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K J Moore

Publications and source records attributed to K J Moore.

At least 37 records · Page 2Linked to original sources
PubMed2000

Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages.

Gene deletion studies indicate that the macrophage scavenger receptor A (SR-A) protects mice from LPS-induced endotoxemia. Paradoxically, cultured human monocyte-derived macrophages down-regulate SR-A expression when exposed to LPS. We found that human THP-1 monocyte/macrophages decrease SR-A expression in response to LPS independent of their differentiation status. In contrast, primary and elicited mouse peritoneal macrophages as well as the J774A.1 and RAW264.7 mouse macrophage lines increase SR-A expression in response to LPS. Exposure to LPS caused J774A.1 and RAW264.7 cells to increase SR-A transcripts by 3- and 5-fold, respectively. LPS caused a concomitant 3-fold increase in SR-A protein levels and increased cell membrane expression of the receptor. RAW264.7 cells increased SR-A transcript levels in response to LPS at concentrations as low as 1 ng/ml, and the response was saturated at 10 ng/ml. The LPS induction of SR-A transcripts required continual protein synthesis and began at 8 h, peaked by 16 h, and persisted for at least 48 h. LPS induction did not increase SR-A gene transcription or affect alternative transcript splicing, but mildly increased mature transcript stability and proceeded in the presence of actinomycin D. Finally, treatment of RAW264.7 cells with TNF-alpha did not induce SR-A transcript levels, indicating that a TNF-alpha autocrine/paracrine signaling mechanism alone is not sufficient to recapitulate the LPS induction of SR-A transcripts. The induction of SR-A expression by LPS-stimulated mouse macrophages is the opposite of the down-regulation of SR-A reported in human monocyte-derived macrophages and may have implications for the observed resistance mice show toward endotoxemia.

Alternative Splicing↗
PubMed2000

Stromelysin-1 (MMP-3) expression driven by a macrophage-specific promoter results in reduced viability in transgenic mice.

Macrophage expression of matrix degrading metalloproteinases (MMPs) in human atheroma has been found to occur in rupture-prone areas of plaques. To investigate the effect of metalloproteinase activity on plaque stability, we attempted to generate mice that expressed a stromelysin-1 (MMP-3) transgene specifically in macrophages. Promoter sequences taken from a macrophage-tropic lentivirus (visna) were used to drive transgene expression. The transgene construct was expressed in macrophages in vitro and its autoactivation was established by casein zymography. Transgenic mice generated with this construct died at or before birth. No gross anatomical changes were observed in these mice. Embryos arising from a second round of oocyte injections with the transgene were examined at day 16 of gestation. Of the products of conception, approximately 40% resulted in vacant conceptuses. Only one animal of 38 examined carried the transgene and its expression of MMP-3 mRNA at E16 was faintly detected by RT-PCR. When a non-toxic reporter gene, luciferase, was substituted for the MMP-3 cDNA, healthy transgenic mice were produced that expressed the reporter gene in a wide variety of tissue macrophages, including those located in the brain, testis, lung, and thymus. These studies suggest that constitutive expression of MMP-3 in diverse populations of tissue macrophages leads to prenatal or neonatal death in the mouse. It appears likely that more sophisticated transcriptional control of MMP-3 expression will be required in order to generate stromelysin-1 transgenic mice that could be useful models for studying overexpression of this metalloproteinase's activity in the lesional macrophages of atherosclerotic plaques.

Animals↗
PubMed2000

Complex trait analysis in the mouse: The strengths, the limitations and the promise yet to come.

In 1990, David Baltimore predicted that the 1990s would be the decade of the mouse (). This certainly proved to be true: The mouse has contributed immensely to biological research through transgenic, embryonic stem cell (ES) knockout, and classical genetic technologies. But its usefulness as a model organism is by no means over; indeed it is still rising to its peak: The mouse as a model mammalian organism still has much to offer. This article reviews use of the mouse to dissect complex genetic traits using quantitative trait analysis, with a particular emphasis on medically important diseases.

Animals↗
PubMed1999

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.

