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J Vacher

Publications and source records attributed to J Vacher.

At least 19 recordsLinked to original sources
PubMed2005

Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice.

To develop specific conditional gene ablation in the hematopoietic myeloid-osteoclast lineage, transgenic mice expressing Cre recombinase under the control of the CD11b promotor were generated on the C57BL/6 background. The cellular specificity of Cre activity following recombination was quantified in the Z/EG reporter transgenic mice by FACS analysis with lineage-specific markers and EGFP coexpression. A high degree of recombination, as evidenced by EGFP-positive cells, was demonstrated in macrophages and granulocytes of bone marrow and spleen by the presence of double-positive cells CD11b/EGFP and Gr1/EGFP, respectively. Interestingly, the peritoneal macrophage population showed almost complete DNA recombination at large. Most important, mature osteoclast cells derived from the double transgenic bone marrow and spleen progenitors were EGFP-positive. Hence, these CD11b-Cre mice will provide a unique tool to unravel novel gene function and activities involved during osteoclast and macrophage differentiation and maturation processes.

Animals↗
PubMed2001

Physical and transcriptional map of the mouse Chromosome 10 proximal region syntenic to human 6q16-q21.

Toward the isolation of the grey-lethal (gl) gene, we have genetically localized this locus on mouse Chromosome (Chr) 10 between the Fyn gene and the D10Mit148 microsatellite marker. Here, we have screened five yeast artificial chromosome (YAC) libraries and isolated more than 100 YAC clones mapping to this region. Forty-two clones were characterized and assembled in an approximately 8.5 megabases (Mb) contig showing high linkage conservation with the human 6q16-q21 interval. During this study, 24 specific novel sequence-tagged sites (STSs) were derived from YAC insert ends, and 15 mouse genes were precisely mapped to the contig. The physical and transcriptional map presented here will provide novel resources to isolate the gl locus associated with osteopetrosis, and will also provide candidate loci for other defects mapped on human Chr 6q.

Animals↗
PubMed2001

The mouse osteopetrotic grey-lethal mutation induces a defect in osteoclast maturation/function.

The osteopetrotic grey-lethal (gl) mouse mutant displays many similarities to the human malignant autosomal-recessive form of osteopetrosis. In this study, we show that the gl osteopetrotic bone phenotype is characterized by the presence of numerous differentiated multinucleated osteoclasts. A significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was detected in vivo, suggesting induction of differentiation in the osteoclast lineage as a compensatory mechanism. These gl osteoclast cells demonstrated a defective cytoskeletal reorganization and an underdeveloped ruffled border, a membrane structure essential for active bone resorption. Accordingly, resorption activity of these cells is markedly impaired by four- to tenfold as evaluated with the pit formation assay. This low bone resorption in gl osteoclasts is highly reminiscent of the loss in key enzymes, V-ATPase or cathepsin-K, and in signaling factors, Src or TRAF-6, which were shown not to be significantly altered in gl osteoclasts. Thus, independently of a deficiency in V-ATPase, Src, cathepsin-K, and TRAF-6, the gl mutation results in increased number of osteoclasts, characterized by a disrupted cytoskeleton and an underdeveloped ruffled border.

Acid Phosphatase↗
PubMed2001

Role of c-Src in the regulation of vascular contraction and Ca2+ signaling by angiotensin II in human vascular smooth muscle cells.

