Biomedical subjects

B Dastugue

Publications and source records attributed to B Dastugue.

At least 19 recordsLinked to original sources

PubMed2005

Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster.

ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 degrees C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.

Animals↗

PubMed2004

DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection.

Endometriosis, a common gynecological disorder that causes infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. However, despite extensive studies its etiology and pathogenesis are not completely understood. Differentially expressed genes were investigated in epithelial and stromal cells from deep endometriosis and matched eutopic endometrium using cDNA microarrays and laser capture microdissection. Validation of results of several up- and down-regulated genes was performed by quantitative real-time RT-PCR. Our data showed that platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta1 (PKC beta1) and janus kinase 1 (JAK1) were upregulated, and Sprouty2 and mitogen-activated protein kinase kinase 7 (MKK7) were downregulated in endometriosis stromal cells, suggesting the involvement of the RAS/RAF/MAPK signaling pathway through PDGFRA in endometriosis pathophysiology. In addition, two potential negative regulators of aromatase expression, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3), were downregulated in endometriosis epithelial cells, which might result in increased local production of estrogen in endometriosis epithelial cells. Furthermore, three potential candidate genes that might be involved in endometriosis related pain were identified: tyrosine kinase receptor B (TRkB) in endometriosis epithelial cells, and serotonin transporter (5HTT) and mu opioid receptor (MOR) in endometriosis stromal cells were all upregulated. One of the candidate genes, MOR, may be involved in a defective immune system in endometriosis. This study has provided new insights into endometriosis pathophysiology.

Endometriosis↗

PubMed2004

[Early recurrence of Groenouw type I corneal dystrophy after phototherapeutic keratectomy. Molecular biology study suggests epithelial genesis].

PURPOSE: Granular corneal dystrophy Groenouw type 1 (GGI) is a rare autosomal dominant disease caused by allelic mutations of the BIGH3 gene. The specific phenotype is characterized by granular opacities (white, sharply demarcated spots resembling bread crumbs) in corneal stroma, which cause recurrent corneal erosions and blurred vision. Phototherapeutic keratectomy (PTK) is an effective procedure that improves visual acuity, but recurrences are unavoidable. Though GGI deposits are well described, their origin is not completely known. The production of mutated keratoepithelin protein (a product of the BIGH3 gene) is the first step necessary for deposits to appear. Molecular biology experiments were conducted to determine the role of corneal cell types in the genesis of early recurrent deposits of post-PTK GGI. METHODS: Tissue specimens from a patient undergoing penetrating keratoplasty for recurrence of GGI (12 months after PTK) and five normal corneas were examined by hybridization in situ and immunohistology to study the expression of BIGH3 and location of keratoepithelin. RESULTS: Only one healthy cornea expressed BIGH3 mainly in the epithelium and less in keratinocytes and endothelial cells. In the GGI corneas, BIGH3 was highly expressed in the modified, hyperplastic epithelium. The keratoepithelin was accumulated under the epithelium where deposits were formed. CONCLUSION: This observation confirms that corneal epithelium is the main producer of mutated keratoepithelin on the cellular scale and thus constitutes the principal source of dystrophic deposit formation during recurrence.

Adult↗

PubMed2003

Alpha1beta1-integrin is an essential signal for neurite outgrowth induced by thrombospondin type 1 repeats of SCO-spondin.