Adenosine Diphosphate↗
PubMed1999

The mahogany protein is a receptor involved in suppression of obesity.

Genetic studies have shown that mutations within the mahogany locus suppress the pleiotropic phenotypes, including obesity, of the agouti-lethal-yellow mutant. Here we identify the mahogany gene and its product; this study, to our knowledge, represents the first positional cloning of a suppressor gene in the mouse. Expression of the mahogany gene is broad; however, in situ hybridization analysis emphasizes the importance of its expression in the ventromedial hypothalamic nucleus, a region that is intimately involved in the regulation of body weight and feeding. We present new genetic studies that indicate that the mahogany locus does not suppress the obese phenotype of the melanocortin-4-receptor null allele or those of the monogenic obese models (Lep(db), tub and Cpe(fat)). However, mahogany can suppress diet-induced obesity, the mechanism of which is likely to have implications for therapeutic intervention in common human obesity. The amino-acid sequence of the mahogany protein suggests that it is a large, single-transmembrane-domain receptor-like molecule, with a short cytoplasmic tail containing a site that is conserved between Caenorhabditis elegans and mammals. We propose two potential, alternative modes of action for mahogany: one draws parallels with the mechanism of action of low-affinity proteoglycan receptors such as fibroblast growth factor and transforming growth factor-beta, and the other suggests that mahogany itself is a signalling receptor.

Amino Acid Sequence↗
PubMed1999

Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

Amino Acid Sequence↗
PubMed1999

Genetic background determines the extent of atherosclerosis in ApoE-deficient mice.

Two strains of ApoE-deficient mice were found to have markedly different plasma lipoprotein profiles and susceptibility to atherosclerosis when fed either a low-fat chow or a high-fat Western-type diet. FVB/NJ ApoE-deficient (FVB E0) mice had higher total cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels when compared with C57BL/6J ApoE-deficient (C57 E0) mice. At 16 weeks of age, mean aortic root atherosclerotic lesion area was 7- to 9-fold higher in chow diet-fed C57 E0 mice and 3.5-fold higher in Western diet-fed C57 E0 mice compared with FVB E0 mice fed similar diets. Lesion area in chow diet-fed first-generation mice from a strain intercross was intermediate in size compared with parental values. The distribution of the lesion area in 150 chow diet-fed second-generation progeny spanned the range of the lesion area in both parental strains. There were no correlations between total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, ApoA2, ApoJ, or anti-cardiolipin antibodies and lesion area in the second-generation progeny. Thus, a genomic approach may succeed in identifying the genes responsible for the variation in atherosclerosis susceptibility in these 2 strains of ApoE-deficient mice, which could not be explained by measured plasma parameters.

Animals↗
PubMed1998

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. I: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques.

The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E.Ile-AMP; Scheme 1). Isoleucine (Ile) binds to the E.ATP complex (K4 = 1.7 +/- 0.9 microM) approximately 35-fold more tightly than to E (K1 = 50-100 microM), primarily due to a reduction in the Ile dissociation rate constant (k-1 approximately 100-150 s-1, cf. k-4 = 3 +/- 1.5 s-1). Similarly, ATP binds more tightly to E.Ile (K3 = approximately 70 microM) than to E (K2 = approximately 2.5 mM). The formation of the E.isoleucyl adenylate intermediate, E.Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k+5 = approximately 60 s-1) and the reverse rate constant (k-5 = approximately 150-200 s-1) to be determined from pyrophosphorolysis of a pre-formed E.Ile-AMP complex (K6 = approximately 0.25 mM). Scheme 1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme 1 could also provide an adequate description of the kinetics of tRNA aminoacylation (kcat,tr = approximately 0.35 s-1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.

Adenosine Triphosphate↗
PubMed1998

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. II. Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics.