OBJECTIVE: Tyrosine kinases, typically associated with growth-signaling pathways, also play a role in Ang II-stimulated vascular contraction. However the specific kinases involved are unclear. We hypothesize here that c-Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascular smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contraction induced by Ang II. METHODS: Cultured VSMCs from resistance arteries of healthy subjects were studied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-deficient VSMCs from small arteries of c-Src knockout mice (Src-/-mVSMCs) were also investigated. Intracellular free Ca2+ concentration ([Ca2+]i), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioimmunoassay respectively. Contraction was examined in intact rat small arteries. RESULTS: Ang II rapidly increased VSMC c-Src activity, with peak responses obtained at 1 min. Ang II induced a biphasic [Ca2+]i response (Emax = 636 +/- 123 nmol/l). The initial [Ca2+]i transient, mediated primarily by Ca2+mobilization, was dose-dependently attenuated by the selective Src inhibitor, PP2, but not by PP3 (inactive analogue). Ang II-elicited [Ca2+]i responses were blunted in cells electroporated with anti-c-Src antibodies and in c-Src-/-mVSMCs. Src inhibition decreased Ang II-induced generation of IP3 in human VSMCs. Ang II dose-dependently increased vascular contraction (Emax = 40 +/- 6.5%). These responses were attenuated by PP2 (Emax = 7.8 +/- 0.08%) but not by PP3 (Emax = 35 +/- 4.5%). CONCLUSIONS: Our findings identify c-Src as an important regulator of VSMC [Ca2+]i signaling and implicate a novel contractile role for this non-receptor tyrosine kinase in Ang II-stimulated vascular smooth muscle.

Angiotensin II↗
PubMed1999

Genetic localization and transmission of the mouse osteopetrotic grey-lethal mutation.

The grey-lethal (gl) mouse is the most relevant animal model for recessive osteopetrosis, a genetic defect affecting bone resorption. To localize the gl gene, two novel backcrosses between the gl mutant strain GL/Le dlJ +/+ gl and with the Mus spretus or the Mus m. molossinus have been generated and typed with 19 DNA markers representative of genes or microsatellites. In the Mus m. molossinus backcross, the gl locus cosegregates with the D10Mit108,109,184,193, 254,255 markers within a 1 centimorgan genetic interval between the markers (D10Mit54,55,215,Cd24a) and D10Mit148. Our results have also eliminated all the five candidate genes previously localized to this region (Braf-rs1, Fyn, Cd24a, Ros1, and Gja1). On the Mus spretus background, segregation distortion due to a approximately threefold differential survival resulted in a severe deficit in gl/gl animals, indicating the presence of modifier genes. We have also characterized nine cosegregating microsatellite markers closely linked to gl as defined by their specific polymorphisms for the Chromosome (Chr) 10 harboring the gl mutation. Screening of several mouse inbred strains for these polymorphic markers revealed an identical pattern between gl and 129/SvEms, suggesting that the gl mutation arose on this genetic background. The linkage between this polymorphic region and the gl locus provides an entry point to produce a physical map to isolate the gl gene.

Alleles↗
PubMed1998

alpha-Fetoprotein gene sequences mediating Afr2 regulation during liver regeneration.

alpha-fetoprotein (AFP) gene expression occurs in the yolk sac, in the fetal liver and gut, and in the adult liver during regeneration and tumorigenesis. Two unlinked genes determine the level of AFP gene expression in adult mice: Afr1 regulates the basal level of expression in the normal adult liver, and Afr2 regulates the increase in expression during liver regeneration. It has been shown that AFP-derived transgenes, including the sequences between -1,010 and -838 bp and between -118 bp and the transcriptional start site were induced appropriately during liver regeneration and were Afr2-regulated. To assess the role of the distal sequence in gene expression during liver regeneration, a new transgene with 7.6 kilobases of 5'-flanking sequence deleted between -1,010 and -838 bp was designed. We show that this transgene was subject to characteristic AFP tissue-specific and developmental regulation, in that it was highly expressed in the yolk sac and the fetal liver and gut but not in normal adult tissues. Expression was induced in response to liver regeneration as observed for the endogenous gene. The genetic regulation of the basal level of AFP gene expression in adult liver by the Afr1 gene was undisturbed. However, transgene expression was not regulated by Afr2 during liver regeneration. Our data suggest that Afr2 regulation of AFP gene expression during liver regeneration requires the sequence between -1,010 and -838 bp and is independent of other regulatory mechanisms.

Animals↗
PubMed1995

Developmental regulation of alpha-fetoprotein expression in intestinal epithelial cells of transgenic mice.

