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Biomedical subjects

A Brehier

Publications and source records attributed to A Brehier.

At least 19 recordsLinked to original sources
PubMed1998

Effect of polyisobutylcyanoacrylate nanoparticles and lipofectin loaded with oligonucleotides on cell viability and PKC alpha neosynthesis in HepG2 cells.

The aim of the present study was to evaluate the inhibitory effect on protein kinase C alpha (PKC alpha) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKC alpha antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKC alpha neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKC alpha neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin). This non-specific effect was only observed in the phase of PKC alpha neosynthesis when the cells were first depleted in PKC alpha by phorbol 12-myristate beta-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.

Adsorption↗
PubMed1993

Estrogen-induced calbindin-D 9k gene expression in the rat uterus during the estrous cycle: late antagonistic effect of progesterone.

Progesterone modulates estrogen-stimulated responses in the uterus. Calbindin-D 9k (CaBP9k), a 17 beta-estradiol-responsive gene expressed in the uterus, was used as a marker to examine the interactions between endogenous progesterone and estradiol in the rat. The variations in uterine CaBP9k messenger RNAs (mRNAs) during the rat estrous cycle indicated that CaBP9k gene expression was greatest during the estrogen-dominated phases (proestrus and estrus) and became totally repressed during diestrus, when progesterone predominates. Estradiol was found to be the major controlling factor of CaBP9k gene expression in vivo, progesterone antagonizing estrogen-induced CaBP9k gene expression. The inhibitory role of progesterone was further examined in two experiments. Mature cyclic rats were injected with the progesterone antagonist RU486 before the progesterone surge of proestrus, and the estrous cycle was mimicked in ovariectomized rats by sequential injections of estrogen and progestin. Progesterone did not appear to be involved in the rapid decrease in CaBP9k mRNA during estrus but was implicated in the down-regulation of the estrogen-stimulated CaBP9k gene expression at the end of estrus and during diestrus. This delayed effect of progesterone was confirmed in the ovariectomized rat model. CaBP9k mRNA accumulation in estrogen-primed ovariectomized rats was suppressed by estrogen followed 1 h later by the progesterone agonist R5020. This effect occurred more than 24 h after progestin treatment. The inhibition of the estrogen-induced CaBP9k gene expression in the rat uterus by progesterone is certainly mediated by the progesterone receptor, because progesterone had no effect without estrogen priming or when the antagonist RU486 was used. The delayed progesterone effect probably does not involve depletion of nuclear estrogen receptors, the major rapid mechanism proposed for estrogen inhibition by progesterone in the rodent uterus, or control of estrogen receptor synthesis, as shown by Northern blot analysis of estrogen receptor mRNA.

Animals↗
PubMed1990

Stimulation of calbindin-D9K (CaBP9K) gene expression by calcium and 1,25-dihydroxycholecalciferol in fetal rat duodenal organ culture.

We have used organ cultures of 18-day fetal rat duodena to study the regulation of calbindin-D9K (CaBP9K) gene expression by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and calcium. The epithelium of the fetal rat duodena maintained in culture for 10 days in a defined serum-free medium with no exogenous 1,25(OH)2D3 contains differentiated enterocytes. Treatment with 10(-8) M 1,25(OH)2D3 resulted in a 2-fold increase in CaBP9K messenger RNA (mRNA) after 1 h; CaBP9K content was increased 2.5-fold in 24 h. Simultaneous treatment of cultures with 1,25(OH)2D3 and 1 microM actinomycin D selectively blocked 1,25(OH)2D3-stimulated CaBP9K mRNA and protein synthesis. Fifty micromolars of cycloheximide significantly decreased CaBP9K protein accumulation but not mRNA. These findings indicate that 1,25(OH)2D3 controls CaBP9K gene transcription. Enterocytes exhibit some CaBP9K gene expression after 10 days under basal culture conditions, suggesting that CaBP9K synthesis is not dependent on 1,25(OH)2D3 alone. This basal expression drops after 24 h in a calcium free medium with 1 mM EGTA, suggesting that extracellular calcium is involved in the control of this gene. Increasing the calcium concentration to 1.2 mM resulted in a 6-fold increase in CaBP9K mRNA after 3 h and a 10-fold increase in CaBP9K protein content after 24 h. Calcium may increase CaBP9K gene transcription, since simultaneous addition of 1 microM actinomycin D and calcium blocked the calcium effect on CaBP9K mRNA. Therefore, this in vitro study demonstrates that both calcium and 1,25(OH)2D3 stimulate CaBP9K gene expression in the rat duodenum.

