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See detailA general method for the chemical synthesis of gamma-P-32-labeled or unlabeled nucleoside 5 '-triphosphates and thiamine triphosphate
Bettendorff, Lucien ULg; Nghiem, Hoang O; Wins, Pierre et al

in Analytical Biochemistry (2003), 322(2), 190-197

Several methods for the chemical synthesis of gamma-P-32-labeled and unlabeled nucleoside 5'-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of ... [more ▼]

Several methods for the chemical synthesis of gamma-P-32-labeled and unlabeled nucleoside 5'-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of low yield, requirement for anhydrous solvents, procedures involving several steps or insufficient specific radioactivity of the labeled triphosphate. In the method described here, all these drawbacks are avoided. The synthesis of [gamma-P-32]TbTP was carried out in one step, using 1,3-dicyclohexyl carbodiimide as condensing agent for thiamine diphosphate and phosphoric acid in a dimethyl sulfoxide/pyridine solvent mixture. Anhydrous solvents were not required and the yield reached 90%. After purification, [gamma-P-32]ThTP had a specific radioactivity of 11 Ci/mmol and was suitable for protein phosphorylation. The method can also be used for the synthesis Of [gamma-P-32]ATP of the desired specific radioactivity. It can easily be applied to the synthesis of unlabeled ThTP or ribo- and deoxyribonucleoside 5'-triphosphates. In the latter case, inexpensive 5'-monophosphate precursors can be used as reactants in a 20-fold excess of phosphoric acid. Deoxyribonucleoside 5'-triphosphates were obtained in 6 h with a yield of at least 70%. After purification, the nucleotides were found to be suitable substrates for Taq polymerase during polymerase chain reaction cycling. Our method can easily be scaled up for industrial synthesis of a variety of labeled and unlabeled triphosphoric derivatives from their mono- or diphosphate precursors. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

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See detailHuman immune cells express ppMCH mRNA and functional MCHR1 receptor
Verlaet, Myriam ULg; Adamantidis, Antoine ULg; Coumans, Bernard ULg et al

in FEBS Letters (2002), 527(1-3), 205-210

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated ... [more ▼]

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT-PCR analysis revealed a low expression of prepro-MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3-stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. [less ▲]

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See detailMethod for estimation of low outer membrane permeability to beta-lactam antibiotics
Lakaye, Bernard ULg; Dubus, Alice ULg; Joris, Bernard ULg et al

in Antimicrobial Agents and Chemotherapy (2002), 46(9), 2901-2907

The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux ... [more ▼]

The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated beta-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of beta-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of beta-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10(-7) cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 X 10(-9) cm/s). Using these values, the MICs for a beta-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method. [less ▲]

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See detailMolecular characterization of a specific thiamine triphosphatase widely expressed in mammalian tissues
Lakaye, Bernard ULg; Makarchikov, Alexander F; Antunes, Adelio F et al

in Journal of Biological Chemistry (2002), 277(16), 13771-13777

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian ... [more ▼]

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank(TM) data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-beta-D-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase. [less ▲]

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See detailStudy of the role of ionogenic amino-acid residues in catalytic activity of thiamine triphosphatase from bovine kidney by means of chemical modification
Makarchikov, Alexander F; Luchko, A. A.; Bettendorff, Lucien ULg et al

in News of Biomedical Sciences (2002), 3

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See detailSteroid receptor coactivator SRC-1 exhibits high expression in steroid-sensitive brain areas regulating reproductive behaviors in the quail brain.
Charlier, Thierry ULg; Lakaye, Bernard ULg; Ball, Gregory F et al

in Neuroendocrinology (2002), 76(5), 297-315

The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including the receptors for sex steroid hormones. Reducing the expression of SRC-1 by ... [more ▼]

