Biomedical subjects

Bernard Dastugue

Publications and source records attributed to Bernard Dastugue.

At least 19 recordsLinked to original sources

PubMed2006

Molecular and metabolic retinoid pathways in human amniotic membranes.

Vitamin A (retinol) and its active derivatives (the retinoids) are essential for the growth and development of the mammalian fetus and placenta. The amniotic membranes are extra-embryonic structures that are indispensable for normal gestation in mammals. Although placental involvement of retinoids is clearly established, little is known about the roles of retinoids for the associated amniotic membranes. The aim of this study was to define the metabolic and molecular pathways of retinoic signaling in human fetal membranes. The expression of retinoid receptors (RARalpha, beta and RXRalpha, beta) was established at transcript and protein levels. Enzymes involved in retinoic acid generation were also detected. The enzymatic generation of functional retinoids was confirmed using specific inhibitors of retinol metabolism. Finally, the functionality of retinoid pathways was demonstrated by inducing established retinoid target gene expression. Our results clearly demonstrated that the molecular and metabolic actors of retinoic signaling pathways are functional in human fetal membranes.

Amnion↗

PubMed2006

Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.

PURPOSE: Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study. METHODS: Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus. RESULTS: A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter. CONCLUSIONS: Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.

Adolescent↗

PubMed2006

Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients.

BACKGROUND: Splanchnic ischemia plays a major role in the development of organ failure during septic shock. Plasma D-lactate has been proposed as a better marker of splanchnic hypoperfusion than L-lactate. We studied the prognostic ability of plasma D- and L-lactate levels. METHODS: A prospective study was performed in an intensive care unit and included patients with septic shock. Two samples for plasma D- and L-lactate determination were collected: the first within 6 h after the patient met the criteria for septic shock (day 1) and the second 24 h later (day 2). RESULTS: In univariate analysis, day 1 plasma D- and L-lactate values were associated with 28-day mortality. For plasma D- and L- lactate, the area under the receiver operating characteristic curve was 0.68+/-0.09 and 0.84+/-0.07 on day 1 (p=0.09), and 0.74+/-0.10 and 0.90+/-0.07 on day 2 (p=0.06), respectively. In survivors, D-lactate levels decreased between day 1 and day 2 (p=0.03), but L-lactate did not (p=0.29). In septic shock patients, plasma D- and L-lactate levels reliably discriminate between survivors and non-survivors. The prognostic ability of plasma L-lactate was better than that of plasma D-lactate. CONCLUSION: A rapid decrease in plasma D-lactate during the course of septic shock could indicate reduced 28-day mortality.

Aged↗

PubMed2006

Drosophila melanogaster p-glycoprotein: a membrane detoxification system toward polycyclic aromatic hydrocarbon pollutants.

Polycyclic aromatic hydrocarbons (PAHs) are well-known ubiquitous environmental contaminants. Permeability glycoprotein (P-gp) is a transmembrane detoxification efflux pump transporting various lipophilic xenobiotics, such as PAHs, out of the cells. The existence of a P-gp detoxification system inducible by PAHs was investigated in Drosophila melanogaster. Western blot experiments showed that D. melanogaster expressed a 140-kDa P-gp in S12 cells, embryos, and adult flies. Permeability glycoprotein was expressed in adult flies in the head, abdomen, and thorax and sublocalized in the sexual and olfactory organs. Flow cytometry experiments using Drosophila S12 cells in the presence of PAHs and target P-gp drug compounds revealed that Drosophila P-gp acted as an efflux detoxification pump. In Drosophila exposed to benzo[a]pyrene or to ambient air polluted by higher or lower PAH concentrations, P-gp expression was clearly showed a dose-dependent increase response. The P-gp induction was detected both in adult flies and in different fly parts, such as the head, thorax, and antennae. Drosophila P-gp acts as a membrane barrier against PAH pollutants.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Congenital diaphragmatic hernia: a retinoid-signaling pathway disruption during lung development?