The alpha-fetoprotein (AFP) gene is transcribed in most epithelial cells lining the fetal mouse small intestine, but transcription persists in only a subset of enteroendocrine cells representing less than 1% of the total intestinal epithelial cells in the adult. The decrease in AFP expression after birth is mediated in part by a repressor element lying between -838 and -250 bp of the AFP gene. Deletion of this element from AFP minigene constructs results in high-level minigene expression in the intestines of adult transgenic mice. Although high levels of AFP minigene RNA are expressed, the fetal pattern of expression is not maintained upon deletion of the repressor element. Instead, the number of cells in which the minigene is expressed increases from less than 1% to approximately 10% of the epithelial cells in the adult small intestine, and includes the majority of the goblet cells in addition to the enteroendocrine cells. In contrast, the pattern of AFP minigene expression in the enterocytes is unaffected by deletion of the repressor element and continues to decrease in the neonate. These studies indicate that the identified AFP repressor is active specifically in goblet cells. The decrease in AFP expression in the enterocytes may be mediated by a separate cis-acting element that is contained in the AFP minigene construct. Alternatively, it is possible that mature enterocytes lack some of the positive factors required for initiation and maintenance of minigene transcription in the absence of the identified negative element.

Animals↗
PubMed1995

Sequence requirements for alpha-fetoprotein gene expression during liver regeneration.

The alpha-fetoprotein (AFP) gene is expressed in fetal liver and in adult liver undergoing regeneration or tumorigenesis. It has been shown previously that three distal enhancers, a proximal promoter, and a dominant negative postnatal repressor element are required for the tissue-specific and developmental regulation of AFP gene expression. Using transgenic mice, we have determined the sequence requirements for AFP gene induction during liver regeneration. Two DNA sequences were found in all transgenes appropriately regulated in response to liver regeneration: a distal sequence between 1010 and 838 bp upstream of the structural gene and a proximal sequence within 118 bp of the transcriptional initiation site. In situ hybridization analysis showed that transgene expression during liver regeneration was first found in all hepatocytes and then localized to perinecrotic hepatocytes surrounding the central vein. This pattern of expression is reminiscent of that observed after birth for the transgenes, suggesting that repression of AFP gene expression after birth and liver injury may be regulated by similar mechanisms.

Animals↗
PubMed1992

The zonal expression of alpha-fetoprotein transgenes in the livers of adult mice.

The developmental regulation of the alpha-fetoprotein (AFP) gene in liver results in high-level expression in the fetus, followed by dramatic transcriptional repression after birth. We have examined the mouse AFP gene for transcriptional control sequences that may be involved in its postnatal repression in liver. We showed previously that removal of a DNA region between positions -250 base pairs (bp) and -838 bp of the AFP gene resulted in the persistence of expression of an AFP minigene in the postpartum liver of transgenic mice (Vacher and Tilghman, Science 250:1732-1735, 1990). This study examines the distribution of these transgene transcripts in liver using in situ hybridization. We show that there is a zonal distribution of minigene transcripts in the adult livers of these animals. Hepatocytes surrounding the central veins express high levels of minigene transcripts, while hepatocytes in the intermediate and portal areas contain few, if any, transcripts. Quantitative RNAse protection analysis shows a decrease in transgene RNA levels after birth, consistent with repression in all but a small subset of hepatocytes. These results indicate that repression in the pericentral hepatocytes is dependent upon the presence of a cis-acting, negative-regulatory domain, which is located between the enhancers and the proximal promoter of the AFP gene. In contrast, this domain is not essential for complete repression of AFP transgenes in the intermediate zone and periportal hepatocytes.

Animals↗
PubMed1992

raf regulates the postnatal repression of the mouse alpha-fetoprotein gene at the posttranscriptional level.

