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J M Dupret

Publications and source records attributed to J M Dupret.

27 records · Page 2Linked to original sources
PubMed1993

Cell- and stage-specific expression of vitamin D receptor and calbindin genes in rat incisor: regulation by 1,25-dihydroxyvitamin D3.

To investigate the extent of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action and its relationships to calbindin gene expression in mineralized tissues, we have analyzed rat incisors with different probes, including a vitamin D receptor (VDR) antibody and specific cDNAs to rat calbindin-D9K and calbindin-D28K. Developmental and hormonal controls of calbindin gene expression were investigated by Northern blot analysis of ameloblast and odontoblast mRNA. Distribution and hormone-induced changes of VDR were also studied by light microscopic immunocytochemistry. A differential tissue- and stage-specific expression of the calbindin genes was observed in microdissected portions of the continuously erupting incisor. The two calbindins were expressed in ameloblasts, whereas only calbindin-D28K was expressed in odontoblasts. Moreover, in ameloblasts, expression of calbindin-D28K preceded that of calbindin-D9K. Immunoreactivity for VDR was present in all progenitor cells and progressively decreased during the differentiation process, whereas, in differentiated tissues, a hormonal upregulation was restricted to hard tissue-forming cells, i.e., ameloblasts and odontoblasts. Furthermore, calbindin gene expression appeared to be regulated by 1,25(OH)2D3. Taken together, these data indicate that ameloblasts and odontoblasts are target cells for 1,25(OH)2D3 and provide the first insights into the hormonal control of tooth genes during development.

Animals↗
PubMed1992

Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2.

The single coding exons of the cloned genes encoding two human arylamine N-acetyltransferases (NAT1 and NAT2) were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based phagemid vector pKEN2 for production of the recombinant proteins in Escherichia coli strain XA90. Induction of cultures grown from selected bacterial transformants resulted in the production of substantial quantities of soluble recombinant human NAT1 and NAT2 with identical electrophoretic, immunologic and catalytic properties to those expressed in mammalian cell culture or in human liver. Oligonucleotide-directed mutagenesis of recombinant human NAT2 was then employed to determine the relative importance of 3 highly conserved cysteine residues in the enzyme's catalytic mechanism. Substitution of cysteine with glycine at position 68 of the 290 amino acid protein molecule (Cys68----Gly) resulted in the production of normal quantities of immunoreactive NAT2 which was completely devoid of enzyme activity, suggesting that the sulfhydryl group of Cys68 is directly involved in the transfer of acetate from the essential cofactor CoASAc to acceptor amine substrates. On the other hand, the mutations producing Cys44----Gly and Cys223----Gly led to the production of enzymatically active NAT2 proteins with markedly reduced in vitro stability, suggesting that substitution of either of these amino acids may cause alterations in the tertiary structure of the native enzyme.

Arylamine N-Acetyltransferase↗
PubMed1992

Calbindin-D9K gene expression in the lung of the rat. Absence of regulation by 1,25-dihydroxyvitamin D3 and estrogen.

Calbindin-D9K (CaBP9K) is classically considered to be the molecular expression of 1,25-dihydroxyvitamin D3. The hormone is known to regulate the rat CaBP9K gene in duodenal tissue at transcriptional and posttranscriptional levels. This study shows that the CaBP9K gene is expressed in the rat lung, and that this expression is probably not vitamin D- or estrogen-dependent. The CaBP9K gene is not expressed in alveolar macrophages, but CaBP9K messenger RNA (mRNA) was localized by in situ hybridization in alveolar epithelial cells. CaBP9K mRNA was detected as early as the 20th day of gestation. The quantity of CaBP9K mRNA gradually increased during growth, from 1-77 days after birth, whereas the CaBP9K concentration dramatically increased from day 19 to day 20 of gestation. Vitamin D-deficient male rats (8 weeks old) were given a single injection of 1,25-dihydroxyvitamin D3 (650 pmol/100 g body wt) and killed 1 h and 24 h after injection. The hormonal treatment resulted in a rise in duodenal CaBP9K mRNA, but no significant change in lung extracted CaBP9K mRNA. Mature ovariectomized rats were injected with 17 beta-estradiol (0.5 microgram/100 g body wt) and killed 24, 48, and 72 h later. The CaBP9K mRNA concentration in the uterus was markedly dependent on estrogen; that of the lung was not. The factors regulating the CaBP9K gene expression in the lung remain to be determined.

