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RT-PCR Experiments (rt-pcr + experiment)
Selected AbstractsPituitary adenylate cyclase-activating polypeptide attenuates streptozotocin-induced apoptotic death of RIN-m5F cells through regulation of Bcl-2 family protein mRNA expressionFEBS JOURNAL, Issue 22 2008Satomi Onoue Oxidative stress, followed by the apoptotic death of pancreatic , cells, is considered to be one of causative agents in the evolution of the type 2 diabetic state; therefore, the protection of , cells can comprise an efficacious strategy for preventing type 2 diabetes. In the present study, RIN-m5F cells (i.e. the rat insulinoma , cell line) were stimulated with streptozotocin, resulting in a time- and concentration-dependent release of lactate dehydrogenase. There appeared to be significant apoptotic cell death after 2 h of treatment with streptozotocin at 10 mm, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining and 2.6-fold activation of cellular caspase-3, an apoptotic enzyme. By contrast, some neuropeptides of the glucagon-secretin family and coenzyme Q10, an endogenous mitochondrial antioxidant, could attenuate streptozotocin cytotoxicity, and especially pituitary adenylate cyclase-activating polypeptide (PACAP), at a concentration of 10,7 m, exhibited 34% attenuation of lactate dehydrogenase release from streptozotocin-treated RIN-m5F cells. Quantitative RT-PCR experiments indicated the inhibitory effect of PACAP on streptozotocin-evoked up-regulation of pro-apoptotic factor (Noxa and Bax) and a 2.3-fold enhancement of Bcl-2 mRNA expression, a pro-survival protein, was also observed after addition of PACAP. The data obtained suggest the anti-apoptotic role of PACAP in streptozotocin-treated RIN-m5F cells through the regulation of pro-apoptotic and pro-survival factors. [source] Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylationGENES TO CELLS, Issue 7 2009Meixiang Sang Plk3, one of Polo-like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study, we found that Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation. During cisplatin (CDDP)-mediated apoptosis, Plk3 was transcriptionally induced, whereas its protein level was kept at basal level, suggesting that Plk3 might rapidly degrade in response to CDDP. Immunoprecipitation and in vitro pull-down experiments demonstrated that Plk3 interacts with p73. Luciferase reporter assays and RT-PCR experiments revealed that Plk3 inhibits p73-mediated transcriptional activity. Consistent with these results, pro-apoptotic activity of p73 was blocked by Plk3. Additionally, Plk3 decreased the stability of p73. Intriguingly, kinase-deficient Plk3 failed to inhibit p73 function, indicating that kinase activity of Plk3 is required for Plk3-mediated inhibition of p73. Indeed, in vitro kinase reaction showed that NH2 -terminal portion of p73 is phosphorylated by Plk3. In accordance with these observations, knocking down of Plk3 increased the stability of p73 and promoted CDDP-mediated apoptosis in association with up-regulation of p73. Collectively, our present findings suggest that Plk3 plays an important role in the regulation of cell fate determination in response to DNA damage through the inhibition of p73. [source] A novel gene, MDS2, is fused to ETV6/TEL in a t(1;12)(p36.1;p13) in a patient with myelodysplastic syndromeGENES, CHROMOSOMES AND CANCER, Issue 1 2002María D. Odero ETV6/TEL is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners of which 16 have been cloned. Fluorescence in situ hybridization (FISH) analysis in a patient with myelodysplastic syndrome (MDS) revealed a t(1;12)(p36;p13) involving ETV6, with the breakpoint in this gene between exon 2 and exon 3. We report here the cloning of a novel ETV6 partner located on 1p36.1, involved in the t(1;12). 3, RACE-PCR from RNA identified a novel sequence fused to exon 2 of ETV6. Database searches localized this sequence in a bacterial artificial chromosome (BAC) mapped to 1p36 by fingerprint analysis. This result was confirmed by FISH using this BAC as probe. 