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M Garnier

Publications and source records attributed to M Garnier.

At least 55 records · Page 3Linked to original sources
PubMed1997

Identification of estrogen-responsive genes in neuroblastoma SK-ER3 cells.

To evaluate the role of estrogen receptor in the differentiation of cells of neural origin, we developed a molecular approach aimed at the identification of estrogen target genes by mRNA differential display PCR (ddPCR) in human neuroblastoma SK-ER3 cells. More than 3000 RNAs were examined, a few of which displayed a differential regulation pattern in response to 17beta-estradiol (E2). Sequence analysis of three differentially amplified ddPCR products showed homology with the growth-associated nuclear protein prothymosin-alpha (PTMA), the Bcl2-interacting protein Nip2, and one mRNA previously described by others in fetal human brain. Two ddPCR products, referred to as P4 and P10, corresponded to new DNA sequences. Northern analysis confirmed that estrogen treatment of SK-ER3 cells resulted in the upregulation and downregulation of expression of these messages. In particular, PTMA was found to accumulate at both 1 and 17 hr after E2 treatment, whereas P10 product accumulated only at 1 hr. Conversely, P4, Nip2, and the fetal brain-related mRNAs were significantly decreased by the treatment. Further time course analysis of PTMA and Nip2 mRNAs levels indicated that the hormone exerted a marked biphasic regulatory effect on expression of both messages during the course of cell differentiation. In the present study we report for the first time the identification of a panel of estrogen target genes in neural cells that provide new insights in the molecular mechanism of action of E2 in cells of neural origin.

Base Sequence↗
PubMed1997

Production and evaluation of non-radioactive probes for the detection of the two 'Candidatus Liberobacter' species associated with citrus huanglongbing (greening).

The production and evaluation of non-radioactive probes for the detection of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter africanum', the two bacterial species associated with citrus huanglongbing (greening) disease is described. Two DNA fagments, In 2.6 and AS 1.7, obtained previously from the beta operons of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter africanum', respectively, were the starting materials for production of the two non-radioactive probes. These digoxigenin (DIG)-labelled probes were generated by PCR incorporation of DIG-11-dUTP, yielding In 1.7-DIG and AS 1.7-DIG. Probe In 1.7-DIG was hybridized with DNAs extracted from 24 field-collected samples in Bali (Indonesia). The membrane on which the DNAs were blotted was first hybridized with radioactive probe 32P-In 2.6. After the hybridization results were recorded, the radioactive probe was removed, and the membrane hybridized with DIG-labelled probe In 1.7-DIG. Identical results were obtained for 23 samples. One sample was positive with the DIG-labelled probe and negative with the 32P-labelled probe. However, cross-hybridization of In 1.7-DIG with DNA from L. africanum was higher than that obtained with the radioactive probe. This cross-hybridization could be eliminated by raising the temperature of the stringent washing step. No field samples from Africa being available, probe AS 1.7-DIG was dot-blot hybridized against DNAs extracted from leaves of greenhouse-kept citrus plants from different geographical origins and infected with one or other Liberobacter species. The data showed that AS 1.7-DIG hybridized with L. africanum with a sensitivity equivalent to that of the radioactive probe.

Citrus↗
PubMed1997

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Animals↗
PubMed1997

SK-ER3 neuroblastoma cells as a model for the study of estrogen influence on neural cells.

The neuroblastoma SK-ER3 cell line obtained by stable transfection of the human SK-N-BE cell line is proposed as a model for the study of estrogen receptor activity in cells of neural origin. In the SK-ER3 cell line the estrogen receptor, once activated, initiates a differentiation program leading to growth arrest, morphological changes, and acquisition of the dopaminergic phenotype. In the absence of estrogens, this program can be triggered by IGF-I, which can activate the unliganded estrogen receptor via the ras-pathway. It is proposed that this model system might recapitulate the events occurring in vivo during the differentiation of the nervous system and that IGF-I may play an important role in the activation of estrogen receptor at the very early stage of brain development affecting the differentiation of a number of hypothalamic and extrahypothalamic brain regions.

