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Biomedical subjects

D Malo

Publications and source records attributed to D Malo.

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

Expression of the human NRAMP1 gene in professional primary phagocytes: studies in blood cells and in HL-60 promyelocytic leukemia.

In the mouse, mutations at the natural resistance-associated macrophage protein 1 (Nramp1) gene abrogate resistance to infection with antigenically unrelated intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Nramp1 expression is restricted to reticuloendothelial organs and peripheral blood leukocytes, where the protein may function as a membrane transporter of an as yet to be identified substrate. To identify the human blood cell type(s) expressing NRAMP1 mRNA and determine how Nramp1 expression is regulated in these cells, we have examined separated populations of peripheral blood leukocytes and in vitro cell lines. We observed that polymorphonuclear leukocytes (PMN) are the major site of NRAMP1 expression, followed to a lesser degree by monocytes (MN). Migration of MN to tissues (alveolar macrophages) or maturation in vitro (long-term culture) was associated with a higher level of NRAMP1 expression compared with blood MN. Northern analyses of RNA from model cultured cells showed absence of NRAMP1 expression in transformed cell lines from either erythroid or lymphoid T or B lineages as well as progenitors of the monocyte/macrophage pathway (KG1, U937, THP1), and the HL-60 promyelocytic leukemia. Induction of differentiation of HL-60 cells toward either the monocyte/macrophage (vitamin D3, phorbol ester) or the granulocyte pathways (DMF, DMSO), as measured by induction of IL8-Rb, c-FMS, and CD14 marker gene expression, was concomitant with a strong induction of NRAMP1 expression. These results suggest that NRAMP1 expression is specific to the myeloid lineage and is acquired during the maturation of PMN and MN. The possibility that NRAMP1 may be a component of the phagosomal/endosomal apparatus common to PMN and MN is discussed.

Base Sequence↗
PubMed1997

Resistance to salmonellosis in the chicken is linked to NRAMP1 and TNC.

Natural resistance to infection with Salmonella typhimurium in mice is controlled by two major loci, Bcg and Lps, located on mouse chromosomes 1 and 4, respectively. Both Bcg and Lps exert pleiotropic effects and contribute to cytostatic/cytocidal activities of the macrophage. Bcg encodes for a membrane phosphoglycoprotein designated Nrampl (natural resistance-associated macrophage protein 1), which belongs to an ancient family of membrane proteins, Lps has not been cloned yet, but its location on mouse chromosome 4 has been refined for positional cloning. As in mice, chicken inbred lines differ in their susceptibility to infection with Salmonella typhimurium. We have tested the candidacy of the chicken homologs of Nrampl and Tnc (a locus closely linked to Lps), in the differential resistance of chicken inbred lines to infection with S. typhimurium. We have first analyzed six inbred chicken lines of Salmonella-resistant or Salmonella-susceptible phenotypes for the presence of nucleotide sequence variations within the coding portion of NRAMP1. We have identified 11 sequence variations within NRAMP1 in the chicken inbred lines tested: 10 of these represented either silent mutations or conservative changes. However, one G-->A substitution at nucleotide 696 resulted in the nonconservative replacement of Arg223 to Gln223 within the predicted TM5-6 region. This allelic variant was specific to the susceptible line C and not observed in any of the resistant strains. To investigate the effect of NRAMP1 and TNC on resistance to infection with S. typhimurium, 425 (W1 x C)F1 x C chicken progeny were examined during a period of 15 days postinfection. Together, NRAMP1 and TNC explain 33% of the early differential resistance to infection with S. typhimurium of parental lines C and W1. Our data established that resistance to infection with S. typhimurium in chickens is inherited as a complex trait and that comparative mapping has proven to be useful to identify Salmonella-resistance genes in the chicken.

Amino Acid Sequence↗
PubMed1996

Fine genetic mapping of the Hyp mutation on mouse chromosome X.

The hypophosphatemic (Hyp) mouse is the murine homolog of hypophosphatemic vitamin-D-resistant rickets (HYP) in human. Despite extensive investigations in the Hyp mouse, the pathophysiology of this X-linked dominant disorder remains unclear. As a first step toward cloning the Hyp gene, we have generated a high-resolution linkage map in the vicinity of the Hyp locus using two independent backcross panels segregating the Hyp mutation, one generated from an interspecific mating between C57BL/6J-Hyp/Hyp and Mus spretus and the other from an intrasubspecific mating between C57BL/6J-Hyp/Hyp and Mus musculus castaneus. Linkage analyses in 1101 backcross progeny using a total of 23 DNA markers favor the following gene order from the centromere: DXMitl3-(DXMit11, DXMit34)-(DXMit36, Alas2)-(Hyp, DXMit8O)-DXMi198-(DXMit28, DXMit33, DXMit7O)-Pdhal-DXMit2O. This study has localized Hyp to a region of approximately 1 cM flanked by the proximal markers DXMit36 and Alas2 and the distal marker DXMit98. One microsatellite marker, DXMit8O, was found to be very tightly linked to Hyp, as it was nonrecombinant with Hyp among all the progeny of both backcrosses corresponding to 1101 meioses.

