PubMed Health⌕ Search

Biomedical subjects

M Thomasset

Publications and source records attributed to M Thomasset.

At least 91 records · Page 5Linked to original sources
PubMed1986

Effects of vitamin D on calcium regulation in vitamin-D-deficient pigs given a phytate-phosphorus diet.

1. Vitamin-D-deficient pigs were fed on a phytate-phosphorus diet and treated with vitamin D3 (+D) to examine the time-course of adaptative changes in plasma minerals, vitamin D metabolites, parathyroid hormone (PTH) and calcium balance and intestinal Ca-binding protein (CaBP). 2. The 5-week vitamin D repletion (25 micrograms cholecalciferol/kg diet) regimen restored plasma Ca, P and alkaline phosphatase (EC 3.1.3.1) to normal, decreased PTH and markedly and rapidly increased plasma 25-hydroxycholecalciferol (25-OHD, sevenfold after 4 d) and 1,25-dihydroxycholecalciferol (1, 25(OH)2D3, 1.8-fold after 4 d). 3. CaBP concentrations were markedly elevated all along the digestive tract, especially in the distal regions. 4. Ca absorption and retention were enhanced (fourfold and sixfold respectively) by the +D diet. 5. The improved Ca absorption, coupled with increased CaBP and 1,25(OH)2D3 levels, suggest that vitamin D metabolism in phytate-P-fed pigs is sensitive to the depressed Ca availability due to phytate feeding. It also indicates that CaBP may play an important role in the adaptation of Ca absorption. 6. Persistent hypercalciuria indicates that mineral metabolism was still affected by the phytate nature of the dietary P in spite of the vitamin D treatment.

Alkaline Phosphatase↗
PubMed1986

In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum.

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 activity.

Animals↗
PubMed1986

Distribution of vitamin D-dependent calcium-binding protein messenger ribonucleic acid in rat placenta and duodenum.

The vitamin D-dependent calcium-binding protein (CaBP), cholecalcin or calbindin, is one of the best documented molecular expressions of 1,25-dihydroxyvitamin D, the hormonal metabolite of vitamin D. In this report, DNA/RNA hybridization assays have been used to examine cholecalcin (CaBP) mRNA production in the placenta and duodenum of 21-day pregnant rats. A cloned CaBP cDNA which codes for the rat intestinal 9000 mol wt cholecalcin (9KCaBP) was radiolabeled and used in hybridization assays to explore 1) the size and relative quantities of CaBP mRNA extractable from placenta and duodenum by molecular hybridization, and 2) the localization and quantification, by in situ hybridization histochemistry, of CaBP mRNA in specific cells in rat placenta and duodenum. Northern hybridization studies show that the [32P]cDNA sequence hybridizes to a single 500- to 600-nucleotide species in the placenta as in the duodenum and, therefore, demonstrate identical 9KCaBP mRNA processing in both tissues. Dot blot hybridization studies show that the concentration of 9KCaBP mRNA was greatest in the duodenum, while that of the inner (fetal) placenta was about 50% the duodenal level. Considerably less CaBP mRNA was found in the outer (maternal) placenta. The observed differences in 9KCaBP mRNA levels correlate well with the in vivo variations in 9KCaBP concentrations. In situ hybridization histochemistry using [3H]cDNA reveals that 9KCaBP mRNA visualized by silver grains was concentrated in the inner placenta over the cytoplasm of syncytial cells in the trophoblastic epithelium of the labyrinth and much less frequently in the cells of the outer placenta. In the duodenum, 9KCaBP mRNA was found only in the absorptive epithelial cells from the crypt region to the upper part of the villi. The silver grains were distributed throughout the cytoplasm of the columnar cells; they were densest in the perinuclear region and rarest in the nuclear region. The concentration was greater in the cells at the villous tips than in those of the crypts. This difference in 9KCaBP mRNA levels correlates well with the distribution of the protein itself along the villi. CaBP mRNA quantities detected by hybridization histochemistry showed greater labeling in the syncytial cells of the trophoblastic epithelium of the labyrinth than in the absorptive epithelial cells of the upper part of the villi (200% less), indicating accumulation of CaBP mRNA at a greater rate in the trophoblastic epithelium than in the absorptive epithelial cells. These results indicate that in the rat, 9KCaBP is synthesized in both the absorptive cells of the duodenum and the cells of the trophoblastic epithelium of the placenta.

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↗
PubMed1985

Purification, immunological and biochemical characterization of rat 28 kDa cholecalcin (cholecalciferol-induced calcium-binding proteins). Identity between renal and cerebellar cholecalcins.

