region, contains three genes G7e, which resembles a viral envelope gene, Bat6 (G7a), which encodes a valyl-tRNA synthetase, and G7c, which has no known function. We cloned a set of overlapping cosmid clones containing 115 kb of DNA surrounding Bat6. Exon trapping has identified a new gene located telomeric of Bat6. Northern blot analysis detected a transcript of 1.7 kb with highest expression in the testis. DNA sequence analysis identified this gene as the mouse homologue of the human gene encoding the p52 subunit of the TFIIH transcription/DNA repair factor. Nucleotide sequence identity was 91% between mouse and human, and the protein sequence was 98% identical. Sequence analysis of p52 cDNA from congenic mouse strains detected an amino acid polymorphism at position 209, which results in the substitution of a threonine in the H2b haplotype to a methionine in the H2a,d haplotypes.

Amino Acid Sequence↗
PubMed1998

Host resistance to mycobacteria is compromised by activation of the hypothalamic-pituitary-adrenal axis.

Host resistance to the growth of Mycobacterium avium and Mycobacterium tuberculosis is controlled by a gene, termed Nramp1, that maps to chromosome 1 in mice. Activation of the HPA axis or treatment of macrophages from susceptible mice with corticosterone suppresses the expression of Nramp1 mRNA and results in an increased susceptibility to mycobacterial growth. In contrast, neither activation of the HPA axis nor treatment of macrophages from resistant mice with corticosterone results in an alteration in their resistance or suppression of Nramp1 expression. Investigation into the mechanism of the differential response of the macrophages to corticosterone indicated that differences were associated with the stability of the mRNA in macrophages from BCG-resistant mice. Thus, corticosterone induced the accelerated degradation of Nramp1 mRNA as well as mRNA of several other macrophage activation genes in macrophages from BCG-susceptible mice. Treatment of macrophages with corticosterone before the induction of Nramp1 resulted in the accelerated degradation of mRNA in macrophages from both resistant and susceptible mice. The Nramp1 gene product appears to protect the mRNA of macrophage activation genes from degradation induced by corticosterone by an iron-dependent mechanism.

Animals↗
PubMed1997

Stabilized expression of mRNA is associated with mycobacterial resistance controlled by Nramp1.

Control of innate resistance to the growth of mycobacteria is mediated by a gene termed Nramp1. Although the role of the protein product of Nramp1 in mediating resistance to mycobacterial growth is not known, the effect of the gene is pleiotropic and it has been suggested that the gene controls macrophage priming for activation. We have found that the functional capacity of macrophages from Mycobacterium bovis BCG-susceptible mice can be suppressed by corticosterone, while the function of macrophages from BCG-resistant mice remains unaffected. In this study, we show that corticosterone differentially affects the stability of mRNAs of several recombinant gamma interferon (rIFN-gamma)-induced genes. Treatment of macrophages from BCG-susceptible mice with corticosterone accelerates the decay of Nramp1 mRNA. The mRNA of IFN-gamma-induced genes of macrophages from BCG-resistant mice was more stable than the mRNA of macrophages from BCG-susceptible mice in the presence or absence of corticosterone. The results of this investigation suggest that Nramp1 acts by stabilizing the mRNA of genes associated with macrophage activation, thus accounting for the functional differences that have been attributed to these macrophage populations.

Animals↗
PubMed1996

Binding of alpha-adrenergic receptors stimulates the anti-mycobacterial activity of murine peritoneal macrophages.

The effects of adrenergic stimulation of the anti-mycobacterial activity of peritoneal macrophages was investigated. We found that epinephrine and norepinephrine stimulated macrophages to suppress the growth of Mycobacterium avium. Stimulation was mediated by binding to the alpha 2 adrenergic receptor. The addition of the alpha 2 agonist clonidine to cultures resulted in an inhibition of mycobacterial growth and the effect of epinephrine was blocked by the alpha-antagonist phentolamine. Treatment of the macrophages with propranolol, a beta-antagonist, potentiated the effect of epinephrine. Epinephrine mediates its effect by stimulating the expression of macrophage activation genes.