The interactions of isoleucyl-tRNA synthetase (IleRS, E) from Staphylococcus aureus with both intermediate analogues and pseudomonic acid (PS-A) have been investigated using transient and steady-state techniques. Non-hydrolyzable analogues of isoleucyl-AMP (I) were simple competitive inhibitors (Ile-ol-AMP, Ki = 50 nM and Ile-NHSO2-AMP, Ki = 1 nM;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E.J (Kj = approximately 2 nM), undergoes an isomerization to form a stabilized E*.J complex (K*j = 50 pM). To overcome tight-binding artifacts when K*j << [E], K*j values were estimated from PPi/ATP exchange where [S] >> Km, thus raising K*j,app well above [E]. Using [3H]PS-A, it was confirmed that binding occurs with 1:1 stoichiometry and is reversible. Formation of inhibitor complexes was monitored directly through changes in enzyme tryptophan fluorescence. For Ile-ol-AMP and Ile-NHSO2-AMP, the fluorescence intensity of E.I was identical to that when E.Ile-AMP forms catalytically. Binding of PS-A induced only a small change in IleRS fluorescence that was characterized using transient kinetic competition. SB-205952, a PS-A analogue, produced a 37% quenching of IleRS fluorescence upon binding as a result of radiationless energy transfer. Inhibitor reversal rates were obtained by measuring relaxation between spectroscopically different complexes. Together, these data represent a comprehensive solution to the kinetics of inhibition by these compounds.

Anti-Bacterial Agents↗
PubMed1998

Effects of substrate and inhibitor binding on proteolysis of isoleucyl-tRNA synthetase from Staphylococcus aureus.

Binding of ligands to isoleucyl-tRNA synthetase (IleRS; E) from Staphylococcus aureus was investigated through effects on proteolytic digestion. Approximately 50-fold higher concentrations of protease (trypsin or chymotrypsin) were required to inactivate IleRS after incubation with substrates and formation of the E. Ile-AMP intermediate compared with free E. Binding of pseudomonic acid A (PS-A) or isoleucynol adenylate (Ile-ol-AMP) also induced resistance to proteolysis and altered the patterns of IleRS cleavage fragments in an inhibitor-class specific manner. The determinants for PS-A binding were investigated via proteolysis of E.[3H]PS-A. Limited proteolysis of E.[3H]PS-A (excising residues 186-407) could be achieved without significant loss of bound inhibitor, eliminating this region as contributing to inhibitor binding. Assays were developed which allowed IleRS proteolysis to be readily followed using fluorescence polarization. Inhibitor-protected IleRS was labeled with fluorescein isothiocyanate with only a small effect upon catalytic activity (Fl-IleRS). The (pseudo) kinetics of proteolytic cleavage of Fl-IleRS could be measured at low nanomolar Fl-IleRS concentrations in 96/384-well microtiter plates, allowing real-time monitoring of dose-dependent protection from proteolysis. Thus, inhibitor (and substrate) binding could be reproducibly assessed in the absence of measurements of catalytic acitvity. This could potentially form the basis of novel screening assays for ligands to other proteins.

Adenosine Triphosphate↗
PubMed1998

Chromosomal localization and genomic characterization of the mouse melastatin gene (Mlsn1).

We recently described a novel gene, melastatin, whose expression is inversely correlated with melanoma aggressiveness. Chromosomal localization of this gene places it on mouse chromosome 7 and in the 15q13-q14 region of the human genome. Although expression patterns and chromosomal localization in the mouse are consistent with involvement of melastatin mutations in the mouse ruby-eye-2 defect, congenic analysis showed genetic segregation of the two loci. Cloning of the full-length human cDNA revealed a much larger transcript than we had previously identified, corresponding to a 1533-amino-acid protein product with homology to members of the transient receptor potential (Trp) family of calcium channels. The mouse melastatin gene contains 27 exons and spans at least 58 kb of genomic DNA. The promoter region of Mlsn1 contains four potential microphthalmia binding sites including an M box, a transcriptional regulatory element unique to genes with a restricted melanocytic expression pattern. A 1-kb PvuII fragment from this region was capable of driving high levels of luciferase expression in B16 melanoma cells.

Amino Acid Sequence↗
PubMed1998

Mahogany (mg) stimulates feeding and increases basal metabolic rate independent of its suppression of agouti.