The cholecalcins are intracellular vitamin D-dependent calcium-binding proteins. High concentrations of 28 kDa cholecalcins have been found in the kidneys and cerebella of birds and mammals. However, whereas the synthesis of the renal protein is vitamin D-dependent, that of the cholecalcin in the cerebella of young growing chicks and rats is apparently not. In the present study a range of immunological, physicochemical and structural characteristics of renal and cerebellar cholecalcins isolated from a single species, the rat, has been examined to ascertain what, if any, differences there are between them, other than the vitamin D-dependence. Both proteins behaved in exactly the same way during purification and showed complete immunocross-reactivity in Ouchterlony double immunodiffusion and radioimmunoassay. No difference could be detected between them on electrophoresis under denaturing conditions or on two-dimensional isoelectric focusing/electrophoresis. Their amino acid compositions were very similar, as were their u.v.-absorption spectra and their chymotryptic and tryptic peptide maps. The N-terminal sequence was found to be Gly-Gly-Val-Ser... for both cholecalcins. We have thus been unable to show any significant difference between the two proteins and suggest that the 28 kDa cholecalcins of the rat cerebellum and kidney are extremely similar, if not identical.

Amino Acids↗
PubMed1985

Purification, calcium-binding properties, and conformational studies on a 28-kDa cholecalcin-like protein from bovine brain.

A large-scale preparation method for bovine brain 28-kDa cholecalcin-like protein is described. Flow dialysis binding studies revealed that the protein binds at least 3 mol of Ca2+/mol of protein. The protein undergoes conformational changes on binding calcium as shown by UV differential absorption spectroscopy, near and far UV circular dichroism, and intrinsic fluorescence. Circular dichroism (CD) studies in the far UV indicate an apparent increase in helical content in the presence of Ca2+. The effect of calcium on the protein structure is nearly maximum for 1 Ca2+ bound/protein molecule. UV differential absorption studies on the binding of the Ca2+ agonist Tb3+ and Tb3+ luminescence induced by energy Trp----Tb3+ transfer indicate that Tb3+ binds to two higher affinity Ca2+-binding sites. These sites are probably very close to the single Trp residue. Analysis of the fluorescence parameters of the single tryptophan residue in the apoprotein and its accessibility to ionic and neutral quenchers suggests that this residue is located in a highly hydrophobic domain on the protein surface.

Animals↗
PubMed1985

28 K cholecalcin (CaBP) levels in abnormal cerebella: studies on mutant mice and harmaline- and 3-acetylpyridine-treated rats.

The cerebellar Purkinje cells of both birds and mammals contain a specific calcium-binding protein, 28 K cholecalcin (CaBP). This is the same protein as the vitamin D-dependent kidney CaBP, but its Purkinje cell level is apparently vitamin D independent. The cerebellar CaBP contents of 3-acetylpyridine- and harmaline-treated rats and 5 mutant mouse strains (Purkinje cell degeneration, reeler, weaver, staggerer and nervous) were measured using a specific radioimmunoassay. The results indicate that the level of cerebellar CaBP is not dependent on the physiological state of the Purkinje cells but is an intrinsic measure of the size of the Purkinje cell population.

Alkaloids↗
PubMed1985

Cholecalcin (a 9-kDa cholecalciferol-induced calcium-binding protein) messenger RNA. Distribution and induction by calcitriol in the rat digestive tract.

In view of the possible physiological importance of the 9-kDa cholecalcin (a 9000-Mr cholecalciferol-induced calcium-binding protein) in the intestinal transport of calcium in mammals, the gene expression of this protein has been analysed. Its regulation in the digestive tract of the growing rat by calcitriol (1,25-dihydroxycholecalciferol) was studied using a specific cloned [32P]cDNA to 9-kDa cholecalcin. Northern hybridisation studies show that the cDNA sequence hybridises to a single 500-600-nucleotide species throughout the digestive tract and therefore demonstrate identical 9-kDa-cholecalcin mRNA processing in the whole of the intestine and caecum. The highest concentrations of cholecalcin mRNA occur in the duodenum, proximal jejunum and caecum. The observed differences in 9-kDa-cholecalcin mRNA levels correlate well with both the in vivo variations in cholecalcin itself and with the known intestinal sites of calcium absorption. The whole intestine is able to respond to exogenous calcitriol but the response of the distal intestine and caecum, as measured by the increase in cholecalcin mRNA and corresponding protein, was proportionally higher than in the duodenum. The rapid production of fully functional cholecalcin mRNA, which was detectable as early as 1 h after a single dose of calcitriol to vitamin-D-deficient rats, provides convincing evidence that calcitriol increases 9-kDa cholecalcin production by increasing cholecalcin gene expression at the transcriptional level.

Animals↗
PubMed1985

Characterisation of rat 9-kDa cholecalcin (CaBP) messenger RNA using a complementary DNA. Absence of homology with 28-kDa cholecalcin mRNA.

