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A Brehier

Publications and source records attributed to A Brehier.

34 records · Page 2Linked to original sources
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

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

Transient biochemical compartmentalization of Purkinje cells during early cerebellar development.

It has recently been observed that during early cerebellar development--from embryonic Day 17 to postnatal Day 3 in the rat--only certain discrete clusters of Purkinje cells (PCs) are immunoreactive to cyclic GMP-dependent protein kinase (cGK). In contrast, at later stages and in the adult, all the PCs are immunoreactive. These results obtained with cGK suggest a transitory intrinsic heterogeneity in the immature cerebellar cortex. It seemed therefore interesting to investigate the distribution of other PC markers during early development in the rat and in other species. The results presented here were obtained with two other antibodies--against vitamin D-dependent calcium binding protein and against Purkinje cell specific glycoprotein--which, like cGK, label all adult PCs. Each antibody gave a different and reproducible mosaic of positive and negative clusters of PCs in the perinatal cerebellum, thus indicating a transient biochemical compartmentalization resulting from the differential expression of parts of the same genotype by clusters of PCs. This compartmentalization in concomitant with the ingrowing of the cerebellar afferents. Once synaptogenesis starts, the biochemical heterogeneity of PCs disappears.

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↗
PubMed1983

Rat duodenal calcium-binding protein messenger RNA: induction by 1,25-dihydroxyvitamin D3.

To extend our previous observations on the regulation of CaBP biosynthesis by 1,25-dihydroxyvitamin D3, we have studied the specific mRNA encoding this protein in vitamin D-deficient and in vitamin D-repleted rats as well as the rate of its induction after a single injection of 1,25(OH)2D3 to vitamin D-deficient animals. The CaBP-mRNA was quantified by translation in a cell-free reticulocyte lysate system. CaBP-mRNA activity and cytoplasmic CaBP (measured by radioimmunoassay), dramatically decreased in rats previously fed a vitamin D-free diet for 5 weeks but neither parameter was zero. In vitamin D-deficient rats, a single injection of 1,25(OH)2D3 led to an increase in CaBP-mRNA activity within 2 h. This CaBP-mRNA activity peaked at about 4-6 h and thereafter declined to low value by 48 h, and the changes in mRNA activity always preceded the changes in cytosolic CaBP concentration. These results indicate that the induction of CaBP biosynthesis results from a 1,25(OH)2D3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH)2D3. This study also shows that the production of many other proteins seem to be under the control of vitamin D3.

Animals↗
PubMed1981

Phosphatidylcholine synthesis and glycogen depletion in fetal mouse lung: developmental changes and the effects of dexamethasone.

We measured the rate of choline incorporation into phosphatidylcholine in lung slices; the glucogen content of the lung; and the activities of pulmonary cholinephosphate cytidylyltransferase, cholinephosphotransferase and phosphatidate phosphatase in the mouse during late fetal and early postnatal development. We also examined the effect of maternal dexamethasone administration on these parameters of fetal lung maturation. There was a development increase in the rate of choline incorporation between 17 days gestation (term is 19 days) and the immediate newborn period. There was also a developmental decrease in the glycogen content of the lung but this did not occur until 18 days. There was a developmental increase in the activities of cholinephosphate cytidylytransferase and phosphatidate phosphatase but little change in the activity of cholinephosphotransferase. Dexamethasone doubled the rate of choline incorporation into phosphatidylcholine at 17 and 18 days gestation. It decreased the glycogen content of the fetal lung by 74% at 18 and 19 days, but had no effect at 16 and 17 days. Dexamethasone increased the activity of pulmonary cholinephosphate cytidylyltransferase by 37% and that of cholinephosphotransferase by 27% at 17 days. It increased the activity of phosphatidate phosphatase by 25% at 16 days and by 32% at 19 days. These data show that the normal development profile of these parameters of fetal lung maturation in the mouse, as well as the effects of glucocorticoids thereon, are generally similar to those in the rabbit and rat. However, stimulation of cholinephosphotransferase by glucocorticoids has not been generally observed in other species. Furthermore, since the changes in the rate of choline incorporation precede those in glycogen depletion, the data suggest that the relationship between phospholipid synthesis and glycogen degradation is not simply that of precursor to product.

Animals↗
PubMed1980

Fetal lung in organ culture. III. Comparison of dexamethasone, thyroxine, and methylxanthines.

Exposure of explants of fetal rat lung to dexamethasone, thyroxine, or the methylxanthines, aminophylline and caffeine, resulted in a significant increase in the rate of choline incorporation into all the choline-containing phospholipids. Dexamethasone, aminophylline, or caffeine treatment also resulted in an increase in the percentage of radioactivity from [3H]acetate in the surfactant-associated phospholipids, disaturated phosphatidylcholine, and phosphatidylglycerol and a corresponding decrease in the membrane phospholipids. Exposure to thyroxine had different effects. Only aminophylline and caffeine produced an increase in the rate of incorporation of acetate into phospholipid. Differences were also observed in the activities of enzymes of phospholipid synthesis. The activity of cholinephosphate cytidylyltransferase was increased by dexamethasone and that of choline kinase and lysolecithin acyltransferase by aminophylline. Thyroxine had no effect on any of the enzymes examined. All these agents produced a significant decrease in lung glycogen content and a small decrease in the protein-to-DNA ratio. These data indicate that corticosteroids, thyroxine, and the methylxanthines act directly on the fetal lung, but produce different effects and presumably act via different mechanisms.