Brain↗
PubMed1997

Quantitation of low abundance mRNAs in glial cells using different polymerase chain reaction (PCR)-based methods.

The conventional methods for mRNA quantitation such as Northern blotting or ribonuclease protection assay sometimes lack enough sensitivity to study low abundance mRNAs or to work with limited amounts of biological samples. The sensitivity of the polymerase chain reaction (PCR) linked to reverse transcription (RT-PCR) has proven useful in amplifying specific mRNAs, especially those present in low copy number. Though, the quantitation of nucleic acids by means of PCR has proven problematic. The main constraint in obtaining quantitative data is inherent in the amplification reaction. Because amplification is an exponential process, small variations in the efficiency of amplification may significantly affect the final yield of the PCR product. The variables that influence the rate of the PCR include the abundance of the mRNA present in the starting material, the concentrations of the Taq DNA polymerase, dNTPs and magnesium ions, the annealing and elongation conditions, the ramping temperatures and the formation of primer secondary structures. Moreover, with the progression of the PCR cycles, reagents are consumed and inhibitors generated, leading to non-linear synthesis of DNA. Finally, tube-to-tube variations sometimes preclude accurate quantitation. Most of the above-mentioned problems can be overcome by the choice of adequate internal controls. The present report reviews two recently developed methods for RNA quantitation, the semi-quantitative PCR and the quantitative PCR illustrated for the measurement of monoamine oxidase (MAO) A and B mRNAs and the estrogen receptor (ER) mRNA respectively, with a particular emphasis on the design of appropriate internal controls to compensate for the intra- and inter-assay variability inherent to RT-PCR.

Animals↗
PubMed1997

Comparison of the 16S/23S ribosomal intergenic regions of "Candidatus Liberobacter asiaticum" and "Candidatus Liberobacter africanum," the two species associated with citrus huanglongbing (greening) disease.

16S/23S intergenic spacer regions from the rRNA operons of two strains of "Candidatus Liberobacter asiaticum" and one strain of "Candidatus Liberobacter africanum" were cloned and sequenced. The intergenic spacers of the two "Candidatus L. asiaticum" strains studied are identical and contain the genes for isoleucine tRNA (tRNA(Ile)) and alanine tRNA (tRNA(Ala)) separated by 11 nucleotides. The intergenic spacer of the "Candidatus L. africanum" strain contains only one tRNA gene (tRNA(Ala)). The level of homology between the intergenic spacers of the two liberobacter species is 79.46%. Ribosomal operons with 16S/23S spacer regions other than those studied might be present in the two "Candidatus Liberobacter" species.

Base Sequence↗
PubMed1996

PCR detection of the two 'Candidatus' Liberobacter species associated with greening disease of citrus.

Greening is a severe and widespread disease of citrus in two main citrus growing areas of the world, Asia and Africa. It is caused by an uncultured phloem restricted bacterium that we have recently characterized from the sequence of its 16S ribosomal DNA. The bacterium is a new "Candidatus' genus, Liberobacter, in the alpha subdivision of the proteobacteria, and two 'Candidatus species' have been recognized: Liberobacter asiaticum and Liberobacter africanum. In this paper we describe a PCR method to detect the two liberobacter species in citrus trees by amplification of a 1160 bp fragment of their 16S rDNA. Distinction between the two species has also been achieved by Xbal digestion of the amplicons.

Citrus↗
PubMed1996

[Erythrocyte rheology and heart hypertrophy in diabetes mellitus].