In the central and peripheral nervous systems a heterogeneous group of proteins constituting the thrombospondin superfamily provides a cue for axonal pathfinding. They either contain or are devoid of the tripeptide RGD, and the sequence(s) and mechanism(s) which trigger in vitro their neurite-promoting activity have remained unclear. In this study, we reconsider the problem of whether sequences present in the thrombospondin type 1 repeats (TSRs), and independent of the well-known RGD-binding site, may activate integrins and account for their neurite-promoting activity. SCO-spondin is a newly identified member of the thrombospondin superfamily, which shows a multidomain organization with a great number of TSR motifs but no RGD sequence. Previous research has implicated oligopeptides derived from SCO-spondin TSRs in in-vitro development of various neuronal cell types. In this study, we investigate whether function-blocking antibodies directed against integrin subunits can block these effects in cell line B104, cloned from a neuroblastoma of the rat central nervous system. By two different approaches: flow cytometry revealing short-term effects and cell cultures revealing long-term effects, we show that: (a). activation of cell metabolism, (b). changes in cell size and structure, and (c). neurite-promoting activity induced by TSR oligopeptides are inhibited by function-blocking antibodies to beta1-subunit. Using a panel of function-blocking antibodies directed against various integrin alpha-subunits we show that the alpha1-subunit might be the partner of the beta1-subunit in B104 cells. Thus, we demonstrate that an original sequence within a TSR motif from SCO-spondin promotes neurite outgrowth through an intracellular signal driven by integrins, independently of an RGD-binding site.

Amino Acid Sequence↗

PubMed2002

Expression of the Idefix retrotransposon in early follicle cells in the germarium of Drosophila melanogaster is determined by its LTR sequences and a specific genomic context.

Retrotransposons are transcriptionally activated in different tissues and cell types by a variety of genomic and environmental factors. Transcription of LTR retrotransposons is controlled by cis-acting regulatory sequences in the 5' LTR. Mobilization of two LTR retroelements, Idefix and ZAM, occurs in the unstable RevI line of Drosophila melanogaster, in which their copy numbers are high, while they are low in all other stocks tested. Here we show that both a full-length and a subgenomic Idefix transcript that are necessary for its mobilization are present in the Rev1 line, but not in the other lines. Studies on transgenic strains demonstrate that the 5' LTR of Idefix contains sequences that direct the tissue-specific expression of the retroelement in testes and ovaries of adult flies. In ovaries, expression occurs in the early follicle and in other somatic cells of the germarium, and is strictly associated with the unstable genetic context conferred by the RevI line. Control of tissue-specific Idefix expression by interactions between cis-acting sequences of its LTR and trans-acting genomic factors provides an opportunity to use this retroelement as a tool for the study of the early follicle cell lineage in the germarium.

Alternative Splicing↗

PubMed2001

The BTB/POZ domain of the regulatory proteins Bric à brac 1 (BAB1) and Bric à brac 2 (BAB2) interacts with the novel Drosophila TAF(II) factor BIP2/dTAF(II)155.

The BTB/POZ domain is an evolutionarily conserved protein-protein interaction domain present in the N-terminal region of numerous transcription factors involved in development, chromatin remodeling, and human cancers. This domain is involved in homomeric and heteromeric associations with other BTB/POZ domains. The Drosophila BTB/POZ proteins Bric à brac 1 (BAB1) and Bric à brac 2 (BAB2) are developmentally regulated transcription factors which are involved in pattern formation along the proximo-distal axis of the leg and antenna, in the morphogenesis of the adult ovaries, and in the control of sexually dimorphic characters. We have identified partners of the BAB1 protein by using the two-hybrid system. The characterization of one of these proteins, called BIP2 for BAB Interacting Protein 2, is presented. BIP2 is a novel Drosophila TATA-box Protein Associated Factor (TAF(II)), also named dTAF(II)155. We show that the BTB/POZ domains of BAB1 and BAB2 are sufficient to mediate a direct interaction with BIP2/dTAF(II)155. This provides a direct link between these BTB/POZ transcription factors and the basal transcriptional machinery. We discuss the implications of the interaction between a BTB/POZ domain and a TAF(II) for the molecular mechanisms of transcriptional control mediated by BTB/POZ transcription factors.

Animals↗

PubMed2001

Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al., 1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at the spindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results strongly suggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

Animals↗

PubMed2001

Effects of SCO-spondin thrombospondin type 1 repeats (TSR) in comparison to Reissner's fiber material on the differentiation of the B104 neuroblastoma cell line.