The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including the receptors for sex steroid hormones. Reducing the expression of SRC-1 by injection of specific antisense oligonucleotides markedly inhibits the effects of estrogens of the sexual differentiation of brain and behavior in rats and inhibits the activation of female sexual behavior in adult female rats. SRC-1 thus appears to be involved in both the development and activation of sexual behavior. In the Japanese quail brain, we amplified by RT-PCR a 3,411-bp fragment extending from the HLH domain to the activating domain-2 of the protein. The quail SRC-1 is closely related to the mammalian (m) SRC-1 and contains a high proportion of GC nucleotides (62.5%). Its amino acid sequence presents 70% identity with mammalian SRC-1 and contains the three conserved LXXLL boxes involved in the interaction with nuclear receptors. In both males and females, RT-PCR demonstrates a similarly high level of expression in the telencephalon, diencephalon, optic lobes, brain stem, spinal cord, pituitary, liver, kidney, adrenal gland, heart, lung, gonads and gonoducts. Males express significantly higher levels of SRC-1 in the preoptic area-hypothalamus than females. In both sexes, lower levels of expression are observed in the cerebellum and muscles. In situ hybridization utilizing a mixture of four digoxigenin-labeled oligonucleotides confirms at the cellular level the widespread distribution of SRC-1 mRNA in the brain and a particularly dense expression in steroid-sensitive areas that play a key role in the control of male sexual behavior. These data confirm the presence and describe for the first time the SRC-1 distribution in the brain of an avian species. They confirm its broad, nearly ubiquitous, distribution in the entire body including the brain as could be expected for a coactivator that regulates to the action of many nuclear receptors. However this distribution is heterogeneous in the brain and sexually differentiated in at least some areas. The very dense expression of SRC-1 in limbic and mesencephalic nuclei that are associated with the control of male sexual behavior is consistent with the notion that this coactivator plays a significant role in the activation of this behavior. [less ▲]

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See detailATP-driven, Na(+)-independent inward Cl- pumping in neuroblastoma cells
Bettendorff, Lucien ULg; Lakaye, Bernard ULg; Margineanu, Ilca et al

in Journal of Neurochemistry (2002), 81(4), 792-801

In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport ... [more ▼]

In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl-](i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl-](i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state [Cl-](i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl-](i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state [Cl-](i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells. [less ▲]

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See detailThe genetic absence epilepsy rat from Strasbourg (GAERS), a rat model of absence epilepsy: Computer modeling and differential gene expression
Lakaye, Bernard ULg; Thomas, E.; Minet, Arlette ULg et al

in Epilepsia (2002), 43(Suppl. 5), 123-129

Purpose: We present results obtained by computer modeling of the thalamic network and differential gene expression analysis in a rat strain with absence epilepsy, the genetic absence epilepsy rat from ... [more ▼]

Purpose: We present results obtained by computer modeling of the thalamic network and differential gene expression analysis in a rat strain with absence epilepsy, the genetic absence epilepsy rat from Strasbourg (GAERS). Methods: (a) Computer modeling used equations from the Hodgking-Huxley model with a circuit of 13 reticular thalamic (nRt) and 39 thalamocortical (TC) neurons; (b) gene-expression analysis using differential mRNA display (DD), in situ hybridization, Northern blotting, and competitive reverse transcriptase-polymerase chain reaction (RT-PCR). Results: (a) Computer modeling showed an increased network synchrony in the thalamic circuit as the value of conductance of low-voltage activated calcium channel (LVACC) is increased. (b) Using differential mRNA display, a 40% upregulation of the H-ferritin mRNA in the hippocampus was demonstrated. Looking for some candidate genes of the VACC family, no difference was found in the alpha1G mRNA expression between GAERS and control animals, whereas a decreased expression of the alpha1E subunit was observed in the cerebellum and the brainstem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed. Conclusions: The use of computer modeling appeared to be an efficient way to evaluate the impacts of electrophysiologic findings in vivo from single cells on an entire circuit. No clear single gene defect was revealed so far in GAERS. More information could arise from linkage analysis. However, some brain structures like hippocampus or cerebellum classically not known to be involved in the production of absence spike-and-wave discharges could in fact participate in the development of this phenotype. [less ▲]

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See detailCloning and distribution of steroid receptor coactivator SRC-1 in quail.
Charlier, Thierry ULg; Lakaye, Bernard ULg; Ball, Gregory F. et al

Poster (2001)

