Biomedical subjects

Virginie Garcia

Publications and source records attributed to Virginie Garcia.

11 recordsLinked to original sources

PubMed2006

Transcriptome analysis of bud burst in sessile oak (Quercus petraea).

Expression patterns of hundreds of transcripts in apical buds were monitored during bud flushing in sessile oak (Quercus petraea), in order to identify genes differentially expressed between the quiescent and active stage of bud development. Different transcriptomic techniques combining the construction of suppression subtractive hybridization (SSH) libraries and the monitoring of gene expression using macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were performed to dissect bud burst, with a special emphasis on the onset of the process. We generated 801 expressed sequence tags (ESTs) derived from six developmental stages of bud burst. Macroarray experiment revealed a total of 233 unique transcripts exhibiting differential expression during the process, and a putative function was assigned to 65% of them. Cell rescue/defense-, metabolism-, protein synthesis-, cell cycle- and transcription-related transcripts were among the most regulated genes. Macroarray and real-time RT-PCR showed that several genes exhibited contrasted expressions between quiescent and swelling buds, such as a putative homologue of the transcription factor DAG2 (Dof Affecting Germination 2), previously reported to be involved in the control of seed germination in Arabidopsis thaliana. These differentially expressed genes constitute relevant candidates for signaling pathway of bud burst in trees.

Cluster Analysis↗

PubMed2005

Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models.

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 (SphK1) activity and subsequent ceramide/sphingosine 1-phosphate (S1P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of the ceramide/S1P ratio only in cell lines sensitive to these drugs. SphK1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S1P ratio. Alternatively, silencing SphK1 by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S1P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK1-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK1 as a sensor of chemotherapy.

Adenocarcinoma↗

PubMed2005

Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato.

The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.

Cell Size↗

PubMed2005

Activation of the {beta}-catenin/T-cell-specific transcription factor/lymphoid enhancer factor-1 pathway by plasminogen activators in ECV304 carcinoma cells.

Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. beta-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non-serine-threonin phosphorylated, nonubiquitinated) form of beta-catenin in ECV304 carcinoma cells. tPA-dependent beta-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of beta-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and beta-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. beta-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3beta through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of beta-catenin/T-cell-specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. beta-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the beta-catenin pathway was triggered by amino-terminal fragment, the NH(2)-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the beta-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.

Active Transport, Cell Nucleus↗

PubMed2004

Role of FAN in tumor necrosis factor-alpha and lipopolysaccharide-induced interleukin-6 secretion and lethality in D-galactosamine-sensitized mice.

Tumor necrosis factor (TNF) alpha-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFalpha receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after d-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFalpha-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFalpha) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFalpha treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that d-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFalpha.

Animals↗

PubMed2004

Automated SNP detection in expressed sequence tags: statistical considerations and application to maritime pine sequences.

We developed an automated pipeline for the detection of single nucleotide polymorphisms (SNPs) in expressed sequence tag (EST) data sets, by combining three DNA sequence analysis programs: Phred, Phrap and PolyBayes. This application requires access to the individual electrophoregram traces. First, a reference set of 65 SNPs was obtained from the sequencing of 30 gametes in 13 maritime pine (Pinus pinaster Ait.) gene fragments (6671 bp), resulting in a frequency of 1 SNP every 102.6 bp. Second, parameters of the three programs were optimized in order to retrieve as many true SNPs, while keeping the rate of false positive as low as possible. Overall, the efficiency of detection of true SNPs was 83.1%. However, this rate varied largely as a function of the rare SNP allele frequency: down to 41% for rare SNP alleles (frequency < 10%), up to 98% for allele frequencies above 10%. Third, the detection method was applied to the 18498 assembled maritime pine (Pinus pinaster Ait.) ESTs, allowing to identify a total of 1400 candidate SNPs, in contigs containing between 4 and 20 sequence reads. These genetic resources, described for the first time in a forest tree species, were made available at http://www.pierroton.inra/genetics/Pinesnps. We also derived an analytical expression for the SNP detection probability as a function of the SNP allele frequency, the number of haploid genomes used to generate the EST sequence database, and the sample size of the contigs considered for SNP detection. The frequency of the SNP allele was shown to be the main factor influencing the probability of SNP detection.