Congenital diaphragmatic hernia (CDH) usually occurs sporadically. The prognosis remains poor, with a 50% perinatal mortality rate. Most deaths result from hypoxemia due to lung hypoplasia and abnormal development of pulmonary vasculature that results in persistent pulmonary hypertension. Our current understanding of the pathogenesis of CDH is based on an assumption linking herniation of abdominal viscera into the thorax with compression of the developing lung. Pulmonary hypoplasia, however, can also result from reduced distension of the developing lung secondary to impaired fetal breathing movements. Moreover, a nitrofen-induced CDH model shows that lung hypoplasia precedes the diaphragmatic defect, leading to a "dual-hit hypothesis." Recent data reveal the role of a retinoid-signaling pathway disruption in the pathogenesis of CDH. We describe the clinical and epidemiological aspects of human CDH, the metabolic and molecular aspects of the retinoid-signaling pathway, and the implications of retinoids in the development of the diaphragm and the lung. Finally, we highlight the existing links between CDH and disruption of the retinoid-signaling pathway, which may suggest an eventual use of retinoids in the treatment of CDH.

Animals↗

PubMed2005

DNA microarray analysis of gene expression in eutopic endometrium from patients with deep endometriosis using laser capture microdissection.

OBJECTIVE: To investigate differentially expressed genes in epithelial and stromal cells of eutopic endometrium from patients with deep endometriosis and women with normal pelvic cavities using laser capture microdissection and complementary DNA microarrays. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with deep endometriosis and fertile women who underwent laparoscopic tubal ligation or reversal of tubal sterilization. INTERVENTION(S): Endometrial tissue biopsies during the late proliferative phase and early, mid-, and late secretory phases. MAIN OUTCOME MEASURE(S): Genes that were regulated with a change greater than threefold were selected as differentially expressed genes. Validation was performed with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULT(S): Microarray analysis identified up-regulation during the late secretory phase (patients with endometriosis vs. controls) of several genes in two important signaling pathways: RAS/RAF/MAPK and PI3K. This included the genes RON, SOS, 14-3-3 protein eta, and uPAR in epithelial cells and KSR and PI3K p85 regulatory subunit alpha in stromal cells; real-time RT-PCR analysis validated up-regulation of all six genes. CONCLUSION(S): The RAS/RAF/MAPK and PI3K pathways may be involved in initial development of endometriosis.

Endometriosis↗

PubMed2005

The Drosophila Toucan protein is a new mitotic microtubule-associated protein required for spindle microtubule stability.

Mitotic spindle dynamics are highly dependent on proteins that interact with microtubules to influence their organization or stability. Here, we show that the Drosophila Toucan protein interacts directly with microtubules. Its localization to the microtubule network when it is expressed in mammalian cells and its direct interaction with microtubules in vitro are dependent on its central basic domain. Moreover, Toc expression in mammalian cells strongly protects microtubules from depolymerization. By using in vivo inducible RNAi in syncytial embryos, we generated a dose-sensitive loss of function of toucan, demonstrating that this technique is an efficient method for inactivating a maternal transcript. This enabled us to accurately characterize several new mitotic defects from the early to the late phases of mitosis, depending on Toucan depletion level. Toucan is required for metaphase spindle formation and centrosome anchoring to the poles. Then, during anaphase, Toc depletion affects kinetochore microtubules and therefore chromosome segregation. Toc is also necessary for central spindle formation by the interpolar microtubules. In contrast, astral microtubules are not disturbed by Toc depletion. Taken together, our results show that Toucan is a microtubule-associated protein specifically required for the stability of spindle microtubules throughout mitosis.

Anaphase↗

PubMed2004

Cell and tissue specific expression of human Krüppel-like transcription factors in human ocular surface.

PURPOSE: The ocular surface, composed of the conjunctiva and the cornea, is essential for vision. Its integrity depends on numerous molecular and cellular processes such as proliferation, differentiation, apoptosis, adhesion, and extracellular matrix homeostasis, whose deregulation can induce ophthalmological pathologies. The Krüppel-like transcription factors (KLFs) family is made up of 15 C2H2 zinc-finger proteins involved in vertebrate development and able to control cell proliferation, growth, and differentiation. In order to better define their respective roles in the human ocular surface, we decided to determine their pattern of expression in ocular tissues. We then focused on the expression of KLF4 and some of its target genes to establish KLF4's biological activities in human ocular surface. METHODS: Firstly, total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells (primary culture and established cell line), corneal keratocytes (primary culture), corneal endothelial cells (established cell line), and conjunctival epithelial cells (established cell line) and submitted to RT-PCR experiments in order to determine the expression patterns of the different KLFs. Secondly, KLF4 protein localization was visualized by immunofluorescence assays at tissue and cell levels. Finally, KLF4 target genes (endoglin, ornithine decarboxylase) mRNA expression levels were determined by semi-quantitative RT-PCR, after KLF4 transient transfection in human corneal epithelium (HCE) cells. RESULTS: We detected the presence of twelve transcripts of KLFs in the cornea (KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF10, KLF11, KLF12, KLF13, and KLF16) and eight in the conjunctiva (KLF2, KLF3, KLF4, KLF6, KLF7, KLF10, KLF11, and KLF12). Under our conditions, the transcripts encoding KLF1 and KLF9 were never detected. Specific expression patterns of each KLF were also determined for the major cellular components of the human cornea and conjunctiva. KLF4 immunolocalization assays indicated its presence in both the cytoplasmic and nuclear compartments of conjunctival and corneal cells. KLF4 transient overexpression in HCE cells down regulated both endoglin and ODC mRNA levels. CONCLUSIONS: For the first time, we established the presence of a KLF network in the human ocular surface and illustrated the conservation of KLF4's biological properties in a corneal derived epithelial cell line.