Reduced red cell filtration and increased aggregation have been reported in diabetic patients. Heart enlargement is a classical complication of high blood pressure but is also frequently encountered in diabetics. The aim of this work was to explore the relationship between rheological disorders and heart enlargement in diabetics and in an animal model of genetically diabetic rats. 1. Among a series of 92 patients with insulin-dependent or non-insulin-dependent diabetes mellitus, silent myocardial ischemia was evidenced in 28. Coronarography in 24 of these patients was strictly normal in 15. In the patients with angiographically normal coronary arteries, the coronary reserve was perturbed by intracoronary injection of papaverine. The left ventricular mass was normal in 79 patients. In patients with silent myocardial ischemia and angiographically normal coronary arteries, there was a positive correlation between left ventricular mass and red cell filtration index using the Hanss hemorheometer (n = 8, r = 0.954, p = 0.001). 2. Red cell stiffness was studied sequentially from 3 to 18 months in diabetic rats Goto Kakisaki (GK). In these rats, cardiac index (heart weight/body weight) was significantly higher than that in Wistors control rats (0.27 +/- 0.02% vs. 0.17 +/- 0.01%, p < 0.001) and was significantly correlated with erythrocyte magnesium (r = 0.920, p 0.026). Overall, the cardiac index in the rats was significantly correlated with red cell filtration (r = 0.370, p = 0.044). In GK rats, heart rate was significantly slower and systolic blood pressure was slightly higher than in control rats (telemetric measures). These findings show that in diabetes mellitus, heart enlargement may be determined by factors other than hypertension or sensitivity to sympathetic stimulation and might be favored by rheological changes and/or excessive intracellular magnesium. In patients with diabetes, the reduced coronary reserve could result in left ventricular hypertrophy but also rheological changes.

Animals↗
PubMed1996

[Urinary screening of drugs of abuse among drivers involved in road accidents].

Driving under the influence of drugs is a growing cause of traffic injuries. Therefore the Abbott Laboratories established a study to estimate the consumption of opiates, cannabinoids, cocaine and amphetamines among young adults involved in a road accident and to compare with a control group. Analytical procedure chosen include an anonymous collection of urines. Drugs are screened by fluorescence polarization immunoassay. Positive samples are confirmed by gas chromatography/mass spectrometry. Results do not show any difference between the two groups.

Accidents, Traffic↗
PubMed1995

Identification of a stimulator of steroid hormone synthesis isolated from testis.

Gonadal steroidogenesis is regulated by pituitary gonadotropins and a locally produced, unidentified factor. A 70-kilodalton (kD) protein complex secreted from rat Sertoli cells was isolated. The complex, composed of 28- and 38-kD proteins, stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine 3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L, respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis and may regulate steroid concentrations and, thus, germ cell development in both males and females.

Amino Acid Sequence↗
PubMed1995

Role of IL-1 beta and corticosteroids in the regulation of the C/EBP-alpha, beta and delta genes in vivo.

We have examined the regulatory effects of interleukin 1 beta (IL-1 beta) on the activation of three different isoforms of the C/EBP family of transcription factors (alpha, beta and delta), in hepatocytes of normal and adrenalectomized (ADX) rats. C/EBP-beta and delta mRNA levels were enhanced by IL-1 beta, whereas that of C/EBP-alpha was not affected by treatment with this interleukin in both normal and adrenalectomized rats. The magnitude of the induction was strikingly higher for C/EBP delta in adrenalectomized animals, indicating a suppressive effect of corticosteroids in the IL-1 beta regulatory pathway. The pattern of C/EBP protein synthesis did not always reflect the mRNA findings. For C/EBP-alpha the protein synthesis was higher than expected in IL-1 beta treated ADX animals compared to normal rats. The pattern of C/EBP synthesis was the one that better reflected the pattern of the mRNA transcription. Differently, the induction of C/EBP-delta was not as pronounced as that of the corresponding mRNA in IL-1 beta treated ADX rats. Hormonal modulation of C/EBP transcription factors was studied in parallel with the hormonal induction of the Alpha-1-Acid Glycoprotein (AGP) gene, which is known to be highly induced in rat liver during the acute phase response. This short report also indicates an important role of corticosteroids in the regulation of transcription factors involved in IL-1 beta signalling during the acute phase response.

Adrenal Cortex Hormones↗
PubMed1995

Detection and characterization of the African citrus greening liberobacter by amplification, cloning, and sequencing of the rplKAJL-rpoBC operon.

