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Biomedical subjects

S Leclerc

Publications and source records attributed to S Leclerc.

At least 19 recordsLinked to original sources
PubMed1999

Juvenile xanthogranuloma presenting as obstructive jaundice.

The authors report the case of a 9-month-old infant presenting with obstructive jaundice. Preoperative investigation findings showed distension of the extrahepatic biliary tract. Urine analysis result was positive for cytomegalovirus. At laparotomy, a 1-cm round mass was found within the head of the pancreas, near its upper border and in the course of the common bile duct. Findings on frozen sections could not rule out a malignant process and an hepaticoduodenostomy was constructed. Final diagnosis was juvenile xanthogranuloma. An association between cytomegalovirus and juvenile xanthogranuloma has been reported previously in the literature.

Cholecystectomy↗
PubMed1999

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors.

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.

CDC2 Protein Kinase↗
PubMed1999

UDP-glucuronosyltransferase in the rat olfactory bulb: identification of the UGT1A6 isoform and age-related changes in 1-naphthol glucuronidation.

Xenobiotic glucuronidation represents a major metabolic protection of the brain against chemical aggressions at blood-brain interfaces. We previously observed that glucuronidation of 1-naphthol was very effective in olfactory bulb, which is a pathway for the entry of foreign molecules into the brain. In this work, we showed that 1-naphthol glucuronidation varied according to age. It was very high at birth, then decreased markedly in 3-month-old rats and increased again significantly during aging. By Western blot and reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the presence in the olfactory bulb of the UDP-glucuronosyltransferase (UGT) 1A6 isoform, which catalyzes the glucuronidation of phenols, such as 1-naphthol. Quantitative RT-PCR indicated that the mRNA levels encoding UGT1A6 did not significantly change according to age, thus suggesting that other differently regulated UGT isoforms were present and would account for the variations of 1-naphthol glucuronidation observed.

Aging↗
PubMed1999

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Animals↗
PubMed1999

Multiple cloning sites from mammalian expression vectors interfere with gene promoter studies in vitro.

When performing transcriptional analyses, reporter gene-expression vectors are used to insert promoter fragments through the selected use of a multiple cloning site (MCS) located upstream of the reporter gene. The MCS from pBluescript has frequently been transferred into reporter plasmids (usually bearing the chloramphenical acetyltransferase reporter gene) and used to subclone various promoter fragments from diverse genes. Analyses in electrophoretic mobility shift assay using this MCS as labeled probe revealed that it specifically binds multiple nuclear proteins from a whole array of widely used cell types. Moreover, the presence of the MCS sequence dramatically altered promoter activity in a totally unpredictable fashion that depends on the distance between the MCS and the basal promoter start site of the gene, leading to severe misinterpretation of the transfection data. Finally, we provide evidence that the BamHI/SmaI/PstI restriction site combination is likely one of the major binding site for nuclear proteins on the pBluescript MCS, therefore suggesting that this particular combination of restriction sites should be avoided in the MCS from plasmids that are to be used in promoter studies.

Binding Sites↗
PubMed1998

Retinol status of newborn infants with congenital diaphragmatic hernia.

The etiology of congenital diaphragmatic hernia (CDH) is not yet known. Studies in the literature from 1941 have reported that nutritional deficiency of vitamin A during pregnancy could lead to CDH, associated or not with other malformations in young rats. More recently, possible correlations between expression patterns of cellular retinoid-binding protein and retinoic-acid receptors and morphologic effects of vitamin A deficiency have been suggested. The purpose of this study was to verify in human newborns the possible link between vitamin A deficiency and CDH previously observed in experimental animals. Blood samples were obtained during the first hours after birth from 11 term CDH newborns and 11 healthy controls matched for gestational age, and also from 7 mothers in each group, for a total of 7 newborn-mother pairs of matched CDH-controls. Plasma retinol was measured by high-performance liquid chromatography and retinol-binding protein (RBP) by nephelometry. In the 11 matched CDH-control newborns, plasma retinol and RBP levels in CDH newborns were 50% less than control values (P< 0.0002 and <0.006, respectively); in contrast, retinol levels in CDH mothers were significantly higher than those of control mothers (P < 0.005). The observation that the plasma concentrations of retinol and RBP are low in infants with CDH relative to controls may be clinically very relevant and may help to elucidate the mechanism of development of this congenital anomaly.

Case-Control Studies↗
PubMed1997

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1.