Aging↗
PubMed1991

[Action of 1,25-dihydroxyvitamin D3 in the tooth germ. Modulations of receptor in the development].

The control of tooth development by 1,25-Dihydroxyvitamin D3 is analyzed by light- and electron-microscope immunocytochemistry and Northern-blotting in vitamin D-deficient rats. The receptor for 1,25-Dihydroxyvitamin D3, immunostained at the light microscope in all stem-cells, became immunodetectable only at the ultrastructural level in the ameloblasts which elaborate enamel and odontoblasts which synthetize dentin. Moreover, 1,25-Dihydroxyvitamin D3 induces an up-regulation specifically in these cells. In parallel, the calbindins-D9k, -D28k and osteocalcin, in contrast to the phosphoprotein, appear sensitive to vitamin D-deficiency. A single injection of 1,25-Dihydroxyvitamin D3 led to the increase of steady-state levels of the corresponding calbindin mRNAs. These data show that tooth constitutes a target-organ for 1,25-Dihydroxyvitamin D3, as other components of the phospho-calcic metabolism.

Animals↗
PubMed1990

[Structure, expression and control of calbindins-D].

The Calbindins-D (CaBP9K and 28K), like calmodulin, belong to a group of intracellular proteins that bind calcium with high affinity. Each protein is encoded by a separate gene and there is no direct filiation between the two genes. We have demonstrated the tissue-specific expression and regulation of CaBP9K gene. This gene is expressed in the intestine, placenta and uterus of the rat as a single 0.5kb long transcript. Exogenous 1,25(OH)2D3 triggers the rapid synthesis of CaBP9K mRNA and accumulation of translatable CaBP9K mRNA in the duodenum of vitamin D-deficient rats. Calcium also stimulates CaBP9K gene expression in this tissue. In contrast 1,25(OH)2D3 does not change the uterine concentration of CaBP9K but estrogen stimulates the transcription of the CaBP9K gene in the uterus. The promoter region of rat CaBP9K gene contains 1 TATA box and 4 CAAT box-type sequences and several steroid hormone regulatory elements. The CaBP9K gene is therefore a suitable model for studying the tissue-specific regulation of gene expression by steroid hormones.

Animals↗
PubMed1987

Transcriptional and post-transcriptional regulation of vitamin D-dependent calcium-binding protein gene expression in the rat duodenum by 1,25-dihydroxycholecalciferol.

Regulation of the expression of vitamin D-dependent calcium-binding protein (Mr 9000 CaBP) gene by 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in the rat duodenum. In vivo stimulation of Mr 9000 CaBP synthesis was analyzed using a complementary DNA probe and by measuring the rate of Mr 9000 CaBP gene transcription in isolated nuclei (run-on assay). A single 1,25-(OH)2D3 injection (650 pmol/100 g of body weight) induced a 2-fold increase in Mr 9000 CaBP gene transcription within 15 min in the duodenum of vitamin D-deficient rats. RNA synthesis was maximal at 1 h, then decreased until 16 h of postinjection. There was an initial transient accumulation of Mr 9000 CaBP mRNA (from 7 to 15 min), which was followed by a second, significant increase, by 3 h which remained elevated until 16 h. The magnitude and time course of the Mr 9000 CaBP increase was similar to that of its mRNA as early as 1 h after 1,25-(OH)2D3 administration. Mr 9000 CaBP gene transcription was not significantly induced by 1,25-(OH)2D3 in vitamin D-replete rats and no transient accumulation of Mr 9000 CaBP mRNA was observed. Thus, 1,25-(OH)2D3 modulates Mr 9000 CaBP gene expression in at least two ways, a rapid transcriptional stimulation and a post-transcriptional effect preventing degradation of Mr 9000 CaBP transcripts and accounting for their accumulation several hours after the hormone treatment.