Animals↗
PubMed1990

17 beta-estradiol stimulates the calbindin-D9k (CaBP9k) gene expression at the transcriptional and posttranscriptional levels in the rat uterus.

The 9 kilodalton vitamin D-dependent calcium-binding protein (CaBP9k), calbindin-D9k, is expressed in the intestine and uterus of mammals. Rat intestinal CaBP9k is a well documented expression of the steroid hormone like action of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In contrast exogenous 1,25-(OH)2D3 does not affect the concentration of uterine CaBP9k which is dependent on estrogen. We have analyzed the effect of 17 beta-estradiol on the regulation of CaBP9k gene expression in the uterus of mature ovariectomized rats. CaBP9k mRNA is undetectable in the uterus of mature ovariectomized rats. A single dose of 17 beta-estradiol results in a detectable level of CaBP9k mRNA at 1 h and a significant increase 3 h after injection. The maximal CaBP9k mRNA level is reached 6 to 12 h post injection. These results show that 17 beta-estradiol increases CaBP9k production by increasing CaBP9k gene transcription. Chronic 17 beta-estradiol administration results in a plateau of CaBP9k mRNA but in a large increase in CaBP9k concentration. The kinetic response to a single estradiol injection was similar in immature rats. This result shows that no cellular differentiation is required for the control of CaBP9k gene expression by 17 beta-estradiol. The uterine cells of immature rats are already competent to respond optimally to estradiol. There is a single 0.5 kilobase CaBP9k gene transcript in the rat duodenum. In contrast there are two estrogen-inducible CaBP9k mRNA species in the uterus of both the mature ovariectomized and the immature rats. The smallest species corresponds to the duodenal CaBP9k mRNA species, while the larger species is at least 50 nucleotides larger. However, a unique CaBP9k identical to that in the duodenum is expressed in the uterus.

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↗
PubMed1989

Immunological characterization, developmental pattern and vitamin-D-dependency of calbindin D-28 K in rat teeth ameloblasts.

It has been suggested that vitamin D is involved in the process of cell differentiation and extracellular mineralization during tooth development. One of the best-defined molecular markers of the action of vitamin D is a calcium-binding protein of Mr 28,000 called calbindin D-28 K (CaBP 28 K). Since this protein is present in growing teeth, we have examined its synthesis in teeth from vitamin D-replete and -deplete rats by Western blotting and immunocytochemistry with an antiserum to CaBP 28 K purified from rat kidney. The CaBP 28 K present in the enamel organ is a single molecular species migrating near 30 k Da, similarly to the kidney protein. The differentiation and maturation of odontogenic cells were followed during early postnatal development (2-12 days) in rat molars. At the light-microscope level, CaBP 28 K was only found in a single cell-type, the ameloblasts. The expression of this protein appeared to be developmentally controlled, since its distribution varied with the cell stage and the functional steps of amelogenesis. The protein was localized in the basal compartment of ameloblasts from the presecretory stage. During the early secretory stage, the concentration of cytoplasmic CaBP 28 K formed a gradient from the apical to the basal pole of the ameloblasts. Staining appeared homogeneous in the cytoplasm of later secretory ameloblasts. CaBP 28 K was discontinuously distributed during the maturation stage. This discontinuity might be related to cyclical changes in mature ameloblasts. In all stages, ameloblasts from vitamin-D-deficient rats appeared depleted of CaBP 28 K.