Animals↗
PubMed1996

A high-resolution map in the chromosomal region surrounding the Lps locus.

The Lps locus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria. The C3H/HeJ inbred mouse strain is characterized by a mutant Lps allel (Lpsd) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects. To identify the Lps gene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus. We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus x C57BL/6J)F1 x C57BL/6J and two novel panels of 597 (DBA/2J x C3H/HeJ)F1 x C3H/HeJ and 748 (C57BL/6J x C3H/HeJ)F1 x C3H/HeJ segregating at Lps. A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping the Lps locus. This positions the Lps locus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three know genes (Cd301, Hxb, and Ambp), and distally by two microsatellite markers (D4Mit7/D4Mit178). The localization of the Lps locus is several centimorgans proximal to that previously assigned.

Animals↗
PubMed1996

Structural organization, sequence, and expression of the chicken NRAMP1 gene encoding the natural resistance-associated macrophage protein 1.

One of the most common causes of food poisoning in humans is salmonellosis, which is frequently caused by ingestion with Salmonella-contaminated poultry products. Several lines of evidence suggest that genetic factors control resistance and susceptibility of chickens to infection with Salmonellae. In the mouse, innate resistance to infection with intracellular pathogens such as Salmonella typhimurium, several species of Mycobacteria, and Leishmania donovani is controlled by the mouse chromosome 1 Nramp1Bcg gene. To investigate the role of NRAMP1 in the differential resistance and susceptibility of chickens to infections with S. typhimurium, we have cloned and characterized cDNA clones corresponding to the chicken NRAMP1 gene. Nucleotide and predicted amino acid sequence analyses indicate that the chicken NRAMP1 polypeptide encodes a 555-amino-acid residue membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif. The peptide sequence identity among chicken, mouse, and human NRAMP1 is 68%. The chicken NRAMP1 gene contains 15 exons and spans 5 kb of genomic DNA. One major and two minor transcription initiation sites were detected using primer extension. Nucleotide sequencing of the promoter region revealed the presence of a classical TATAA element and consensus sequences for binding the myeloid specific PU.1 factor and several lipopolysaccharide (LPS) (NF-IL6 and NF-kappa B) and interferon-gamma (IFN-gamma)-inducible response elements. Similar regulatory elements are found in the promoters of mouse and human NRAMP1. Northern blot analyses revealed NRAMP1 expression in reticuloendothelial organs (spleen and liver), lung, and thymus. As demonstrated in mice and humans, the macrophage is also a major site of NRAMP1 mRNA expression in chickens. However, the high levels of expression detected in chicken thymus contrast with the absence of expression of the mammalian Nramp1 gene in this tissue.

Amino Acid Sequence↗
PubMed1996

The Bcg/Ity/Lsh locus: genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1 Gly169 allele.

The murine Bcg/Ity/Lsh locus determines the susceptibilities of inbred strains to infection with unrelated intracellular parasites, such as Mycobacterium bovis, Salmonella typhimurium, and Leishmania donovani. A candidate for Bcg/Ity/Lsh, designated Nramp1, has been recently identified and shown to encode a novel integral membrane protein that is expressed exclusively in professional phagocytes but whose function remains unknown. In inbred strains, the susceptibility to infection is associated with a single glycine-to-aspartic acid substitution at position 169 (G169D) in the predicted TM4 of the protein. To confirm the candidacy of Nramp1 as Bcg/Ity/Lsh and to determine the importance of the G169D mutation on Nramp1 function, we constructed transgenic mice in which the G169 allele of Nramp1 was transferred onto the background of a homozygous D169 allele. These transgenic mice were analyzed for their sensitivity to infections under the control of Bcg/Ity/Lsh. The transgene constructed for these studies contained the entire Nramp1G169 gene together with approximately 5 kb of sequences upstream of the transcription initiation site of this gene. We observed that these sequences were sufficient to direct Nramp1G169 expression in transgenic macrophages, resulting in the appearance of a mature protein of 90 to 100 kDa over a background of Nramp1G169 characterized by the complete absence of the mature Nramp1 polypeptide. The appearance of the Nramp1G169-encoded protein in transgenic macrophages was concomitant with the emergence of resistance to infection by M. bovis BCG, as measured by the extent of bacteria] replication in the spleen, and by S. typhimurium, as measured by survival after an intravenous challenge. The gain of function detected in transgenic Nramp1G169 animals establishes unambiguously that Nramp1 and Bcg/Ity/Lsh are allelic.