Antigens, CD↗

PubMed2004

Coordinated development of muscles and tendons of the Drosophila leg.

Since Miller's morphological description, the Drosophila leg musculature and its formation has not been revisited. Here, using a set of GFP markers and confocal microscopy, we analyse Drosophila leg muscle development, and describe all the muscles and tendons present in the adult leg. Importantly, we provide for the first time evidence for tendons located internally within leg segments. By visualising muscle and tendon precursors, we demonstrate that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg.

Animals↗

PubMed2004

Intercellular communication between germ line and somatic line is utilized to control the transcription of ZAM, an endogenous retrovirus from Drosophila melanogaster.

ZAM is an long terminal repeat (LTR) retrotransposon from Drosophila melanogaster that bears striking resemblance to the vertebrate retroviruses, in their structure and replication cycle. This element transposes via an RNA intermediate and its reverse transcription, and ultimately inserts copies within the germ line. In this paper, we show that intercellular communication established between the germ line cells and the somatic follicle cells is used to initiate the replication cycle of ZAM. ZAM has been shown to be transcribed in the follicle cells located at the posterior pole of the oocyte. Here, we determine the cis-regulatory elements necessary for its somatic expression, and show that they respond to the EGF-receptor signaling pathway and its activation by the ligand Gurken emitted by the germ line. We further show that the ETS-transcription factor Pointed2 acting downstream of this pathway acts as a trans-regulatory factor and targets a specific cis-regulatory binding site located within the ZAM LTR. Our data give an insight into the molecular mechanism for how intercellular communications between germ cells and somatic cells may be used by endogenous retroviruses to control their replication, and thereby specify their intrinsic and highly restricted expression in the reproductive apparatus.

Animals↗

PubMed2004

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells.

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.

Animals↗

PubMed2004

Placental expression of SCO-spondin during mouse and human development.

During mammalian development, the placenta is a transitory but indispensable structure for a harmonious gestation involving several biological processes, such as adhesion, differentiation, apoptosis or cellular guidance. Nevertheless, the molecular pathways implicated during the placentation are still not totally understood. We previously described, the subcommissural organ (SCO)-spondin, a member of the 'thrombospondin' super-family, which is strongly expressed during mammalian central nervous system development. This extra-cellular matrix glycoprotein shows a unique arrangement of several conserved domains, including thrombospondin type 1 repeats, low-density lipoprotein receptor type A domains, two epidermal growth factor-like domains, and N- and C-terminal von Willebrand factor cysteine-rich domains. The presence of these domains strongly suggests the SCO-spondin involvement in cellular events occurring during placental development and physiology. In order to define this new role of SCO-spondin during development, we demonstrated its expression at relevant steps of gestation in human and mouse placenta, using RT-PCR, immunohistochemistry and Western-blot experiments. These data initiate further insights into the molecular and genetic functions of the neuronal gene SCO-spondin during trophoblastic and more globally during placental physiology and development.

Animals↗

PubMed2003

Patterning of the cardiac outflow region in Drosophila.

Specification of bilateral cardiac primordia and formation of the linear heart tube are highly conserved from Drosophila to humans. However, subsequent heart morphogenesis involving nonmesodermal neural crest cells was thought to be specific for vertebrates. Here, we provide evidence that a group of nonmesodermal cells that we have named heart-anchoring cells (HANCs) contribute to heart morphogenesis in Drosophila. We show that the homeobox genes ladybird (lb) known to be involved in diversification of cardiac precursors are expressed in HANCs and required for their specification. Interestingly, the HANCs selectively contact the anterior cardiac cells, which express lb as well. Direct interaction between HANCs and cardiac cells is assisted by a pair of cardiac outflow muscles (COMs), each of which selectively attaches to both the lb-expressing cardiac cells and HANCs. COM muscles seem to ensure ventral bending of the heart tip and together with HANCs determine the spatial positioning of the cardiac outflow region. Experimentally depleted cardiac lb expression leads to the disruption of the contact between the tip of the heart and either the COM muscles or the HANC cells, indicating a pivotal morphogenetic role for the lb expression within the heart.