SCO-spondin is a newly identified protein that is strongly expressed in the subcommissural organ (SCO), an ependymal differentiation of the brain. When released into the cerebrospinal fluid at the entrance to the Sylvian aqueduct, the glycoproteins condense and form a thread-like structure, Reissner's fiber (RF). To analyze the role of SCO-spondin on neuronal development, we studied the effects induced by an oligopeptide derived from a thrombospondin type 1 repeat (TSR) of SCO-spondin on neuroblastoma B104 cells and compared them with the effects of soluble RF material containing complete SCO-spondin proteins. In low density cell culture, the TSR peptide first induced a notable flattening of cells accompanied by increased neurite outgrowth. Grouping of these differentiated B104 cells, which later formed dense aggregates, was then observed with increasing time in culture. Soluble RF material induced similar morphological changes and neurite-promoting effects on B104 cells, although the cells remained evenly distributed throughout the culture time and no aggregates were visible. In high-density cell culture, both TSR peptide and RF material induced prominent neurite outgrowth and subsequent rapid cell aggregation. Whereas soluble RF material inhibited cell proliferation, no respective effect was observed in the presence of the TSR peptide. A direct interaction of TSR peptide and soluble RF material with a B104 cell binding site was revealed by increased B104 cell metabolic activity by flow cytometry.

Amino Acid Motifs↗

PubMed2001

Co-localization of KLF6 and KLF4 with pregnancy-specific glycoproteins during human placenta development.

Pregnancy-specific glycoproteins (PSGs) are major placental proteins essential for the maintenance of normal gestation. However, little is known about their gene expression regulation during placentation. It was previously demonstrated that the human core promoter binding protein recently renamed Krüppel-like factor (KLF) 6 binds to a highly conserved sequence within the PSG promoters and is mainly expressed in human term placenta. Here, we determined the expression pattern of the 13 other KLFs during human placental development. We demonstrate that eight KLFs exhibit specific expression patterns in human placental tissues and membranes, in favor of a functional cooperation of specific KLFs during placentation. In addition, we demonstrate that KLF6, KLF4 and PSG proteins are co-expressed in same cell types of placental villi and membranes. This experimental evidence further strengthens the potential cross talk of both transcription factors for PSG gene regulation in vivo.

DNA-Binding Proteins↗

PubMed2001

Use of transgenic mice model for understanding the placentation: towards clinical applications in human obstetrical pathologies?

The mammalian embryo and fetus are unable to develop without a well-established, functional placenta. This transitory yet indispensable structure attaches the conceptus to the uterus and establishes the vascular connections necessary for nutrient and gaseous exchange between maternal and fetal compartments. Genetic targeting strategy allows the generation of mice lacking a specific gene. Such approaches reveal: (i) the high incidence of mutant embryonic or fetal death in utero, and (ii) the extraembryonic (placental) causes of these deaths. Due to the similarities presented between mouse and human placenta, we propose to use the potential of mouse targeting experiments as a model in order to understand human obstetrical pathologies. In this paper, we first review genes that have been demonstrated to be required in mice for implantation, choriovitelline and chorioallantoic placentation. Using examples (integrins, homeoboxs, hepatocyte growth factor or epidermal growth factor receptor...) we demonstrate the reality and efficiency of such an approach. Other candidate genes (receptor of leukemia inhibitory factor, Wnt2 or retinoic acid receptor alpha...) in order to understand, prevent and treat human obstetrical pathologies.

Animals↗

PubMed2000

SCO-spondin and RF-GlyI: two designations for the same glycoprotein secreted by the subcommissural organ.

SCO-spondin and RF-GlyI are two designations for cDNAs strongly expressed in the bovine subcommissural organ (SCO), characterized, respectively, in 1996 and 1998 by two different research groups. Because both cDNAs were partial sequences and exhibited close similarities in their nucleotide and deduced amino acid sequences, it was thought that they might be part of the same encoding sequence. To find out, we performed 3'RACE using a SCO-spondin-specific upstream primer. From the RT-PCR product generated and by nested PCR techniques, we amplified both SCO-spondin and RF-GlyI specific products with the expected length. Also, probes generated from both PCR products hybridized to the same major 14 kb transcript in Northern blot analyses, clearly showing that SCO-spondin and RF-GlyI cDNAs do belong to the same encoding sequence. In addition, we amplified, cloned, and sequenced a PCR product of 3 kb spanning both the known SCO-spondin and RF-GlyI sequences. The deduced amino acid sequence contains nine thrombospondin type 1 repeats that alternate with sequences sharing similarities with the D-domain of von Willebrand factor. Taken together, these findings show that SCO-spondin and RF-GlyI are two designations of the same gene encoding proteins secreted by the bovine SCO and forming Reissner's fiber. In addition, compared to the sequence provided by Nualart et al. (1998), we extended the reading frame and identified new conserved domains in the 3' end of SCO-spondin. The putative function of SCO-spondin on axonal pathfinding is discussed regarding the presence of a great number of thrombospondin type 1 repeats.