Ameloblasts↗
PubMed1989

Calbindins D-9kDa and -28kDa and enamel secretion in vitamin D-deficient and control rats.

The present study focuses on the ultrastructure of enamel organ cells and the immunolocalization of calbindins D-9kDa and -28kDa during enamel secretion in Vitamin D-deficient rats. Vitamin D-deficiency disturbed the deposition of the layer of inner enamel and depleted the calbindins-content of ameloblasts. These data raise the possibility of a direct action of Vitamin D on the physiology of ameloblasts through ionic calcium homeostasis.

Ameloblasts↗
PubMed1989

[The systemic environment and dental development. From the clinical to the molecular approach].

Clinical observation of patients with disordered phosphocalcium metabolism has demonstrated that dyschromia and/or dental dysplasias systematically accompany such disorders. A certain action of this steroid on dental buds has been demonstrated after analysis of the effects of experimental vitamin D deficiency in the rat: vitamin D would seem to control the behaviour of cells undergoing differentiation and also after this process is complete. Dentinogenesis and amelogenesis would appear principally to be affected. Two proteins, calbindins D-9K and -28K, may constitute the molecular mediators of this ameloblastic regulation.

Animals↗
PubMed1988

Appearance and distribution of neuron-specific enolase and calbindin (CaBP 28 kDa) in the developing human inner ear.

The onset and development of neuron-specific enolase (NSE) and calbindin immunoreactivities were studied in the inner ear of human fetuses aged from 6-7 to 14 weeks of gestation. NSE occurred very early in ganglion neurons. Its appearance in vestibular sensory cells at 8 weeks coincided with the formation of the first afferent synapses, and showed an apex/base gradient in the cristae. Calbindin was found in vestibular ganglion neurons at 6-7 weeks and in the cochlear ganglion neurons at 8-9 weeks. Vestibular sensory cells and the whole ventral wall of the cochlear duct were stained from 8-9 weeks. At 14 weeks, calbindin staining occurred only in the sensory cells of the cochlear neuroepithelium. Non-neuronal secretory structures, i.e. Kölliker's organ and some cells of the transitional zone of the utricle, were also reactive. Staining appeared in Kölliker's organ with a base to apex gradient and disappeared from it with an internal to external gradient. Calbindin appeared in vestibular sensory cells later than NSE staining, synapse formation and sensory hair bundle differentiation. By contrast in the cochlea, calbindin staining appeared in the neuroepithelium before sensory cell differentiation, but remained only in the hair cells after they had differentiated and been contacted by the afferent fibers.

Antibodies↗
PubMed1988

Human colon cell line HT-29: characterisation of 1,25-dihydroxyvitamin D3 receptor and induction of differentiation by the hormone.

The human colon carcinoma cell line HT-29 differentiates into functional enterocytes upon replacement of glucose by galactose in the culture medium. Since the differentiation of other types of cells is associated with the modulation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) receptor concentrations and since enterocytes are classical target cells for 1,25(OH)2D3 we have examined the HT-29 cells to determine whether the differentiated and undifferentiated stages could be directly linked to the presence of 1,25(OH)2D3 receptors. HT-29 cells were grown in Dulbecco's modified medium containing 10% fetal calf serum (FCS) and glucose or galactose. Cell differentiation was assessed by measuring the brush border hydrolase, maltase. 1,25(OH)2D3 receptors were studied in the cells after 48 h without FCS. Nuclear uptake was measured in intact dispersed cells and the receptor protein was further characterized by vitamin D metabolite binding specificity, sucrose density gradient analysis and binding to DNA-cellulose. Maltase activity was 5-fold greater in differentiated HT-29 cells than in undifferentiated cells. Scatchard analysis showed a highly specific saturable (9500 sites per cell) high affinity (2 x 10(-10) M), binding of 1,25(OH)2D3 in undifferentiated cells. This receptor-like protein sedimented at 3.3S, bound to and eluted from DNA-cellulose and had all the characteristics of a 1,25(OH)2D3 receptor. No specific binding was detected in differentiated HT-29 cells. The presence of 1,25(OH)2D3 receptors in undifferentiated HT-29 cells implies that these cells are targets for vitamin D. The maltase activity increased significantly when undifferentiated cells were exposed to 1,25(OH)2D3 for 5-6 days, indicating that the hormone can promote differentiation of HT-29 cells. These results demonstrate that HT-29 cells can provide a new model for studying steroid receptor regulation and cell differentiation.