Expression of the gene encoding poly(ADP-ribose) polymerase (PARP), although ubiquitous, nevertheless varies substantially between tissues. We have recently shown that Sp1 binds five distinct target sequences (US-1 and F1-F4) in the rat PARP (rPARP) gene promoter. Here we used deletion analyses and site-directed mutagenesis to address the regulatory function played by these Sp1 sites on the basal transcriptional activity directed by the rPARP promoter. Transfection experiments revealed that the most proximal Sp1 site is insufficient by itself to direct any promoter activity. In addition, a weak negative regulatory element was identified between positions -101 and -60. The rPARP promoter directed high levels of chloramphenicol acetyltransferase activity in Jurkat T-lymphoblastoid and Ltk- fibroblast cells but only moderate levels in pituitary GH4C1 and liver HTC cells, correlating with the amounts of PARP detected in these cells by western blot analysis. However, the reduced promoter efficiency in HTC and GH4C1 cells did not result from the lack of Sp1 activity in these cells but suggested that yet uncharacterized regulatory proteins might turn off PARP gene expression by binding negative regulatory elements from the rPARP promoter. Similarly, site-directed mutagenesis on the three most proximal Sp1 elements suggested the influence exerted by Sp1 on the rPARP promoter activity to vary substantially between cell types. It also provided evidence for a basal rPARP promoter activity driven through the recognition of unidentified cis-acting elements by transcription factors other than Sp1.

Animals↗
PubMed1997

Molecular and functional characterization of a 5-HT4 receptor cloned from human atrium.

5-Hydroxytryptamine (5-HT) has been shown to exert positive inotropic, chronotropic, and lusitropic effects and to stimulate the L-type calcium channel current (I(Ca)) in human atrial tissue through activation of the pharmacologically defined 5-HT4 receptor subtype. However, the molecular nature of the receptor(s) involved in these effects is still unknown. In the present study, we report the molecular nature of a 5-HT4 receptor cloned from human atrium, h5-HT4A. Sequence analysis reveals that h5-HT4A displays a 93% protein identity with the short form of the 5-HT4 receptor recently isolated from rat brain. h5-HT4A mRNA is expressed in human atrium but not ventricle, and is also found in brain and GI tract. h5-HT4A transiently expressed in COS-7 cells displays a classical 5-HT4 pharmacological profile. However, affinities of the h5-HT4A receptor for agonists such as ML10302, BIMU1, renzapride or zacopride were 4-10-fold lower than the ones found in brain. Moreover, the stimulatory patterns of cAMP formation by h5-HT4A in response to the 5-HT4 agonists ML10302 and renzapride were very similar to the patterns of stimulation of I(Ca) obtained in response to these compounds in human atrial myocytes. We conclude that h5-HT4A likely mediates the effects of 5-HT in human atrium and may differ from 5-HT4 receptor isoforms present in the brain and GI tract.

Adolescent↗
PubMed1997

Morphometrics of normal and hypoplastic lungs in preterm lambs with gas and partial liquid ventilation

To test the hypothesis that perfluorochemical (PFC) instillation may reduce the pulmonary trauma commonly associated with conventional gas ventilation, we studied 12 lambs with normal lungs and 10 with pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH). We used mechanical ventilation for up to 3.5 h, with and without tracheal instillation of LiquiVent PFC liquid. At the end of experimentation lungs were fixed for morphometrical analysis of their components and pulmonary trauma was evaluated by measurement of the perivascular compression index (PCI = % perivascular emphysema/% vessels). In normal lungs good gas exchange and respiratory mechanics were obtained with all modes of ventilation, with no statistical difference in the index of pulmonary trauma with or without instillation of LiquiVent (P >0.05). In the hypoplastic lungs, tracheal instillation of PFC liquid after 30 min of conventional gas ventilation significantly improved PaCO2 (from 107+/-8 to 55+/-6 mmHg, P <0.05), pH (from 7.00+/-0.03 to 7.29+/-0.04, P <0.05), compliance (from 0.08+/-0.01 to 0.25+/-0.03 ml/cmH2O . kg, P <0.05), and ventilatory index (from 1,445+/-148 to 794+/-139, P <0.05). Survival was 6/6 animals with PFC ventilation compared to 1/4 with conventional gas ventilation with no more pulmonary trauma (mean PCI 12.6+/-1.8 vs. 11.4+/-4.0%, P >0.05) for a longer mean ventilatory period in the PFC group. We conclude that the PFC liquid technique of ventilation can improve respiratory physiology when conventional gas ventilation alone is proving inefficient. There was no significant difference in pulmonary trauma at morphometrics between gas and partial liquid ventilation.

Journal Article↗
PubMed1997

Cytokinin-derived cyclin-dependent kinase inhibitors: synthesis and cdc2 inhibitory activity of olomoucine and related compounds.