Amino Acid Motifs↗

PubMed2000

Subcommissural organ/Reissner's fiber complex: characterization of SCO-spondin, a glycoprotein with potent activity on neurite outgrowth.

In the developing vertebrate nervous system, several proteins of the thrombospondin superfamily act on axonal pathfinding. By successive screening of a SCO-cDNA library, we have characterized a new member of this superfamily, which we call SCO-spondin. This extracellular matrix glycoprotein of 4,560 amino acids is expressed and secreted early in development by the subcommissural organ (SCO), an ependymal differentiation located in the roof of the Sylvian aqueduct. Furthermore, SCO-spondin makes part of Reissner's fiber (RF), a thread-like structure present in the central canal of the spinal cord. This novel protein shows a unique arrangement of several conserved domains, including 26 thrombospondin type 1 repeats (TSR), nine low-density lipoprotein receptor (LDLr) type A domains, two epidermal growth factor (EGF)-like domains, and N- and C-terminal von Willebrand factor (vWF) cysteine-rich domains, all of which are potent sites of protein-protein interaction. Regarding the huge number of TSR, the putative function of SCO-spondin on axonal guidance is discussed in comparison with other developmental molecules of the CNS exhibiting TSR. To correlate SCO-spondin molecular feature and function, we tested the effect of oligopeptides, whose sequences include highly conserved amino acids of the consensus domains on a neuroblastoma cell line B 104. One of these peptides (WSGWSSCSRSCG) markedly increased neurite outgrowth of B 104 cells and this effect was dose dependent. Thus, SCO-spondin is a favorable substrate for neurite outgrowth and may participate in the posterior commissure formation and spinal cord differentiation during ontogenesis of the central nervous system.

Age Factors↗

PubMed2000

Differential expression of retinoic acid-inducible (Stra) genes during mouse placentation.

Several retinoid binding proteins and nuclear receptors are specifically expressed in murine placenta. However, little is known about molecular events and target genes regulated by retinoids during placentation. Here, we report that several retinoic acid-inducible (Stra) genes, originally isolated by a differential screening procedure, exhibit specific expression patterns in mouse placental tissues. Three Stra genes, including the ephrinB1 receptor tyrosine kinase ligand, are prominently expressed in the regions of exchanges between maternal and embryonic circulations, i.e. the yolk sac and/or the labyrinthine zone of the mature placenta. The Meis2 homeobox gene appears to be specifically expressed in maternally-derived cell populations. Three other Stra genes, including the AP-2-related gene AP-2gamma, are differentially expressed in the trophoblastic cell lineage. Thus, retinoids may regulate various signaling pathways in specific placental cell-types.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Expression and cellular localization of the Toucan protein during Drosophila oogenesis.

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.

Animals↗

PubMed2000

Impact of multiple insertions of two retroelements, ZAM and Idefix at an euchromatic locus.

Transposable elements represent a large fraction of eukaryotic genomes and they are thought to play an important role in chromatin structure. ZAM and Idefix are two LTR-retrotransposons from Drosophila melanogaster very similar in structure to vertebrate retroviruses. In all the strains where their distribution has been studied, ZAM appears to be present exclusively in the intercalary heterochromatin while Idefix copies are mainly found in the centromeric heterochromatin with very few copies in euchromatin. Their distribution varies in a specific strain called RevI in which the mobilization of ZAM and Idefix is highly induced. In this strain, 15 copies of ZAM and 30 copies of Idefix are found on the chromosomal arms in addition to their usual distribution. Amongst the loci where new copies are detected, a hotspot for their insertion has been detected at the white locus where up to four elements occurred within a 3-kb fragment at the 5' end of this gene. This property of ZAM and Idefix to accumulate at a defined site provides an interesting paradigm to bring insight into the effect exerted by multiple insertions of transposable elements at an euchromatic locus.