Calcitriol↗
PubMed1988

Calbindin (CaBP 28 kDa) localization in the peripheral vestibular system of various vertebrates.

Previous reports on calbindin, a 28 kDa vitamin D-induced calcium-binding protein, located in the mammalian peripheral vestibular system indicated that it is specifically distributed and postulated that it could play a role in the electrophysiological functioning of the sensory cells. This immunocytochemical investigation of the distribution of calbindin in the vestibular system of various vertebrates: fishes (goldfish and sea-perch), amphibia (frog), birds (chicken) and mammals (mouse, cat and baboon), was performed to verify these observations. In the vestibular ganglion, only a few neurons were faintly immunoreactive in the fishes and the frog, while the staining was more intense but still not present in all neurons of the chicken, the mouse and the cat. All the neurons were immunoreactive in the baboon. No immunoreactivity was observed in the sensory epithelia of the fishes. All hair cells were strongly immunoreactive in the frog. In the other species, most of the hair cells in the cristae were immunostained except those situated in the peripheral areas. In the maculae, the hair cells of the striola were either the only ones stained or were more intensely stained or were more intensely stained than the others. The localization of calbindin in specific cellular types and its increasing abundance from the fishes to the mammals suggest that calbindin is associated with the capacity of sensory and nerve cells to analyze precise mechanical or biochemical stimulations.

Animals↗
PubMed1987

Immuno-electronmicroscopic localization of 'vitamin D-dependent' calcium-binding protein (CaBP-28k) in the vestibular hair cells of the cat.

The PAP immunohistochemical method was used to carry out a light- and electronmicroscopic study of the distribution of the vitamin D-dependent calcium-binding protein (CaBP-28k, calbindin, cholecalcin) in the vestibule of the young cat. It was found that the two types of hair cells, types I and II, were stained differently. Type II cells were intensely immunoreactive and their staining did not vary with the location of the cells within the crista ampullaris. Type I cells at the top of the cristae were lightly stained, or unstained, while the type I cells laterally or basally were frequently intensely stained. The nerve fibers arriving at the top of the cristae are highly immunoreactive while the fibers of the base are not stained. Immunostaining for CaBP was correlated with differences in the innervation of hair cells at the top and base of the cristae. This differential CaBP-immunostaining may reflect differences in the physiological activity of the cells. The electronmicroscopic study showed that CaBP is present throughout the cytoplasm of the hair cells but that its concentration was particularly high in the cuticular plate and stereocilia. This specific intracellular distribution of CaBP is discussed with the possible role of Ca2+ in the physiology of the vestibular hair cells.

Animals↗
PubMed1987

Cerebellar mutations affecting the postnatal survival of Purkinje cells in the mouse disclose a longitudinal pattern of differentially sensitive cells.