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See detailIncreased Expression of mRNA Encoding Ferritin Heavy Chain in Brain Structures of a Rat Model of Absence Epilepsy
Lakaye, Bernard ULg; de Borman, B.; Minet, Arlette ULg et al

in Experimental Neurology (2000), 162(1), 112-20

Differential mRNA display was carried out to find genes that are differentially regulated in the brain of a rat strain with absence epilepsy, the genetic absence epilepsy rats from Strasbourg (GAERS ... [more ▼]

Differential mRNA display was carried out to find genes that are differentially regulated in the brain of a rat strain with absence epilepsy, the genetic absence epilepsy rats from Strasbourg (GAERS). Among the 32 differentially displayed cDNA fragments actually cloned and sequenced, one shows 100% identity with the rat heavy chain ferritin (H-ferritin) mRNA. Northern blot analysis confirmed the up-regulation of the H-ferritin mRNA. Using dot blotting, a 40% increase in expression was reported in the subcortical forebrain of the adult GAERS, while cortex, brain stem, and cerebellum appeared unmodified. This change was not observed in the brain of 25-day-old rats, an age at which the epileptic phenotype is not present. By in situ hybridization, the enhanced expression was localized in the hippocampus. The increase in mRNA encoding H-ferritin was not immunodetected at the protein level by Western blotting. These results are not apparently related to the neural substrate of SWD or to the distribution of local increase in glucose metabolism previously described in the GAERS. It is hypothesized that the up-regulation of the H-ferritin mRNA is part of a mechanism protecting the hippocampus, a seizure-prone area, against a possible overactivation during absence seizures. [less ▲]

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See detailEstrogen Receptor-Beta in Quail: Cloning, Tissue Expression and Neuroanatomical Distribution
Foidart, Agnès ULg; Lakaye, Bernard ULg; Grisar, Thierry ULg et al

in Journal of Neurobiology (1999), 40(3), 327-42

A partial estrogen receptor-beta (ERbeta) cDNA had been previously cloned and sequenced in Japanese quail. The 3'- and 5'-rapid amplification of cDNA ends techniques were used here to identify a cDNA ... [more ▼]

A partial estrogen receptor-beta (ERbeta) cDNA had been previously cloned and sequenced in Japanese quail. The 3'- and 5'-rapid amplification of cDNA ends techniques were used here to identify a cDNA sequence of the quail ERbeta that contains a complete open reading frame. For the first time in an avian species, this cDNA sequence and the corresponding amino acid sequence are described. They are compared with the known ERbeta sequences previously described in mammals and with the ERalpha sequences identified in a selection of mammalian and avian species. The analysis by Northern blotting of the ERbeta mRNA expression in the brain and kidneys revealed the presence of several transcripts. The presence of ERbeta identified by reverse transcriptase-polymerase chain reaction demonstrated a widespread distribution quite different from the distribution of ERalpha. The complete neuroanatomical distribution of ERbeta mRNA as determined by in situ hybridization with 35S- and 33P-labeled oligoprobes is also presented. Transcripts are present in many nuclei implicated in the control of reproduction such as the medial preoptic nucleus, the nucleus striae terminalis, and the nucleus taeniae, the avian homologue of the amygdala. These data demonstrate the presence of ERbeta in a nonmammalian species and indicate that the (neuro)-anatomical distribution of this receptor type has been conserved in these two classes of vertebrates. The role of this receptor in the control of reproduction and other physiological processes should now be investigated. [less ▲]

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See detailExpression of Mrna Encoding Alpha1e and Alpha1g Subunit in the Brain of a Rat Model of Absence Epilepsy
de Borman, B.; Lakaye, Bernard ULg; Minet, Arlette ULg et al

in Neuroreport (1999), 10(3), 569-74

Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding ... [more ▼]

Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding calcium channel alpha1E and alpha1G subunits was measured in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Using quantitative RT-PCR and in situ hybridization, no difference was found in alpha1G mRNA expression between GAERS and control animals, while a decreased expression of alpha1E was seen in the cerebellum and the brain stem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed. [less ▲]