Animals↗

PubMed2000

Genotype-phenotype correlation in inherited brain myelination defects due to proteolipid protein gene mutations. Clinical European Network on Brain Dysmyelinating Disease.

Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2 (SPG2) are X-linked developmental defects of myelin formation affecting the central nervous system (CNS). They differ clinically in the onset and severity of the motor disability but both are allelic to the proteolipid protein gene (PLP), which encodes the principal protein components of CNS myelin, PLP and its spliced isoform, DM20. We investigated 52 PMD and 28 SPG families without large PLP duplications or deletions by genomic PCR amplification and sequencing of the PLP gene. We identified 29 and 4 abnormalities respectively. Patients with PLP mutations presented a large range of disease severity, with a continuum between severe forms of PMD, without motor development, to pure forms of SPG. Clinical severity was found to be correlated with the nature of the mutation, suggesting a distinct strategy for detection of PLP point mutations between severe PMD, mild PMD and SPG. Single amino-acid changes in highly conserved regions of the DM20 protein caused the most severe forms of PMD. Substitutions of less conserved amino acids, truncations, absence of the protein and PLP-specific mutations caused the milder forms of PMD and SPG. Therefore, the interactions and stability of the mutated proteins has a major effect on the severity of PLP-related diseases.

Adolescent↗

PubMed2000

Effect of vitamin A status at the end of term pregnancy on the saturation of retinol binding protein with retinol.

BACKGROUND: Vitamin A (retinol), which is required for normal fetal development and successful gestation, circulates in the blood bound to a specific protein, the retinol binding protein (RBP). Little is known about the transport and metabolism of this complex protein or about retinol status during normal human pregnancy. OBJECTIVE: The aim of this study was to assess retinol status and transport modalities of retinol in well-nourished women with normal pregnancies, a population poorly investigated compared with pathologic and malnourished pregnant women. DESIGN: The maternal blood and cord blood concentrations of retinol, vitamin E, beta-carotene, RBP, and transthyretin of pregnant French women at term (n = 27) were measured and compared with values from a nonpregnant control group (n = 27). In addition, holo-RBP (retinol bound), apo-RBP (retinol free), and total protein were assessed in both groups to enable the hemodilution occurring during pregnancy to be taken into consideration and to evaluate the extent of saturation of RBP with retinol. RESULTS: Healthy pregnant women at term had normal serum circulatory amounts of retinol, vitamin E, binding proteins, and beta-carotene. However, they had less binding of retinol to RBP (holo-RBP: 49.9% in pregnant women, 54.0% in cord blood, and 77.5% in the control group). CONCLUSION: The results of this study suggest that retinol homeostasis and transport are modified during normal human pregnancy.

Adult↗

PubMed2000

Life cycle of an endogenous retrovirus, ZAM, in Drosophila melanogaster.

ZAM is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. In this paper, we traced ZAM mobilization to get information about a potential path a retroelement may take to reach the germ line of its host. In situ hybridization on whole-mount tissues and immunocytochemistry analyses with antibodies raised against ZAM Gag and Env proteins have shown that all components necessary to assemble ZAM viral particles, i.e., ZAM full-length RNAs and Gag and Env polypeptides, are coexpressed in a small set of follicle cells surrounding the oocyte. By electron microscopy, we have shown that ZAM viral particles are indeed detected in this somatic lineage of cells, which they leave and enter the closely apposed oocyte. Our data provide evidence that the vesicular traffic and yolk granules in the process of vitellogenesis play an important role in ZAM transfer to the oocyte. Our data support the possibility that vitellogenin transfer to the oocyte may help a retroelement pass to the germ line with no need of its envelope product.

Animals↗