Greening disease of citrus is caused by a phloem-restricted, uncultured bacterium, recently characterized and named Liberobacter. As shown previously, a probe encoding ribosomal protein genes (rplKAJL-rpoBC operon) from an Asian liberobacter could detect all Asian liberobacter strains tested, but not African strains. Using the sequence of the rplKAJL-rpoBC operon of the Asian liberobacter strain from Poona (India), we have defined primers for PCR amplification of the equivalent genes of an African liberobacter strain. The amplified fragment was cloned in pUC18 and successfully used as a probe to detect African liberobacter strains by Southern and dot hybridizations. Sequence comparisons of the African and Asian liberobacter operons indicate that they represent two different species in the proposed genus Liberobacter.

Base Sequence↗
PubMed1995

Characterization of the mycoplasmalike organism associated with witches'-broom disease of lime and proposition of a Candidatus taxon for the organism, "Candidatus phytoplasma aurantifolia".

Witches'-broom disease of small-fruited acid lime (WBDL) is a severe disease caused by a mycoplasmalike organism (MLO) in the Sultanate of Oman and the United Arab Emirates. The WBDL MLO was characterized by studying its genome size, the sequences of its 16S ribosomal DNA and the 16S-23S ribosomal DNA spacer region, and hybridization profiles obtained by using WBDL MLO-specific probes. The size of the WBDL MLO genome is 720 kbp. Genomic similarities with the MLOs of sunhemp, sesame, and alfalfa phyllodies were demonstrated, and we found that the WBDL MLO belongs to the sunhemp phyllody phylogenetic subgroup.

Animals↗
PubMed1995

Adrenergic control of alpha-melanocyte-stimulating hormone release in frog pituitary is mediated by both beta- and a nonconventional alpha 2-subtype of adrenoreceptors.

Previous studies have shown that melanotrope cells of the pars intermedia of Rana ridibunda are inhibited by dopaminergic D2 agonists and stimulated by beta-adrenergic agonists. In the present study, we have examined the possible involvement of alpha-adrenoreceptors in the regulation of frog melanotrope cells. Reversed-phase HPLC analysis combined with electrochemical detection revealed the presence of both dopamine and noradrenaline in pars intermedia extracts (74.1 and 3.2 ng/mg protein, respectively), while adrenaline was undetectable. Administration of graded doses of noradrenaline and adrenaline (from 0.1 to 10 microM) to perifused frog neurointermediate lobes induced a dose-dependent inhibition of alpha-MSH release. The inhibitory effect of adrenaline was partially blocked by the D2-dopaminergic antagonist sulpiride and totally suppressed by concomitant administration of sulpiride and yohimbine (an alpha 2-adrenergic antagonist). Conversely, in the presence of sulpiride, noradrenaline provoked a strong stimulation of alpha-MSH secretion which was totally blocked by the beta-adrenergic antagonist propranolol. Taken together, our results indicate that endogenous catecholamines may exert a complex regulatory action on frog melanotrope cells through activation of dopaminergic D2, alpha 2- and beta-adrenergic receptors.

Adrenergic Agents↗
PubMed1994

Functional characterization of a nonclassical nicotine receptor associated with inositolphospholipid breakdown and mobilization of intracellular calcium pools.

Classical nicotinic receptors are neurotransmitter-gated channels that, upon activation by acetylcholine, induce the opening of an intrinsic cationic channel. We have recently observed that, in frog pituitary melanotrophs, nicotine stimulates alpha-melanocyte-stimulating hormone (alpha-MSH) release through a noncholinergic mechanism. In the study reported here, we investigated the intracellular events that mediate the response of frog melanotrophs to nicotine. Nicotine was capable of stimulating alpha-MSH release in the absence of Ca2+ and/or Na+ in the extracellular medium. A short pulse of nicotine induced a rapid and transient increase of cytosolic free Ca2+ concentration ([Ca2+]i). The effect of nicotine on Ca2+ mobilization was not affected in the absence of Na+ and Ca2+ in the extracellular medium, indicating that the nicotine-evoked increase in [Ca2+]i did not result from Na+ or Ca2+ influx. Nicotine induced both an increase in inositol trisphosphate and a reduction in phosphaditylinositol bisphosphate concentrations but did not affect cAMP production. The present results indicate that nicotine-induced stimulation of alpha-MSH release in frog melanotrophs can be explained by activation of inositolphospholipid breakdown and mobilization of inositol triphosphate-dependent intracellular Ca2+ pools. These data provide evidence for the existence of an unusual type of noncholinergic nicotine receptor positively coupled to phospholipase C.