5, and 3, RACE experiments with primers from this novel sequence were carried out on RNA from a healthy donor and identified a novel full-length mRNA, which we named MDS2 (myelodysplastic syndrome 2). RT-PCR experiments were performed on a panel of human cDNAs to analyze the expression pattern of this gene and they revealed four splicing variants. RT-PCR analysis showed that ETV6-MDS2, but not the reciprocal MDS2-ETV6 fusion transcript, was expressed in the bone marrow of the patient. The product of the ETV6-MDS2 fusion transcript predicts a short ETV6 protein containing the first 54 amino acids of ETV6 plus four novel amino acids, lacking both the PTN and the DNA-binding domains. Possible mechanisms to account for the development of MDS in this patient are discussed. © 2002 Wiley-Liss, Inc. [source] Expression of Fas and Fas ligand in human testicular germ cell tumoursINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2009E. Baldini Summary In the present study, we analysed the expression of Fas ligand (FasL) and its cognate receptor Fas in 14 seminomatous testicular germ cell tumours (TGCT) and six normal testicular tissues obtained following orchiectomy. Tissue samples have been processed to prepare either total RNA or protein extracts or fixed and embedded in paraffin for immunohistochemistry (IHC) experiments. Quantitative RT-PCR experiments demonstrated in TGCT a significant (p < 0.01) increase of the FasL mRNA expression of 21.1 ± 5.4 fold, with respect to normal tissues. On the contrary, in the same cancer tissues, the levels of Fas mRNA were significantly (p < 0.01) reduced to 0.27 ± 0.06 fold. These observations were confirmed in western blot experiments showing a significant increase of FasL and a concomitant decrease of Fas proteins in testicular cancer tissues, with respect to normal testis. Moreover, IHC experiments showed a strong FasL immuno-reactivity in six out of eight TGCT samples analysed, while Fas immuno-positivity was found in cancer cells of only two TGCT tissues. In addition, in all tumour samples, infiltrating lymphocytes were Fas positive. However, no correlation could be observed between Fas or FasL mRNA variations and clinical parameters such as patient's age, TNM stage or tumour size. We also compared the serum levels of soluble FasL (sFasL) of 15 patients affected by seminomatous TGCT, of four patients with non-seminomatous TGCT and six age-matched healthy males. No significant differences in sFasL serum level could be identified. In conclusion, our data demonstrated that the majority of seminomas are characterized by an increased expression of FasL and a concomitant reduction of Fas, with respect to human normal testis, and that sFasL serum level is not a tumour marker for patients affected by TGCT. [source] Expression and identification of a new splice variant of neuroglycan C, a transmembrane chondroitin sulfate proteoglycan, in the human brainJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2006Sachiko Aono Abstract Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan with an EGF module. We studied the expression of NGC in the human brain, mainly in the hippocampus, and confirmed some observations by conducting experiments using rat brain. In humans, NGC mRNA was expressed exclusively in the brain, especially in the immature brain. The telencephalon, including the hippocampus and neocortex, showed strong mRNA expression. NGC was immunolocalized to neuropils in the hippocampus and neocortex of the adult rat. RT-PCR experiments showed that four splice variants (NGC-I, -II, -III, and -IV) were expressed in the adult human hippocampus. By Western blotting, the expression as proteins of all splice variants except NGC-II was confirmed in the adult rat hippocampus. NGC-IV, which was first found in the present study, had the shortest cytoplasmic domain among the four variants. NGC-IV mRNA was expressed by neurons, but not by astrocytes, in culture prepared from the fetal rat hippocampus, suggesting that NGC-IV plays a role specific to neurons. In addition, the human NGC gene, which is registered as CSPG5, comprised six exons and was approximately 19 kb in size. In exon 2, a single nucleotide polymorphism resulting in Val188Gly in the NGC ectodomain was observed. © 2005 Wiley-Liss, Inc. [source] Genes Differentially Expressed By Schwann Cells Of Motor Versus Sensory NervesJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001D Imperiale Charcot-Marie-Tooth (CMT) disease includes a heterogeneous group of inherited demyelinating peripheral neuropathies related to genetic defects of myelin-forming Schwann cells (SC). In CMT, as in other common acquired demyelinating neuropathies (Guillain Barré syndrome, chronic inflammatory demyelinating polyneuropathy), motor nerves are invariably more involved than sensory nerves. Also in transgenic mouse models of peripheral neuropathy, there is a preferential demyelination of motor districts independent of the type of genetic alteration. The basis for differential susceptibility to demyelination is unknown. The aim of this study was to identify differences in gene and