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See detailWhen Drug Inactivation Renders the Target Irrelevant to Antibiotic Resistance: A Case Story with Beta-Lactams
Lakaye, Bernard ULg; Dubus, Alice ULg; Lepage, Sylvie ULg et al

in Molecular Microbiology (1999), 31(1), 89-101

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to ... [more ▼]

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a beta-lactamase-overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor beta-lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents. [less ▲]

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See detailSteroid sensitive sites in the avian brain: does the distribution of the estrogen receptor alpha and beta types provide insight into their function?
Ball, G. F.; Bernard, D. J.; Foidart, Agnès ULg et al

in Brain, Behavior & Evolution (1999), 54

Studies in avian species have often been useful in elucidating basic concepts relevant to the regulation of reproductive behaviors by sex steroid hormones. Once a link between a steroid hormone and a ... [more ▼]

Studies in avian species have often been useful in elucidating basic concepts relevant to the regulation of reproductive behaviors by sex steroid hormones. Once a link between a steroid hormone and a behavioral response has been established, one can use the localization of steroid hormone receptors in the brain to facilitate the identification of neural circuits that control behavior. The recent identification of a second type of estrogen receptor called estrogen receptor beta or ERbeta has raised new issues about the action of steroid hormones in the brain. A hypothesis has been proposed by Kuiper et al. [1998] based on studies in mammalian species suggesting that ERalpha (the name given to the ER that was previously described) is important for reproduction while ERbeta is more important for non-reproductive functions. In this paper we apply this hypothesis more generally by examining possible functions of ERbeta in avian species. We have initiated studies of the ERbeta in the brain of two avian species, the Japanese quail (Coturnix japonica) and the European starling (Sturnus vulgaris). ERbeta was cloned in both species and the mRNA for this receptor type was localized in the brain employing in situ hybridization histochemistry methods. In both species ERbeta was found to be diffusely present in telencephalic areas consistent with a role for this receptor subtype in cognitive functions. However, ERbeta mRNA was also found in many brain areas that are traditionally thought to be important in the regulation of reproductive functions such as the preoptic region, the bed nucleus of the stria terminalis and the nucleus taeniae. Of the two receptor types, only mRNA for ERalpha was observed in the telencephalic vocal control nucleus HVc of male starlings. Steroid receptors in this nucleus are thought to be an example of an evolutionary specialization that has evolved to coordinate the production of courtship vocalizations with other aspects of reproduction. The lack of ERbeta mRNA expression in HVc is consistent with the hypothesis that ERalpha is preferentially involved in reproductive behaviors while ERbeta is involved in the steroid regulation of other neural functions. However, the widespread occurrence of ERbeta in other nuclei involved in reproductive function suggests that one must be cautious about the general applicability of the above hypothesis until more is known about ERbeta function in these other nuclei [less ▲]

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See detailThe Molecular Neuron-Glia Couple and Epileptogenesis
Grisar, Thierry ULg; Lakaye, Bernard ULg; Thomas, E. et al

in Advances in Neurology (1999), 79

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See detailThe molecular neuron-glia couple and epileptogenesis
Grisar, Thierry ULg; Lakaye, Bernard ULg; Thomas, Elizabeth et al

in Delgado-Escueta, A. V.; Wilson, W. A.; Olsen, R. W. (Eds.) et al Jasper's Basic Mechanisms of the Epilepsies, 3rd edition (1999)

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See detailHousekeeping Genes as Internal Standards: Use and Limits
Thellin, Olivier ULg; Zorzi, Willy ULg; Lakaye, Bernard ULg et al

in Journal of Biotechnology (1999), 75(2-3), 291-5

Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their ... [more ▼]

Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed. [less ▲]

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See detailPartial Cloning and Distribution of Estrogen Receptor Beta in the Avian Brain
Lakaye, Bernard ULg; Foidart, Agnès ULg; Grisar, Thierry ULg et al

in Neuroreport (1998), 9(12), 2743-8

A partial estrogen receptor beta (ER-beta) cDNA was isolated from testicular quail RNA by RT-PCR with degenerate primers specific to the rat ER-beta sequence. A high expression of ER-beta was demonstrated ... [more ▼]