The rat possesses two cholecalciferol-induced calcium-binding proteins, the cholecalcins (CaBP). The 9-kDa CaBP is mainly concentrated in the duodenum while 28-kDa CaBP is located in the kidney and cerebellum. The mRNA encoding 9-kDa CaBP has been characterised using the cloned cDNA, pC109, synthesised from rat duodenal 9-kDa CaBP mRNA [Desplan et al. (1983) J. Biol. Chem. 258, 13502-13505]. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3' untranslated region. The cDNA-hybridised mRNA, isolated from rat duodenum, directs the cell-free synthesis of two proteins precipitable by antisera to 9-kDa intestinal CaBP. A major protein comigrates with 9-kDa CaBP whereas a minor product corresponds to a protein which is larger by 2000 Da. The minor protein appears to result from read-through of the 'leaky' UGA stop signal. No protein band which was immunoprecipitable with 28-kDa CaBP antiserum was detected when cDNA-hybridized mRNA from rat kidney and cerebellum was translated in a cell-free system. Northern blots show that the cDNA pC109 sequence hybridizes to a homogeneous mRNA species 500-600 nucleotides long from rat duodenum. Larger mRNA species encoding 28-kDa CaBP are undetectable in rat kidney and cerebellum even under low stringency conditions. All these findings demonstrate that there is no cross-hybridisation between 9-kDa and 28-kDa CaBP mRNAs. Southern blot analysis of rat genomic DNA, that shows only one homologous 9-kDa gene, is consistent with these findings. Thus, all our data indicate that there are distinct genes coding for each rat cholecalcin.

Animals↗
PubMed1985

[Molecular approach to the action of vitamin D in man].

Some applications to man of specific markers of the molecular action of vitamin D (1.25(OH)2D3 receptors and antibodies to hormone-dependent proteins (CaBP and cDNA] are reported in this study. On case of type II vitamin-dependent rickets was characterized by 1.25(OH)2D3 plasma level greater than 250 pg/ml and a ten-fold decrease of the number of binding sites of the hormone in cultured skin fibroblasts. We propose that CaBP 28K and/or 9K-containing cells, such as Purkinje's cells and chondroblasts may be targets for vitamin D action. Detection in fetuses, from the 20th week of gestation, of CaBP 9K messenger RNA in the duodenum and sternum and presence of CaBP 28K and 9K in the chondroblasts of the upper extremity of tibia, suggest that vitamin D acts on the nucleus of its target-cells during fetal development. Finally, discovery of the gene of CaBP 9K in man opens the prospect of studies which will improve the understanding of the mechanism of action of vitamin D.

Adolescent↗
PubMed1985

[Comparative effects of magnesium deficiency and overload on calcium and phosphorus metabolism in the growing pig].

This study was designed to assess the influence of dietary magnesium on calcium and phosphorus metabolism in growing pigs. 18 4-month-old pigs received either an Mg-deficient (40 ppm), an Mg overload (5,600 ppm) or a control Mg diet (1,000 ppm) for 70 days. The following parameters were measured: kinetics of plasma concentrations of Ca, phosphates, Mg and alkaline phosphatase (AP), absorptions, urinary and fecal excretions, retentions of Ca, P and Mg, soft tissue and bone mineral contents (BMC), and intestinal mucosal CaBP (calcium-binding protein) and AP activities. Dietary Mg level had no effect on calcium and phosphorus metabolism as far as Ca and P absorptions and retentions, BMC, intestinal CaBP and plasma levels of Ca, P and AP are concerned. Mg overload significantly decreased urinary P excretion and increased kidney P content, but did not change fecal P excretion. Jejunal AP activity was slightly decreased in Mg-deficient pigs. The plasma, bone, urinary, fecal, absorbed and retained Mg varied linearly with Mg intake (r: 0,85-0.96). In Mg-deficient pigs, hypomagnesemia appeared after 1 week and its severity did not increase with time. In Mg-overloaded pigs, hypermagnesemia occurred very late and was limited. This suggests that, in the growing pig, dietary Mg has little or no effect on Ca-P metabolism. It may also be concluded that growing pigs are rather resistant to Mg deficiency since no clinical symptoms were observed.

Alkaline Phosphatase↗
PubMed1984

In vitro stimulation of articular chondrocyte differentiated function by 1,25-dihydroxycholecalciferol or 24R,25-dihydroxycholecalciferol.