Animals↗
PubMed1994

The polypeptide diazepam-binding inhibitor and a higher affinity mitochondrial peripheral-type benzodiazepine receptor sustain constitutive steroidogenesis in the R2C Leydig tumor cell line.

The polypeptide diazepam binding inhibitor (DBI) and drug ligands for the mitochondrial peripheral-type benzodiazepine receptor (PBR) have been shown to regulate cholesterol transport, the rate-determining step in steroidogenesis, in hormone-responsive steroidogenic cells including the MA-10 Leydig tumor cells. The present study was designed to characterize the role of DBI and PBR in the R2C rat Leydig tumor constitutive steroid-producing cell model. Both DBI and PBR were present in R2C cells. R2C cell treatment with a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to DBI, but not sense, resulted in the reduction of DBI levels and a concomitant dramatic decrease of the amount of progesterone produced. These observations strongly suggested that DBI was important in maintaining constitutive steroidogenesis in R2C cells. Radioligand binding assays revealed the presence of a single class of PBR binding sites with an affinity 10 times higher (Kd approximately 0.5 nM) than that displayed by the MA-10 PBR (Kd approximately 5 nM). Photolabeling of R2C and MA-10 cell mitochondria with the photoactivatable PBR ligand [3H]1-(2-fluoro-5-nitrophenyl)-N-methyl-N-(1-methyl-propyl)-3- isoquinolinecarboxamide showed that the M(r) 18,000 PBR protein was specifically labeled. This indicates that the R2C cells express a PBR protein which has properties similar to the MA-10 PBR. Chemical crosslinking studies of purified metabolically radiolabeled DBI to mitochondria provided direct evidence that DBI specifically binds to the M(r) 18,000 PBR protein. Moreover, DBI and a PBR synthetic ligand were able to increase steroid production in isolated R2C cell mitochondria which express the 5 nM affinity receptor. However, mitochondrial PBR binding was increased by 6-fold upon addition of the post-mitochondrial fraction, suggesting that a cytosolic factor modulates the binding properties of PBR in R2C cells and is responsible for the 0.5 nM affinity receptor seen in intact cells. In conclusion, these data demonstrate that DBI plays a key role in maintaining R2C constitutive steroidogenesis by binding to the mitochondrial higher affinity PBR which promotes a continuous supply of cholesterol to the inner mitochondrial side chain cleavage cytochrome P450.

Animals↗
PubMed1994

The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria.

Using the PCR, we amplified the 16S ribosomal DNAs (rDNAs) of an Asian strain and an African strain of the uncultured, gram-negative, walled, phloem-limited bacterium-like organism (BLO) associated with citrus greening disease. We evaded coamplification of chloroplast 16S rDNA by using restriction enzymes; the chloroplast 16S rDNA was sensitive to BclI digestion and resistant to EcoRI digestion, while the 16S rDNA of the BLO was resistant to BclI digestion and sensitive to EcoRI digestion. The 16S rDNA of the African BLO strain was amplified from BclI-digested DNA extracted from infected periwinkle leaf midribs. The Asian strain was isolated from plant extract by using a specific monoclonal antibody coated onto the surface of a PCR tube. The 16S rDNAs of the two BLO strains were cloned and sequenced. Comparisons with sequences of 16S rDNAs obtained from the GenBank data base revealed that the two citrus greening disease BLOs belong to the alpha subdivision of the class Proteobacteria. Even though their closest relatives are members of the alpha-2 subgroup, these BLOs are distinct from this subgroup as we observed only 87.5% homology between the 16S rDNAs examined. Therefore, the two BLOs which we studied probably are members of a new lineage in the alpha subdivision of the Proteobacteria. We propose the trivial name "liberobacter" for this new group of bacteria and will wait until additional characteristics have been determined before we propose a formal name.

Base Sequence↗