A partial estrogen receptor beta (ER-beta) cDNA was isolated from testicular quail RNA by RT-PCR with degenerate primers specific to the rat ER-beta sequence. A high expression of ER-beta was demonstrated by RT-PCR in the telencephalon, diencephalon, pituitary, testis and kidneys of male quail but little or no expression was detected in the cerebellum, pectoral muscle and adrenal gland. In situ hybridization with a 35S-labelled oligoprobe in sections through the preoptic area-rostral hypothalamus identified high expression in the medial preoptic nucleus, bed nucleus striae terminalis and nucleus taeniae. These data demonstrate the presence of an ER-beta in brain areas implicated in the control of reproduction in a non-mammalian species. [less ▲]

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See detailCloning of the Rat Brain Cdna Encoding for the Slc-1 G Protein-Coupled Receptor Reveals the Presence of an Intron in the Gene
Lakaye, Bernard ULg; Minet, Arlette ULg; Zorzi, Willy ULg et al

in Biochimica et Biophysica Acta (1998), 1401(2), 216-20

In order to isolate new G protein-coupled receptors expressed in the cerebral cortex, a set of degenerate oligonucleotides corresponding to the third and seventh transmembrane segment were synthetized ... [more ▼]

In order to isolate new G protein-coupled receptors expressed in the cerebral cortex, a set of degenerate oligonucleotides corresponding to the third and seventh transmembrane segment were synthetized. Their use in PCR on rat brain cortex mRNA amplified several cDNA fragments. One of them, a 526 bp sequence, encoded for what was at that time an unknown G protein-coupled receptor. An oligonucleotide derived from the sequence was then used as a probe to isolate the receptor cDNA from a rat brain cDNA library. It encodes for a 353aa protein with seven transmembrane segments, three consensus N-glycosylation sites at the amino terminus and several potential phosphorylation sites in the intracellular loops. This protein shares 91% overall identity with a recently cloned human somatostatin-like receptor of 402aa named SLC-1. This suggests that we have cloned the rat orthologue of the human SLC-1. However, the extracellular N-terminus of the human receptor is 49 amino acids longer and shows 50% identity with the rat one. Because the human sequence was deduced from genomic DNA, we suspected the presence of an intron in the gene. This was confirmed by PCR using primers spanning the intron. On the basis of the sequence of a 128 kb fragment of chromosome 22 encompassing the SLC-1 gene, we were able to deduce a corrected amino acids sequence for the human receptor. So both rat and human SLC-1 receptors are 353aa long, with three consensus N-glycosylation sites. They share 96% identity at the amino acid level and are encoded by a gene containing one intron in the coding sequence. [less ▲]

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See detailDemonstration of the expression of CD95 ligand transcript and protein in human placenta
Zorzi, Willy ULg; Thellin, Olivier ULg; Coumans, Bernard ULg et al

in Placenta (1998), 19(4), 269-277

Tolerance of the fetal allograft enables the human conceptus to implant itself into the maternal uterus and survive and grow there. This tolerance phenomenon remains largely obscure, notably because it ... [more ▼]

Tolerance of the fetal allograft enables the human conceptus to implant itself into the maternal uterus and survive and grow there. This tolerance phenomenon remains largely obscure, notably because it appears to be controlled by multiple mechanisms. CD95 ligand (CD95-L), which can trigger death of CD95-positive cells by apoptosis, may participate in inducing anti-fetus-sensitized CD95-positive T lymphocytes to enter apoptosis. Using immunohistochemistry (first trimester and term placentae), FAGS assays (term placenta) and RT-PCR assays (term placenta), the presence of CD95-L protein and mRNA has been shown in crude placental tissue preparations and isolated placental cells. Among the latter, CD95-L expression was detected in trophoblastic cells, fetal blood cells (mRNA only) and also the Hofbauer macrophages. No CD95-L was detected in fibroblasts or fetal endothelial cells. Thus trophoblastic cells, Hofbauer macrophages, and perhaps also fetal blood cells could form a sequential barrier blocking maternal activated defence cells bearing CD95 molecules. (C) 1998 W. B. Saunders Company Ltd. [less ▲]

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