protein expression that may underlie the differential susceptibility to demyelination of motor and sensory myelin-forming SC. Since spinal roots are the only portion of mammalian PNS in which motor and sensory axons are segregated, we extracted RNA from adult rat dorsal (sensory) and ventral (motor) spinal roots and compared corresponding cDNAs by an RNA fingerprint approach. Four differentially displayed bands were isolated. We first characterized the most differentially expressed band, which was highly enriched in sensory roots. Sequence analysis showed that the band encoded a portion of rat sarco/endoplasmic reticulum calcium transporting ATPase type 1 coding sequence (SERCA1). RT-PCR experiments confirmed SERCA1 enrichment in dorsal sensory roots. SERCA enzymes are ubiquitous calcium regulatory systems in muscle and non-muscle cells and SERCA1 is selectively enriched in skeletal muscle. To our knowledge, no studies have investigated SERCA isoform expression in peripheral nerve. Identification of a calcium regulatory molecule in SC is interesting, as calcium is essential for the proper structure and function of the nodal and paranodal portions of SC, as well as the myelin sheath. However, calcium homeostasis in SC is relatively unexplored. Experiments to localize SERCA1 transcript and protein in different PNS districts and to clarify its functional role in peripheral nerve are underway. [source] Identification and molecular analysis of candidate genes homologous to HcrVf genes for scab resistance in applePLANT BREEDING, Issue 1 2009A. Boudichevskaia Abstract The genetic locus for resistance to apple scab most frequently used in apple breeding is Vf, derived from Malus floribunda 821. For the Vf locus a cluster of four resistance gene paralogs (called as HcrVf genes) encoding receptor-like proteins (RLP) with similarity to the tomato Cf resistance genes is known. Based on published sequences for HcrVf1 and HcrVf2 PCR primers were designed from the domain B and the variable leucine-rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85,100%) to HcrVf1 and HcrVf2 were obtained not only from M. floribunda 821 and Vf cultivars but also from other apple scab resistance sources, such as ,Russian Seedling' R12740-7A (Vr resistance) or ,Antonovka polutorafuntovaya' (VA resistance). A series of 13 HcrVf candidate genes have been partly cloned from the PCR fragments spanning N-terminal LRRs 20,30. A considerable number of amino acid exchanges within the solvent-exposed xxLxLxx structural motives were detected among the homologous sequences. Expression analyses and mapping focused on a selected Vf- homologous candidate gene (called Vf2ARD) identified in resistant Malus genotypes known for carrying other scab resistance genes than Vf. RT-PCR experiments showed that Vf2ARD is expressed under pathogen-free conditions. The results of a quantitative PCR-based transcription profiling suggest that this gene is scab-inducible in some resistant cultivars. Vf2ARD has been mapped on linkage group LG 1. It is separated from the Vf gene cluster with a genetic distance of about 2 cM and might be a member of a second Vf - like locus on apple linkage group LG 1. [source] Placental expression of the nuclear receptors for oxysterols LXR, and LXR, during mouse and human developmentTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 1 2005Geoffroy Marceau Abstract Ontogenesis and localization expression of genes encoding for the nuclear receptors for oxysterols (lxr, and lxr,) were investigated in human and mouse placenta during gestation. Both mRNAs were detectable in the placentation throughout the gestation (from 7 days postcoitum and 6th week of gestation in mouse and human, respectively) by Northern blots or RT-PCR experiments. Lxr, showed a higher accumulation in the mouse yolk sac. In situ hybridization pointed a specific expression of lxr, in the human amniotic membranes. © 2005 Wiley-Liss, Inc. [source] Apical SK potassium channels and Ca2+ -dependent anion secretion in endometrial epithelial cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Melissa L. Palmer Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (Isc) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K+ dependent. The current,voltage relationship was slightly inwardly rectifying with a reversal potential of ,52 ± 2 mV, and the PK/PNa ratio was 14, indicating high selectivity for K+. Concentration,response relationships for apamin and dequalinium had IC50 values of 0.5 nm and 1.8 ,m, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 ,m BAPTA-AM completely blocked the