Animals↗
PubMed1987

Vitamin D-dependent calcium-binding proteins (CaBPs) in human fetuses: comparative distribution of 9K CaBP mRNA and 28K CaBP during development.

The vitamin D-dependent calcium-binding protein (CaBP) cholecalcin or calbindin, has been used as a molecular marker of 1,25-dihydroxyvitamin D3 action. Mammals possess two CaBPs: a 9,000 mol wt (9K CaBP) and a 28,000 mol wt (28K CaBP). The distinct localization of each protein in the rat has been previously described with the aid of specific radioimmunoassays developed for each CaBP. Antibodies raised against the rat 28K CaBP can be used to detect this protein in a number of mammalian species including humans. In contrast, antibodies against rat 9K CaBP do not cross react with human 9K CaBP, but human 9K CaBP mRNA can be analyzed using a cDNA probe for rat 9K CaBP mRNA. Such a cross-hybridization between the rat cDNA probe and human CaBP mRNA was demonstrated by Northern analysis. We have documented the distribution and evolution of 28K CaBP and 9K CaBP mRNA in human tissues during fetal development from 14 to 32 wk of gestation. 28K CaBP was only present in kidney and cerebellum, and not detectable in duodenum. There was a 2-fold increase of 28K CaBP in the cerebellum between 14 and 24 wk of gestation. The 9K CaBP mRNA was unevenly distributed in human fetal tissues. 9K CaBP mRNA was present in classical vitamin D target tissues such as duodenum and placenta; high levels of 9K CaBP mRNA also were found in thymus and lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

In vivo effects of transcriptional and translational inhibitors on duodenal vitamin D-dependent calcium-binding protein messenger ribonucleic acid stimulation by 1,25-dihydroxycholecalciferol.

The in vivo stimulation of vitamin D-dependent calcium-binding protein (9 K CaBP) synthesis by 1,25-dihydroxycholecalciferol [1,25(OH)2D3] in the rat duodenum has been analyzed using a specific [32P]complementary DNA probe for rat 9 K CaBP and inhibitors of RNA transcription (actinomycin D, alpha-amanitin) or protein synthesis (cycloheximide). The relative amounts of 9 K CaBP messenger RNA (mRNA) were assayed by dot-blot hybridization and the relative amounts of 9 K CaBP by RIA. Both inhibitors were injected at doses which significantly inhibited by 80-95% [35S]methionine or [3H]uridine incorporation into protein and RNA, respectively. In vitamin D-deficient rats, a single 1,25(OH)2D3 injection (650 pmol/100 g BW) resulted in a rapid production of 9 K CaBP mRNA which was significantly detectable as early as 3 h, and was followed by an increase of 9 K CaBP levels. Injection of actinomycin D (25 micrograms/100 g BW) 1 h before 1,25(OH)2D3 treatment and repeated every 4 h did not prevent the hormone-induced elevation of duodenal CaBP mRNA, even when the actinomycin dose was doubled and given 2 h before hormonal treatment. alpha-Amanitin (2 micrograms/100 g BW) also failed to block the hormonal stimulation. The protein synthesis inhibitor cycloheximide (25 micrograms/100 g BW) did not cause any change in the 1,25(OH)2D3-induced CaBP mRNA but blocked the CaBP increase after hormone injection. Thus, transcription inhibitors did not prevent the in vivo hormone-induced elevation of 9 K CaBP mRNA, which suggests that 1,25(OH)2D3 increases 9 K CaBP synthesis by increasing 9 K CaBP gene expression at one or more posttranscriptional steps. More precise data will be obtained by measuring the rate of 9 K CaBP gene transcription on isolated nuclei from rat duodenum.

Amanitins↗