The effects of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) (10(-13)M-10(-8) M) and 24R ,25-dihydroxycholecalciferol ( 24R ,25-(OH)2D3) (10(-12)M-10(-7) M) on cell proliferation and proteoglycan deposition were examined in our newly developed multilayer culture system for rabbit and human articular chondrocytes. The cells are embedded in an extracellular matrix similar to that seen in vivo and maintain their in vivo phenotype. We extracted and purified native proteoglycans and degraded material from three culture compartments: the medium, intercellular matrix, and cells. Proteoglycan synthesis and deposition were analyzed by measuring 35SO4 incorporation, hexuronic acid, and galactose contents. In both rabbit and human chondrocyte cultures, chronic 1,25-(OH)2D3 treatment inhibited chondrocyte proliferation and stimulated proteoglycan synthesis and accumulation in the three compartments at 10(-12)-10(-8) M; maximal effect was at 10(-10)M. Cell proliferation was reduced by 55% and the content of hexuronic acid (or galactose) was increased to about three times that of controls in all compartments. 1,25-(OH)2D3 did not alter the proteoglycan composition. Chronic 24R ,25-(OH)2D3 treatment induced comparable effects with a maximum at 10(-8)M. When human dermal fibroblasts were treated as above both vitamin D metabolites increase mitosis. 1,25-(OH)2D3 mainly reduced the pericellular deposition of proteoglycans, while 24R ,25-(OH)2D3 appeared to reduce their synthesis and deposition in both medium and pericellular compartments. These results suggest that both 1,25-(OH)2D3 and 24R ,25-(OH)2D3 act specifically on articular chondrocytes to promote phenotype expression.

Animals↗
PubMed1984

Tissue distribution of human calcium-binding protein (28 000 g mol-1).

Immunoreactive calcium-binding protein (28 000 g mol-1, L-CaBP) has been quantified using a specific radioimmunoassay to human cerebellar L-CaBP. The level of L-CaBP in post-mortem tissue samples varied from undetectable in muscle to 8 micrograms/mg-1 protein in the cerebellum and 16 micrograms mg-1 protein in the dentate nucleus. L-CaBP was found to be widely distributed throughout the nervous system while the only non-nervous tissue which contained appreciable levels was the renal cortex.

Aged↗
PubMed1983

Sequence of rat intestinal vitamin D-dependent calcium-binding protein derived from a cDNA clone. Evolutionary implications.

We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and II) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.

Amino Acid Sequence↗
PubMed1983

Synthesis, molecular cloning, and restriction analysis of DNA complementary to vitamin D-dependent calcium-binding protein mRNA from rat duodenum.

The mRNA coding for rat intestinal calcium-binding protein, a vitamin D3-induced protein (Mr 7500), has been partially purified from growing rat duodenum. Double-stranded DNA synthesized from the purified mRNA preparation was inserted into the PstI site of pBR322, using the oligo(dG-dC) tailing procedure. Clones containing DNA complementary to vitamin D-dependent calcium-binding protein mRNA were selected by differential colony hybridization with [32P] cDNA synthesized from enriched or low vitamin D-dependent calcium-binding protein mRNA preparations. Plasmid DNAs from the selected clones were each verified by both a solution hybrid-arrest assay and a filter hybrid-selection assay. Four recombinant clones showed identical endonuclease restriction maps and contained inserts ranging from 250 to 380 base pairs.

Animals↗
PubMed1983

Rat vitamin-D-dependent calcium-binding proteins. Specificity of mRNAs coding for the 7500-Mr protein from duodenum and the 28000-Mr protein from kidney and cerebellum.

mRNA extracted from rat duodenum, kidney and cerebellum was translated in a cell-free reticulocyte lysate system in the presence of L-[35S]methionine. Vitamin-D-dependent calcium-binding proteins (D-CaBPs) were identified by immunoprecipitation using antibodies specific to duodenal D-CaBP (7500 Mr) and cerebellar D-CaBP (28000 Mr). When duodenal mRNA was translated, the immunoprecipitated polypeptide, obtained using antibodies to duodenal D-CaBP, comigrated with the pure small D-CaBP. Only the addition of unlabeled small duodenal D-CaBP prevented the immunoprecipitation of the major protein. Likewise, when mRNA extracted from the kidney and cerebellum was translated, the product immunoprecipitated by antibodies specific to large mammalian D-CaBP was electrophoretically similar to pure 28000-Mr protein, being displaced only by the addition of unlabeled large D-CaBP. The yield of the duodenal D-CaBP synthesized in the reticulocyte lysate assay was remarkably high (about 10%) compared to that of the large D-CaBP with renal (1%) or cerebellar (0.4%) mRNA. In the absence or presence of microsomal membranes, proteins of similar molecular weight were synthesized, suggesting that the biosynthesis of both large and small D-CaBPs do not involve the processing of leader sequences. Moreover in our experimental conditions duodenal poly(A)-rich RNA was unable to direct the synthesis of large D-CaBP while the mRNAs extracted from kidney and cerebellum did not code for the small D-CaBP. Our data indicate that two distinct mRNAs, coding for small and for large vitamin-D-dependent CaBPs, are expressed in specific tissues of the rat.

Animals↗