Alleles↗
PubMed1995

The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.

In mice, natural resistance or susceptibility to infection with intracellular parasites is determined by a locus or group of loci on chromosome 1, designated Bcg, Lsh, and Ity, which controls early microbial replication in reticuloendothelial organs. We have identified by positional cloning a candidate gene for Bcg, Nramp1, which codes for a novel macrophage-specific membrane transport protein. We have created a mouse mutant bearing a null allele at Nramp1, and we have analyzed the effect of such a mutation on natural resistance to infection. Targeted disruption of Nramp1 has pleiotropic effects on natural resistance to infection with intracellular parasites, as it eliminated resistance to Mycobacterium bovis, Leishmania donovani, and lethal Salmonella typhimurium infection, establishing that Nramp1, Bcg, Lsh, and Ity are the same locus. Comparing the profiles of parasite replication in control and Nramp1-/- mice indicated that the Nramp1Asp169 allele of BcgS inbred strains is a null allele, pointing to a critical role of this residue in the mechanism of action of the protein. Despite their inability to control parasite growth in the early nonimmune phase of the infection, Nramp1-/- mutants can overcome the infection in the late immune phase, suggesting that Nramp1 plays a key role only in the early part of the macrophage-parasite interaction and may function by a cytocidal or cytostatic mechanism distinct from those expressed by activated macrophages.

Alleles↗
PubMed1995

Genomic structure, promoter sequence, and induction of expression of the mouse Nramp1 gene in macrophages.

A candidate gene for the mouse chromosome 1 host resistance locus Bcg/Ity/Lsh was recently cloned and designated Nramp (natural resistance-associated macrophage protein). Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. Primer extension and cDNA cloning were used to isolate the complete 5' end of the Nramp1 mRNA. Analysis of genomic cosmid and bacteriophage clones overlapping the complete Nramp1 gene revealed that the gene was composed of 15 exons and spanned 11.5 kb of genomic DNA. Positioning of introns on the coding portion of the mRNA revealed a modular relationship between coding exons and predicted structural domains of the protein, with 8 of the 12 transmembrane (TM) domains encoded by individual exons. Northern blotting analysis indicated that Nramp1 expression was restricted to J774A.1 and RAW 264.7 macrophage lines and was dramatically increased by treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Primer extension and S1 nuclease mapping experiments were used to locate the transcription initiation site of Nramp1 and revealed the presence of one major and several minor initiation sites. Nucleotide sequencing of the corresponding region failed to detect classical TATA and CAAT elements, but identified two putative initiator sequences located near the major initiation site. Consensus sequences for binding of the macrophage and B-cell-specific transcription factor PU.1, as well as several LPS (NF-IL6) and IFN-gamma response elements, were also identified.

Amino Acid Sequence↗
PubMed1995

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken.

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp 1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.

Animals↗
PubMed1994

Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites.

The mouse chromosome 1 locus Bcg (Ity, Lsh) controls the capacity of the tissue macrophage to restrict the replication of antigenically unrelated intracellular parasites and therefore determines the natural resistance (BCG-R, dominant) or susceptibility (BCG-S, recessive) of inbred mouse strains to infection with diverse pathogens, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Bcg (Nramp) that encodes a predicted macrophage-specific transport protein. We have analyzed a total of 27 inbred mouse strains of BCG-R and BCG-S phenotypes for the presence of nucleotide sequence variations within the coding portion of Nramp and have carried out haplotype typing of the corresponding chromosome 1 region in these mice, using 11 additional polymorphic markers mapping in the immediate vicinity of Nramp. cDNA cloning and nucleotide sequencing identified 5 nucleotide sequence variations within Nramp in the inbred strains; while 4 of these represented silent sequence polymorphisms, one G to A substitution at nucleotide position 783 resulted in the non-conservative replacement of Gly105 to Asp105 within the second predicted transmembrane domain (TM2) of the Nramp protein. An absolute association of this allelic variation and Bcg phenotype was observed in the 20 BCG-R strains (Gly105) and 7 BCG-S strains (Asp105) tested. Moreover, sequence analysis of the corresponding region of the Nramp gene from distantly related species indicated strong amino acid sequence conservation of TM2, including an invariant glycine at position 105. Haplotype mapping using sequence polymorphism identified within Nramp and additional RFLPs and SSLPs from the region revealed that although the 20 BCG-R strains analyzed showed diverse allelic combinations for these markers, the 7 BCG-S strains tested share a conserved core haplotype of 2.2 Mb overlapping and including Nramp. Taken together, these results suggest that (1) Gly105 is the wildtype form of Nramp and that the nonconservative substitution to Asp105 underlies the BCG-S phenotype, and (2) Bcg8 alleles carry the same Gly105-->Asp105 mutation and are identical by descent.

Alleles↗