Animals↗
PubMed1980

Transplacental stimulation of lung development in the fetal rabbit by 3,5-dimethyl-3'-isopropyl-L-thyronine.

The effect of thyroid hormone on maturation of fetal rabbit lung was studied with maternal treatment using 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT), a synthetic analogue of triiodothyronine. To investigate the in vivo kinetics and distribution of DIMIT, we prepared [3H]DIMIT and injected both pregnant rats (18-21 d gestation) and rabbits (25 d gestation). In the rat, maximal concentrations of radioactivity in maternal plasma, fetal plasma, and amniotic fluid occurred within 10 min, 1-2 h, and 4-6 h, respectively, after intramuscular injection. After 7 h the concentration of radioactivity in fetal plasma was 163 and 71% of the maternal level in rats and rabbits, respectively, indicating that DIMIT readily crosses the placenta. We treated pregnant rabbits for 1-2 d with DIMIT in doses of 0.5-3 mg/kg per d and examined the fetuses at 26 and 27 d gestation. Treatment did not affect fetal growth or viability. In fetal liver, DIMIT increased the activity of NADPH cytochromeac reductase by 64% and decreased the glycogen content by 73% compared to controls. The rate of choline incorporation by lung minces increased in dose-dependent manner to a maximum of +104% at 3 mg/kg DIMIT; this does stimulated by 38% the activity of lung phosphatidic acid phosphatase (PAPase), a corticosteroid-responsive enzyme, but there was no increase in tissue PAPase activity at most lower doses of DIMIT that enhanced choline incorporation. Treated lungs had 38% less glycogen tha controls, but there was no effect on tissue levels of DNA, protein, or phospholipid. DIMIT treatment increased the amount of total phospholipid (+163%). saturated phosphatidylcholine (+330%), and PAPase activity (+134%) in lung lavage fluid. The DIMIT effects on both choline incorporation by lung minces and phospholipid content of lavage fluid were substantially greater than what had occurred with an optimal dose of betamethasone. DIMIT also increased corticosteroid binding capacity in fetal plasma and produced a dose-dependent increase (maximal threefold) in total and free corticoids of both maternal and fetal plasma. It is estimated that elevated endogenous corticoids probably account for less than one-third of the increases in phospholipid synthesis and secretion observed at the higher doses of DIMIT. These data indicate that administration of DIMIT to pregnant rabbits accelertes maturation of the surfactant system in fetal lung. The magnitude of the effects on phospholipid synthesis and secretion, along with the minimal effect of PAPase activity in fetal lung tissue, suggest that thyroid hormones affect different biochemical processes from those influenced by glucocorticoids.

Adrenal Cortex Hormones↗
PubMed1978

Failure to detect an effect of prolactin on pulmonary surfactant and adrenal steroids in fetal sheep and rabbits.

Recent reports have indicated an association between low cord prolactin (PRL) and the occurrence of respiratory distress syndrome in premature infants, and it is reported that PRL administration increases the lecithin content of fetal rabbit lung. We administered 1 mg ovine PRL to 32 rabbit fetuses on day 24 of gestation and evaluated lung phospholipid synthesis and content on day 26. Compared with diluent-injected littermates, PRL had no effect on the rate of choline incorporation into lecithin, tissue content of phospholipid and disaturated lecithin, or plasma corticoids. However, both choline incorporation and corticoids were increased in all animals undergoing surgery compared with unoperated controls. We also infused PRL (1 mg/day, i.v.) into three fetal sheep continuously over five periods of 5-8 days. Although supraphysiologic concentrations of PRL were achieved in plasma and amniotic fluid, there was no effect of this treatment on the flux of tracheal fluid surfactant or on plasma concentrations of corticoids of dehydroepiandrosterone sulfate. Thus, in this study, we failed to detect either a stimulation of the surfactant system or an adreno-corticotropic effect by PRL as previously postulated. This suggests that the relationship between PRL and respiratory distress sundrome is an indirect association.

Adrenal Cortex Hormones↗
PubMed1978

Triiodothyronine nuclear binding in fetal and adult rabbit lung and cultured lung cells.

To investigate the possible mechanism of thyroid hormone action in the lung, we examined fetal and adult rabbit lung, and cell lines derived from lung, for specific nuclear binding sites for T3. Using incubation of isolated nuclei with L-[125I]T3 at 37 C, we found approximately 2400 specific binding sites/cell in fetal lung and 1120 sites/cell in adult lung, with a similar dissociation constant (approximately 500 pm) for both tissues. The L-2 and A549 cell lines, which may have originated from pulmonary type II alveolar cells, contained 2280 and 1580 nuclear sites/cell, and the dissociation constants were 280 and 200 pm, respectively. In fetal lung, the ability of various analogs to compete for L-T3 (100%) binding was: 3,5-diiodo-3'-isopropylthyronine, 81%; D-T3, 73%; L-T4, 6.7%; 3,3'-diiodothyronine, 0.19%; 3,5-dimethyl-3'-isopropyl-L-thyronine, 0.15%; and rT3, 0.08%. These findings indicate that both fetal and adult lung, and cultured lung cells, contain specific nuclear binding sites for T3, suggesting that these tissues and their type II alveolar cells may be directly influenced by thyroid hormones.

Animals↗