Cyclin-dependent kinases (cdk) have recently raised considerable interest in view of their essential role in the regulation of the cell division cycle. The structure-activity relationships of cdk inhibition showed that the 1, 3; and 7 positions of the purine ring must remain free, probably for a direct interaction, in which it behaves as a hydrogen bond acceptor. Olomoucine (6-(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine, OC), roscovitine (6-(benzylamino)-2(R)-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpur ine), and other N6,2,9-trisubstituted adenines were found to exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. Removal or change of the side chain at position 2 or the hydrophobic group at position 9 dramatically decreased the inhibitory activity of olomoucine or roscovitine. Inhibition of cdk with OC and related compounds clearly arrests cell proliferation of many tumor cell lines at G1/S and G2/M transitions and also triggers apoptosis in the target tumor cells in vitro and in vivo. Thus, from a pharmacological point of view, OC may represent a model compound for a new class of antimitotic and antitumor drugs.

Animals↗
PubMed1997

Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine.

Cyclin-dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurin e], was identified by screening a series of C2,N6,N9-substituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A., Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J.-G. & Moulinoux, J.-P. (1997) Eur. J. Biochem. 243, 527-536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24-nm (2.4 A) resolution and refined to an Rfactor of 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP-complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)-stereoisomer of roscovitine is bound to cdk2. The (R)-isomer is about twice as potent in inhibiting cdc2/cyclin B than the (S)-isomer. Results from structure/activity studies and from analysis of the cdk2/roscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.

Adenosine Triphosphate↗
PubMed1997

Oriented macroporous polyacrylamide gels.

Macroporous gels with huge cavities and partition walls result from controlled microsyneresis during gelation. In this report we show that the microsyneresis process can be further controlled: it is possible to orient the partition walls of macropores by the use of an electric field throughout polymerization. Our oriented macroporous polyacrylamide gels offer a specific structure: a set of parallel channels of about 2 microm inner diameter, with polyacrylamide walls including acidic groups linked to the aggregated matter.

Acrylic Resins↗
PubMed1997

The rat growth hormone and human cellular retinol binding protein 1 genes share homologous NF1-like binding sites that exert either positive or negative influences on gene expression in vitro.

High levels of expression for the rat growth hormone (rGH) gene are restricted to the somatotroph cells of the anterior pituitary. Previously, we have shown that rGH cell-specific repression results in part from the recognition of negatively acting silencers by a number of nuclear proteins that repress basal promoter activity. Examination of these silencers revealed the presence of binding sites for proteins that belong to the NF1 family of transcription factors. Indeed, proteins from this family were shown to bind the rGH proximal silencer (designated silencer-1) in in vitro assays. Furthermore, this silencer site is capable of repressing chloramphenicol acetyltransferase (CAT) gene expression driven by an heterologous promoter (that of the mouse p12 gene), even in pituitary cells. Recently, we identified in the 5' untranslated region of the gene encoding human cellular retinol binding protein 1 (hCRBP1) a negative regulatory element (Fp1) that also bears an NF1 binding site very similar to that of rGH silencer-1. However, although deletion of Fp1 in the hCRBP1 gene yielded increased CAT activity, pointing toward a negative regulatory function exerted by this element, its insertion upstream of the p12 basal promoter results in an impressive positive stimulation of CAT gene expression. By exploiting NaDodSO4 gel protein fractionation and renaturation, we identified a 40-kD nuclear protein (designated Bp1) present in GH4C1 cells that binds very strongly to rGH silencer-1 but only weakly to hCRBP1 Fp1. Similarly, we also detected a 29-kD nuclear factor (designated Bp2) that recognizes exclusively the Fp1 element as its target site, therefore suggesting that different, but likely related, proteins bind these homologous elements to either activate or repress gene transcription. Although they bind DNA through the recognition of the NF1-like target sequence contained on these elements, competition and supershift experiments in electrophoretic mobility shift assays provided evidence that neither of these proteins belong to the NF1 family.

Animals↗
PubMed1997

Morphometrics of normal and hypoplastic lungs in preterm lambs with gas and partial liquid ventilation.