Animals↗
PubMed1995

Cloning and characterization of a novel cDNA that is IFN-gamma-induced in mouse peritoneal macrophages and encodes a putative GTP-binding protein.

Macrophage activation by IFN-gamma results in a cascade of gene expression. To identify genes activated in mouse peritoneal macrophages by IFN-gamma, we created a cDNA subtraction library of IFN-gamma-induced genes. We have isolated from this subtraction library a novel cDNA clone, called Mg21, whose mRNA is absent in unstimulated mouse peritoneal macrophages and is induced to high levels within 4 h following the addition of IFN-gamma. Induction of Mg21 mRNA by IFN-gamma occurred in the presence of cycloheximide, indicating that expression of Mg21 mRNA does not require protein synthesis. A small amount of Mg21 mRNA was also induced by LPS, but not by IL-2, IL-4, IL-10, or TNF-alpha. The DNA sequence of Mg21 is 1617 nucleotides and contains an open reading frame that codes for a protein of 415 amino acids with a predicted molecular weight of 47,106 Da. The predicted amino acid sequence lacks a signal sequence or transmembrane segments, indicating that the protein is an intracellular protein. Computer search of the GenBank and EMBL databases indicates that this cDNA clone is unique but has 57% sequence identity with IRG-47, which is a mouse gene induced by IFN-gamma in pre-B and B lymphocyte cell lines. IRG-47 encodes an intracellular protein that contains three conserved protein motifs present in GTP-binding proteins. Analysis of the protein sequence of Mg21 showed that these three conserved protein motifs are also present in Mg21.

Amino Acid Sequence↗
PubMed1995

IFN-gamma increases cathepsin H mRNA levels in mouse macrophages.

Expression of major histocompatibility complex (MHC) class II molecules and ability to present antigen to T lymphocytes is acquired upon activation of the macrophage by interferon-gamma (IFN-gamma). Little information is available concerning immune regulation of protease gene expression in mouse macrophages. We have isolated a cDNA clone for cathepsin H, a lysosomal cysteine proteinase from a cDNA subtraction library of mouse macrophage genes induced by IFN-gamma, and have characterized its expression. The level of cathepsin H mRNA increased in mouse peritoneal macrophages following addition of IFN-gamma. Cathepsin H mRNA levels began to increase 8 h after the addition of IFN-gamma and was maximal at 24-48 h. This increase was concordant in time with appearance of MHC class II E beta mRNA and Ia invariant chain mRNA. The increase in cathepsin H mRNA levels by IFN-gamma was dose dependent. Cycloheximide treatment of peritoneal macrophages inhibited the increase in cathepsin H mRNA levels induced by IFN-gamma, suggesting that the increase in cathepsin mRNA levels requires de novo protein synthesis. Lipopolysaccharide and cytokines interleukin-2 (IL-2), IL-4, IL-10, and tumor necrosis factor alpha were found to have no effect on cathepsin H mRNA levels in mouse peritoneal macrophages.

Amino Acid Sequence↗
PubMed1995

The cytotoxic T lymphocyte gene FIBLP with homology to fibrinogen beta and gamma subunits is also induced in mouse macrophages by IFN-gamma.

To identify genes induced in mouse macrophages by IFN-gamma, a cDNA subtraction library of IFN-gamma-induced genes was screened. One of the clones, 36F2, was identified by DNA sequencing as the FIBLP gene. The FIBLP (fibrinogen-like protein) gene is a T-lymphocyte-specific gene that is expressed in mouse cytotoxic T lymphocytes but not in helper T lymphocytes or B lymphocytes. The protein sequence shows a high homology to fibrinogen beta and gamma subunits. The FIBLP gene is not expressed in unstimulated mouse peritoneal macrophages but is induced by IFN-gamma to high levels. FIBLP mRNA is detected by 1 hr after the addition of IFN-gamma and maximal levels are reached by 12 hr. Expression of FIBLP mRNA was not induced by IL-2, IL-4, IL-10, or TNF-alpha. Though the function of this gene is unknown, its expression in both cytotoxic T lymphocytes and activated macrophages suggests that FIBLP may play an as yet undefined role in the cytotoxic function of these cells.

Animals↗