The mahogany (mg) locus originally was identified as a recessive suppressor of agouti, a locus encoding a skin peptide that modifies coat color by antagonizing the melanocyte-stimulating hormone receptor or MC1-R. Certain dominant alleles of agouti cause an obesity syndrome when ectopic expression of the peptide aberrantly antagonizes the MC4-R, a related melanocyte-stimulating hormone receptor expressed in hypothalamic circuitry and involved in the regulation of feeding behavior and metabolism. Recent work has demonstrated that mg, when homozygous, blocks not only the ability of agouti to induce a yellow coat color when expressed in the skin of the lethal yellow mouse (AY), but also the obesity resulting from ectopic expression of agouti in the brain. Detailed analysis of mg/mg AY/a animals, presented here, demonstrates that mg/mg blocks the obesity, hyperinsulinemia, and increased linear growth induced by ectopic expression of the agouti peptide. Remarkably, however, mg/mg did not reduce hyperphagia in the AY/a mouse. Furthermore, mg/mg induced hyperphagia and an increase in basal metabolic rate in the C57BL/6J mouse in the absence of AY. Consequently, although mahogany is broadly required for agouti peptide action, it also appears to be involved in the control of metabolic rate and feeding behavior independent of its suppression of agouti.

Agouti Signaling Protein↗
PubMed1998

Neurofibromatosis pseudogene amplification underlies euchromatic cytogenetic duplications and triplications of proximal 15q.

Cytogenetically visible interstitial duplications of proximal 15q, which lack the Prader-Willi Angelman critical region (PWACR) frequently segregate in families without phenotypic effect, but the nature of the extra euchromatin has remained unclear. We used comparative genome hybridisation to confirm that the extra material in a cytogenetic triplication originated from proximal 15q. A PAC clone containing sequences specific for the type-1 neurofibromatosis (NF-1) pseudogenes, which map to 15q11.2, hybridised along the length of the enlarged region between the PWACR and the centromere. Computerised measurement of the fluorescent signal from the enlarged and normal chromosomes gave an average ratio of 9.85:1, consistent with amplification. In a second family, an amplified P1-4 signal co-segregated with a cytogenetic duplication and the average ratio between amplified and normal signals in the proband was 8.22:1. Ratios in noncarrier family members and control individuals were close to unity in most cases, but significantly greater than one in at least one instance. Our results provide a novel explanation for cytogenetic variation in 15q11.2. They also suggest that NF-1 pseudogene copy number may be polymorphic in the normal population, and that high copy numbers can produce G bands which do not reflect those of the normal constitutional karyotype.

Adolescent↗
PubMed1998

Gene transfer of RANTES elicits autoimmune renal injury in MRL-Fas(1pr) mice.

We report that the beta-chemokine RANTES, a chemoattractant for macrophages and T cells, is up-regulated in the MRL-Fas(1pr) kidney prior to injury, but not normal kidneys (MRL-++, C3H-++) and increases with progressive injury. Furthermore, we establish an association between RANTES expression in the kidney and renal damage using a gene transfer approach. Tubular epithelial cells genetically modified to secrete RANTES infused under the renal capsule incites interstitial nephritis in MRL-Fas(1pr), but not MRL-++ or C3H-++ mice. RANTES recruits predominantly macrophages (M phi) and CD4+ and CD8+ T cells. In contrast, gene transfer of CSF-1, another molecule up-regulated simultaneously with RANTES in MRL-Fas(1pr) kidneys, promotes the influx of M phi, CD4+ T cells and the unique double-negative (DN) T cells (CD4-, CD8-), which are prominent in diseased MRL-Fas(1pr) kidneys. Thus, RANTES and CSF-1 recruit distinct T cell populations into the MRL-Fas(1pr) kidney. In addition, delivery of RANTES and CSF-1 into the kidney of MRL-Fas(1pr) mice causes an additive increase in pathology. We suggest that the complementary recruitment of T cell populations by RANTES (CD4, CD8) and CSF-1 (CD4, DN) promotes autoimmune nephritis in MRL-Fas(1pr) mice.

Animals↗