Animals↗

PubMed2003

Detection of trisomy 21 by quantitative fluorescent-polymerase chain reaction in uncultured amniocytes.

Prenatal diagnosis of fetal trisomy 21 is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. Today, trisomy 21 can be rapidly diagnosed within 24 h by molecular analysis of uncultured fetal cells using the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. The aim of our study was to test a chromosome quantification method on the basis of the analysis of fluorescent PCR products derived from non-polymorphic target genes. Co-amplification of a portion of DSCR1 (Down syndrome Critical Region 1) and the reference gene, CFTR (cystic fibrosis transmembrane regulator) enabled molecular detection of trisomy 21. Our method was successfully tested on a total of 154 amniotic fluids in a blind prospective study. Calculation of the DSCR1/CFTR ratio allowed us to distinguish between 152 normal amniotic fluids (mean ratio 0.99) and 2 amniotic fluids presenting a trisomy 21 status (DSCR1/CFTR ratio of 1.53 and 1.61, respectively). The results obtained by conventional cytogenetic analysis and our quantitative PCR method were concordant in every case. Our gene-based fluorescent PCR approach represents an alternative molecular method for rapid and reliable detection of trisomy 21, which can be helpful in the prenatal diagnosis of women at high risk of fetal trisomy 21.

Adult↗

PubMed2003

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach.

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.

Biomarkers↗

PubMed2003

Trophoblastic remodeling in normal and preeclamptic pregnancies: implication of cytokines.

OBJECTIVE: To summarize the recent knowledge on the implications of placenta and cytokines in normal and preeclamptic pregnancies. DATA SOURCES: A literature search was conducted of applicable articles related to interactions between trophoblast and cytokines in generating preeclampsia. CONCLUSIONS: The initiating event in preeclampsia has been postulated to be the reduced uteroplacental perfusion as a result of abnormal extravillous cytotrophoblast invasion and remodeling of the uterine spiral arteries. Focal ischemia and hypoxia, deportation of hypoxemic trophoblast cells and abnormal expression of various placental biologic molecules, particularly the cytokines, are thought to lead to widespread dysfunction of the maternal vascular endothelium resulting in overproduction of endothelin and thromboxane, enhanced vascular sensitivity to angiotensin II, and reduced secretion of vasodilators such as nitric oxide and prostacyclin. These alterations, in turn, cause hypertension, proteinuria and edema, and pathologies in many organ systems (kidney, lung, liver, brain).

Cytokines↗

PubMed2003

The thrombospondin type 1 repeat (TSR) and neuronal differentiation: roles of SCO-spondin oligopeptides on neuronal cell types and cell lines.

SCO-spondin is a large glycoprotein secreted by ependymal cells of the subcommissural organ. It shares functional domains called thrombospondin type 1 repeats (TSRs) with a number of developmental proteins expressed in the central nervous system, and involved in axonal pathfinding. Also, SCO-spondin is highly conserved in the chordate phylum and its multiple domain organization is probably a chordate innovation. The putative involvement of SCO-spondin in neuron/glia interaction in the course of development is assessed in various cell culture systems. SCO-spondin interferes with several developmental processes, including neuronal survival, neurite extension, neuronal aggregation, and fasciculation. The TSR motifs, and especially the WSGWSSCSVSCG sequence, are most important in these neuronal responses. Integrins and growth factor receptors may cooperate as integrative signals. We discuss the putative involvement of the subcommissural organ/Reissner's fiber complex in developmental events, as a particular extracellular signaling system.

Amino Acid Sequence↗

PubMed2003

Modifiers of muscle and heart cell fate specification identified by gain-of-function screen in Drosophila.

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors and are both implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, we have performed a gain-of-function (GOF) screen for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Amongst the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of our screen. Observed GOF phenotypes have led us to identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Amongst them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Our analysis of rac2 phenotypes clearly demonstrates that the altered mesodermal level of Rho-GTPase Rac2 can influence specification of a number of cardiac and muscular cell types including those expressing ladybird. Finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicate a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinases (RTKs) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types.

Animals↗