To test the hypothesis that perfluorochemical (PFC) instillation may reduce the pulmonary trauma commonly associated with conventional gas ventilation, we studied 12 lambs with normal lungs and 10 with pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH). We used mechanical ventilation for up to 3.5 h, with and without tracheal instillation of LiquiVent PFC liquid. At the end of experimentation lungs were fixed for morphometrical analysis of their components and pulmonary trauma was evaluated by measurement of the perivascular compression index (PCI = % perivascular emphysema/% vessels). In normal lungs good gas exchange and respiratory mechanics were obtained with all modes of ventilation, with no statistical difference in the index of pulmonary trauma with or without instillation of LiquiVent (P >0.05). In the hypoplastic lungs, tracheal instillation of PFC liquid after 30 min of conventional gas ventilation significantly improved PaCO2 (from 107+/-8 to 55+/-6 mmHg, P <0.05), pH (from 7.00+/-0.03 to 7.29+/-0. 04, P <0.05), compliance (from 0.08+/-0.01 to 0.25+/-0.03 ml/cmH2O . kg, P <0.05), and ventilatory index (from 1,445+/-148 to 794+/-139, P <0.05). Survival was 6/6 animals with PFC ventilation compared to 1/4 with conventional gas ventilation with no more pulmonary trauma (mean PCI 12.6+/-1.8 vs. 11.4+/-4.0%, P >0.05) for a longer mean ventilatory period in the PFC group. We conclude that the PFC liquid technique of ventilation can improve respiratory physiology when conventional gas ventilation alone is proving inefficient. There was no significant difference in pulmonary trauma at morphometrics between gas and partial liquid ventilation.

Animals↗
PubMed1996

Multiple nuclear regulatory proteins bind a single cis-acting promoter element to control basal transcription of the human alpha 4 integrin gene in corneal epithelial cells.

Expression of the fibronectin-binding integrin alpha 4 beta 1 has been postulated to be an important event in the process of corneal epithelial wound healing. In a previous study, we identified upstream positive and negative cis-acting regulatory elements that are needed to modulate the transcriptional activity of the human alpha 4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells. We have shown that most of the basal activity directed by this promoter was dependent on the presence of a cis-acting DNA sequence designated the alpha 4.1 element, centered at position -45 relative to the human alpha 4 mRNA start site. Here, we demonstrate that five distinct nuclear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the alpha 4.1 element in a specific manner in vitro. However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence in in vitro assays. The apparent molecular masses of the Bp1 to Bp5 proteins were determined and found to be of 91, 74, 59, 45, and 39 kD, respectively. Electrophoretic mobility-shift assays (EMSAs) indicated that only Bp2 also possesses the ability to bind the alpha 4.2 element, a site homologous to alpha 4.1 which plays a minor role in alpha 4 gene expression. Despite the presence of three Ets binding sites in the immediate vicinity of alpha 4.1, competition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp5 do not belong to the Ets family of transcription factors. Insertion of both alpha 4.1 and alpha 4.2 upstream from the basal promoter of the mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter. alpha 4.1 was shown to function as an activator, whereas alpha 4.2 acted as a repressor in a manner that is dependent on its orientation, further stressing the critical regulatory function played by these two elements on alpha 4 gene basal expression.

Animals↗
PubMed1996

Optimization of competitor poly(dI-dC).poly(dI-dC) levels is advised in DNA-protein interaction studies involving enriched nuclear proteins.

Procedures used for investigating DNA-protein interactions, such as the electrophoretic mobility shift assay (EMSA) or DNasel footprinting, require that exogenous nucleic acids (or synthetic equivalents) be added to the reaction mixture to prevent or reduce the nonspecific interaction of nuclear proteins with the labeled probe of choice, especially when proteins are obtained from crude nuclear extracts. One of the most potent, and likely the most widely used, non-specific competitor is the synthetic polymer poly(dI-dC).poly(dI-dC). Its addition to the reaction mixture prior to crude nuclear proteins has unquestionably proven very efficient in reducing nonspecific interactions by facilitating detection of the complexes of interest. However, in certain instances, the use of crude extracts alone does not provide adequate answers and the need to further enrich such extracts becomes absolutely necessary. In this study, we provide evidence that amounts of poly(dI-dC).poly(dI-dC) well below those currently described in the literature substantially impair, or even totally prevent, the detection of specific DNA-protein complexes in EMSA when enriched, gel-fractionated or commercially purified nuclear proteins are used, therefore indicating the need to precisely optimize the amount of such a competitor in DNA-protein interaction studies.

Animals↗
PubMed1995

Imperforate anus, malrotation and Hirschsprung's disease: a rare association.

The association of imperforate anus with anomalies of rectal innervation is very rare. Moreover, malrotation only rarely accompanies these malformations. We report 2 patients with this unusual triple association and its impact on their management. Retrospectively both patients presented at birth with the same radiological picture of distended distal colon and right-sided small bowel location. We discuss the importance of a systematic search for associated intestinal malformations in cases of imperforate anus in order to avoid serious complications and to choose the optimal operative strategy.

Anastomosis, Surgical↗