The mouse alpha-fetoprotein (AFP) gene is transcribed at a high rate in liver during the second half of gestation. Its steady-state mRNA levels decrease 10(4)-fold shortly after birth, at least in part as the consequence of a dramatic decrease in its transcription rate. The final basal level of AFP mRNA in adult liver is influenced by a trans-acting locus on chromosome 15 termed raf. Two strategies were used to demonstrate that the raf gene acts posttranscriptionally to affect the processing and/or stability of AFP transcripts. Transgenic mouse studies demonstrated that raf gene action is independent of both positive and negative transcription control elements of the AFP gene. Nuclear run-on analysis was used to confirm that transcriptions of both AFP transgenes and another endogenous raf-responsive gene, H19, are invariant with respect to the raf genotype. Thus, the postnatal repression of the AFP gene is mediated by both transcriptional and posttranscriptional mechanisms.

Albumins↗
PubMed1991

Sensitivity evaluation of positron emission tomographs.

Applying the recommended guidelines of the Instrumentation Group of the EEC Concerted Action, the sensitivity of an ECAT 933/4-16 scanner was determined. Several filters were used to reconstruct the images. On the images regions of interest (ROIs) were defined, and the ROI-counts were compared to the specific activity in the phantom. Whereas the uncorrected sensitivity was not influenced by the reconstruction filters, the sensitivity corrected for the scatter fraction appeared to be dependent on the reconstruction filter. Images reconstructed with a non-smoothing filter (Ramp filter, Shepp filter) yielded a higher sensitivity due to the lower reconstructed scatter fraction.

Algorithms↗
PubMed1991

Interest of TOF PET for clinical applications.

The interest of PET is now well established in medical research. Recently PET has proved to be also useful in the clinical domain e.g. for the diagnosis of epilepsy, myocardial disease etc... Its use has been so far limited by the cost of the radionuclide production and the cost of the PET itself. However, inexpensive, compact cyclotrons and centralized distribution of F18 are beginning to facilitate the availability of radionuclides. Thanks to its performances in terms of high random rate, sensitivity gain, random reduction, TOF is becoming practical for clinical use, especially with new types of architecture allowing the reduction of the cost of the detection head, so far the main obstacle to the use of TOF PET in clinical domain.

Algorithms↗
PubMed1990

Dominant negative regulation of the mouse alpha-fetoprotein gene in adult liver.

Transcription of the mouse alpha-fetoprotein gene is activated in the developing fetal liver and gut and repressed in both tissues shortly after birth. With germline transformation in mice, a cis-acting element was identified upstream of the transcription initiation site of the alpha-fetoprotein gene that was responsible for repression of the gene in adult liver. This negative element acts as a repressor in a position-dependent manner.

Aging↗
PubMed1987

Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase.

Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).

Amino Acyl-tRNA Synthetases↗
PubMed1985

Functional mutants of phenylalanine transfer RNA from Escherichia coli.

The gene pheV from Escherichia coli, coding for tRNAPhe and carried on a plasmid, has been mutagenised with hydroxylamine. Mutants in the structural gene have been identified using two criteria: (i) de-attenuation of beta-galactosidase expression, while under the control of the attenuator region of the pheS,T operon by means of an operon fusion; (ii) loss of ability to complement thermosensitivity of a mutant Phe-tRNA synthetase. Mutants showing de-attenuation were sequenced and two nucleotide changes identified: G44----A44 (found five times) and m7G46----A46 (found once). Sequencing of mutants that lost complementation identified two further tRNA mutants, C2---U2 and G15----A15; the mutant m7G46----A46 was also re-isolated by this criterion. Three of the mutants involve bases implicated in tertiary rather than secondary structure hydrogen bonding. One hypothesis for the mechanism of de-attenuation is that mutant tRNAPhe molecules compete with the wild-type tRNAPhe on the ribosome but are inefficient at some step in the elongation process.

Base Sequence↗
PubMed1984

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression.

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.

Anticodon↗
PubMed1984

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys.

In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in beta-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACys structure may be unadapted to efficient UCA anticodon function.

Animals↗