The pattern of surviving Purkinje cells (PCs) was investigated in three cerebellar mutant mice with severe postnatal PC death. Two of these mutations, nervous (nr) and Purkinje cell degeneration (pcd) mutations are already well characterized. The third mutation is a new one, which appeared spontaneously in DW/J-Pas mice and was called tambaleante (tbl). PCs were identified by immunocytochemistry using an antibody against vitamin D-dependent calcium-binding protein which labels all the PCs in adult control mice. In each of the three mutations, surviving PCs are arranged according to a different and reproducible pattern which is symmetric relative to the midline. In NR and young PCD mutants, PCs are closely packed in broad sagittal bands. In TBL, they are more loosely arranged in a rather patchy pattern. In PCD and in TBL mutants the death of resistant PCs is only shortly delayed but in NR there is little change in the number of surviving PCs after 3 months. The differential sensitivity of subsets of PCs to the effect of nr, pcd, and tbl mutations is topographically determined. These results provide a new evidence of the PC heterogeneity which has been previously demonstrated by histochemical and immunohistochemical techniques. Moreover, in the anterior vermis of control mice, three thin sagittal bands of PCs are labeled by the Q113 monoclonal antibody. Similarly, in the anterior lobe of the NR cerebellum, the thin longitudinal strips of missing PCs coincide with the absence of Q113 immunoreactivity: in this region the nr mutation affects specifically the survival of Q113 positive cells. However, other clusters of Q113 immunoreactive PCs do survive in NR mice suggesting that susceptibility to the nr mutation and Q113 positivity are two independent markers of the underlying PC compartmentalization.

Animals↗
PubMed1987

Ontogenesis of 28 kDa vitamin D-induced calcium-binding protein in human kidney.

The kidney distribution of 28 kDa vitamin D-induced calcium binding protein (CaBP) was studied in 15 fetuses (11 to 33 weeks old), six children and adults (12 days to 32 years old) by immunocytochemistry using a specific antibody to rat renal 28 kDa CaBP. Similar results were obtained on frozen and fixed tissues. Kidneys from one adult and three fetuses were studied by immunoelectronmicroscopy for antigen localization at the subcellular level using the indirect immunoperoxidase technique. The 28 kDa CaBP was present in all kidneys from the eleventh week of gestation. At that stage, all deep parts of collecting ducts were homogeneously stained and a few distal tubules located in the deep cortex were intensely labeled. No labeling was observed in the early stage of nephron differentiation (S-body). 28 kDa CaBP distribution changed with kidney maturation. There was a progressive reduction of the deep part of collecting duct labeling and a concomitant increase in the number and intensity of stained distal tubular cells. At the ultrastructural level, 28 kDa CaBP was observed in the cytosol and the nuclear euchromatin. Our study demonstrates the early cellular synthesis of 28 kDa CaBP and its transient expression by deep collecting duct cells during early fetal life, at a time when only a few distal convoluted tubular cells synthetize it.

Adult↗
PubMed1986

Immunocytochemical detection of vitamin D-dependent calcium-binding protein (CaBP-28K) in vestibular sensory hair cells and vestibular ganglion neurones of the cat.

Vestibular sensory hair cells, afferent fibres and vestibular ganglion neurones of the cat are intensely labelled by a specific antibody to rat kidney vitamin D-dependent calcium-binding protein (CaBP-28K). Type I hair cells are more weakly CaBP immunoreactive than type II hair cells. Ganglion neurones also present a differential staining. The presence of calcium-binding protein in sensory hair cells could be of interest for the understanding of transductional mechanisms.

Animals↗
PubMed1986

Evidence for the presence of Calbindin-D 28K (CaBP-28K) in the tibial growth cartilages of rats.

The distribution of the vitamin-D dependent calcium-binding protein (Calbindin-D 28K) (CaBP-28K) in the tibial growth plate cartilage of the rat has been studied immunohistochemically using an antibody raised against rat renal CaBP-28K. The protein was detected mainly in the nuclei of chondrocytes and occasionally in the juxtanuclear cytoplasm. The distribution was not uniform throughout the growth plate, but concentrated in the proliferatively active chondrocytes of the resting and proliferative zones. These findings raise the possibility that CaBP-28K may be involved in the mitotic activity of the chondrocytes, acting as a regulator of the proliferative process, perhaps via intranuclear calcium.

Animals↗
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↗