effects of UTP on K+ channel activation, indicating that the apamin-sensitive current was also Ca2+ dependent. Moreover, channel activation was blocked by calmidazolium (IC50= 5 ,m), suggesting a role for calmodulin in Ca2+ -dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17, produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K+ channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca2+ near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation. [source] Identification of distinct gene expression profiles in the synovium of patients with systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 5 2007A. Nzeusseu Toukap Objective Synovitis is a common feature of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but the pattern of joint involvement differs in each disease. This study was undertaken to investigate the global gene expression profiles in synovial biopsy tissue from the swollen knees of untreated SLE patients (n = 6), RA patients (n = 7), and osteoarthritis (OA) patients (n = 6). Methods Synovial biopsy samples were obtained from the affected knees of patients in the 3 groups by needle arthroscopy. Half of the material was used for extraction of total RNA, amplification of complementary RNA, and high-density oligonucleotide spotted hybridization arrays. On the remaining tissue samples, real-time reverse transcription,polymerase chain reaction (RT-PCR) and immunohistochemical experiments were performed to confirm the microarray data. Results SLE synovial biopsy tissue displayed a significant down-regulation of genes involved in extracellular matrix (ECM) homeostasis and a significant up-regulation of interferon-inducible (IFI) genes. Real-time RT-PCR experiments confirmed the up-regulation of selected IFI genes (IFI27, IFI44, and IFI44L) in the SLE synovial tissue. Immunohistochemical analyses showed that 3 molecules involved in ECM regulation, chondroitin sulfate proteoglycan 2, latent transforming growth factor , binding protein 2, and fibroblast activation protein ,, were significantly down-regulated in SLE synovium. In contrast, immunostaining for IFI27, Toll-like receptor 4, and STAT-1 resulted in higher quantitative scores in SLE synovial tissue, which could be attributed to the fact that the RA samples had a large population of inflammatory cell infiltrates that were negative for these markers. Conclusion Arthritis in SLE has a very distinct molecular signature as compared with that in OA and RA, characterized by up-regulation of IFI genes and down-regulation of genes involved in ECM homeostasis. [source] Gene expression profiling of advanced-stage serous ovarian cancers distinguishes novel subclasses and implicates ZEB2 in tumor progression and prognosisCANCER SCIENCE, Issue 8 2009Kosuke Yoshihara To elucidate the mechanisms of rapid progression of serous ovarian cancer, gene expression profiles from 43 ovarian cancer tissues comprising eight early stage and 35 advanced stage tissues were carried out using oligonucleotide microarrays of 18 716 genes. By non-negative matrix factorization analysis using 178 genes, which were extracted as stage-specific genes, 35 advanced stage cases were classified into two subclasses with superior (n = 17) and poor (n = 18) outcome evaluated by progression-free survival (log rank test, P = 0.03). Of the 178 stage-specific genes, 112 genes were identified as showing different expression between the two subclasses. Of the 48 genes selected for biological function by gene ontology analysis or Ingenuity Pathway Analysis, five genes (ZEB2, CDH1, LTBP2, COL16A1, and ACTA2) were extracted as candidates for prognostic factors associated with progression-free survival. The relationship between high ZEB2 or low CDH1 expression and shorter progression-free survival was validated by real-time RT-PCR experiments of 37 independent advanced stage cancer samples. ZEB2 expression was negatively correlated with CDH1 expression in advanced stage samples, whereas ZEB2 knockdown in ovarian adenocarcinoma SKOV3 cells resulted in an increase in CDH1 expression. Multivariate analysis showed that high ZEB2 expression was independently associated with poor prognosis. Furthermore, the prognostic effect of E-cadherin encoded by CDH1 was verified using immunohistochemical analysis of an independent advanced stage cancer samples set (n = 74). These findings suggest that the expression of epithelial,mesenchymal transition-related genes such as ZEB2 and CDH1 may play important roles in the invasion process of advanced stage serous ovarian cancer. (Cancer Sci 2009) [source] | |||||||||||||