Algorithms↗

PubMed2003

IPPRED: server for proteins interactions inference.

SUMMARY: IPPRED is a web based server to infer protein-protein interactions through homology search between candidate proteins and those described as interacting. This simple inference allows to propose or to validate potential interactions. AVAILABILITY: IPPRED is freely available at http://cbi.labri.fr/outils/ippred/.

Algorithms↗

PubMed2003

The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs.

Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short-chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin-treated GM95/GCS cells, metabolism of drug-induced ceramide did not appear to be instrumental in cell survival. Furthermore, N-(n-butyl)deoxynojirimycin, a potent and non-toxic GCS inhibitor, had no chemosensitizing effect on wild-type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug-resistance mechanisms.

ATP-Binding Cassette Transporters↗

PubMed2003

HDL counterbalance the proinflammatory effect of oxidized LDL by inhibiting intracellular reactive oxygen species rise, proteasome activation, and subsequent NF-kappaB activation in smooth muscle cells.

Oxidized low-density lipoproteins (oxLDL) exhibit proinflammatory properties and play a role in atherosclerosis plaque formation, rupture, and subsequent thrombosis. OxLDL alter the activity of the transcription factor NF-kappaB that is involved in the expression of immune and inflammatory genes. In contrast, high-density lipoproteins (HDL) are anti-atherogenic and exhibit anti-inflammatory properties. This work aimed to investigate how oxLDL activate NF-kappaB and whether and how HDL may prevent NF-kappaB activation. In cultured rabbit smooth muscle cells, mitogenic concentrations of mildly oxLDL trigger a rapid and transient NF-kappaB activation, which is strongly inhibited by HDL. Growth factors, phosphatidylinositol 3-kinase/Akt, and sphingosine kinase pathways are not implicated in the oxLDL-induced NF-kappaB activation and are not targets of HDL. OxLDL induce reactive oxygen species (ROS) generation and proteasome activation, which are implicated in NF-kappaB activation, as suggested by the inhibitory effect of the antioxidants N-acetyl-L-cysteine and pyrrolidinedithiocarbamate and the proteasome inhibitor PSI. HDL were able to prevent the intracellular ROS rise triggered by oxLDL or H2O2, thereby inhibiting the subsequent proteasome activation, IkappaB degradation, and NF-kappaB activation. In conclusion, the oxLDL-induced NF-kappaB activation involves ROS generation and proteasome activation, both events being inhibited by HDL. This 'antioxidant' and potentially anti-inflammatory effect of HDL may participate in their general anti-atherogenic properties.

Animals↗

PubMed2002

Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways.

OBJECTIVE: Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL. METHODS AND RESULTS: Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutant showed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominant-negative Deltap85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of N,N-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in Deltap85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway. CONCLUSIONS: SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL.

1-Phosphatidylinositol 4-Kinase↗

PubMed2002

Danon's disease (X-linked vacuolar cardiomyopathy and myopathy): a case with a novel Lamp-2 gene mutation.

Herein, we report a new case of Danon's disease in a 41-year-old Frenchman. This patient displays the typical clinical triad, with cardiomyopathy, mental retardation and myopathy, and a vacuolar myopathy without acid alpha-glucosidase deficiency. He has also developed a diffuse chorio-capillary ocular atrophy, and represents the second case of successful heart transplantation in this lysosomal disease. Interestingly, analysis of LAMP-2 protein expression in cultured fibroblasts revealed a primary deficiency of this lysosomal membrane protein. This defect resulted from a yet undescribed deletion in exon 7 of lamp-2 gene.

Adult↗