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qRT-PCR Analysis (qrt-pcr + analysis)
Selected AbstractsDROUGHT STRESS: Role of Carbohydrate Metabolism in Drought-Induced Male Sterility in Rice Anthers,JOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 5 2010G. N. Nguyen Abstract Rice plants exposed to three consecutive days of water stress (,0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT-PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers. [source] OCIA domain containing 2 is highly expressed in adenocarcinoma mixed subtype with bronchioloalveolar carcinoma component and is associated with better prognosisCANCER SCIENCE, Issue 1 2007Tadashi Ishiyama Although lung adenocarcinoma is a major cause of cancer death worldwide, details of its molecular carcinogenesis and stepwise progression are still unclear. To characterize the sequential progression from bronchioloalveolar adenocarcinoma of the lung (BAC, in situ carcinoma) to adenocarcinoma mixed subtype with BAC component, polymerase chain reaction-based cDNA suppression subtractive hybridization (SSH) was carried out using two representative cases of BAC (non-invasive tumors) and adenocarcinoma mixed subtype with BAC (invasive tumors). Through differential screening, virtual reverse northern hybridization and quantitative real-time reverse-transcription,polymerase chain reaction (qRT-PCR) we selected five genes (TncRNA, OCIAD2, ANXA2, TMED4 and LGALS4) that were expressed at significantly higher levels in invasive adenocarcinoma mixed subtype with BAC than in BAC. After in situ hybridization and qRT-PCR analyses, we confirmed that only the OCIAD2 gene showed significantly higher expression in the tumor cells of invasive adenocarcinoma mixed subtype with BAC than in BAC (P = 0.026). We then carried out in situ hybridization of OCIAD2 in 56 adenocarcinoma mixed subtype with BAC component and assessed the correlation between OCIAD2 expression and clinicopathological features. In contrast to our expectation, the patients with OCIAD2 expression showed a better clinical outcome than those without OCIAD2 expression, and OCIAD2 expression showed an inverse correlation with lymphatic invasion, blood vessel invasion and lymph node metastasis. These results suggest that OCIAD2 begins to express at the progression from in situ to invasive carcinoma, and is associated with the favorable prognosis of adenocarcinoma mixed subtype with BAC component. (Cancer Sci 2007; 98: 50,57) [source] Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathwayMOLECULAR CARCINOGENESIS, Issue 11 2007Xingya Wang Abstract Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1,4). In this study, we investigated the role of EP receptors in PGE2 -induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM,10 µM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2 -induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2,5,-dideoxyadenosine, at concentrations that inhibited PGE2 -induced cAMP, significantly blocked PGE2 -induced VEGF secretion in PC-3 cells. We conclude that PGE2 -induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. © 2007 Wiley-Liss, Inc. [source] Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycinaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2010Giuseppe Gallo Abstract A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially and constitutively expressed protein isoforms, which were associated with 203 ORFs in the A. balhimycina genome. These data, providing insights on the major metabolic pathways/molecular processes operating in this organism, were used to compile 2-DE reference maps covering 3,10, 4,7 and 4.5,5.5 pH gradients available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhimycin precursors, such as amino acids, amino sugars and central carbon metabolism intermediates, were upregulated during antibiotic production. qRT-PCR analysis revealed that 8 out of 14 upregulated genes showed a positive correlation between changes at translational and transcriptional expression level. Furthermore, proteomic analysis of two nonproducing mutants, restricted to a sub-set of differentially expressed proteins, showed that most proteins required for the biosynthesis of balhimycin precursors are downregulated in both mutants. These findings suggest that primary metabolic pathways support anabolic routes leading to balhimycin biosynthesis and the differentially expressed genes are interesting targets for the construction of high-yielding producer strains by rational genetic engineering. [source] Deregulation of Aurora kinase gene expression in human testicular germ cell tumoursANDROLOGIA, Issue 4 2010E. Baldini Summary The Aurora kinases regulate chromosome segregation and cytokinesis, and alterations in their expression associate with cell malignant transformation. In this study, we demonstrated by qRT-PCR analysis of 14 seminomas that Aurora-A mRNA was, with respect to control tissues, augmented in five of 14 tumour tissues by 2.17 ± 0.30 fold (P < 0.05) and reduced in 9 to 0.38 ± 0.10 (P < 0.01). Aurora-B mRNA was increased in 11 tumour tissues by 4.33 ± 0.82 fold (P < 0.01) and reduced in 3 to 0.41 ± 0.11 fold. Aurora-C mRNA was reduced to 0.20 ± 0.32 fold (P < 0.01) in 13 seminomas and up-regulated in one case. Western blot experiments, performed on protein extracts of nine seminomas and six normal testes, showed an up-regulation of Aurora-B protein by 10.14 ± 3.51 fold (P < 0.05), while Aurora-A protein was found increased in four seminomas by 2.16 ± 0.43 (P < 0.05), unchanged in three and reduced in two tumour tissues. Aurora-C protein was increased by 9.2 ± 2.90 fold (P < 0.05), suggesting that post-transcriptional mechanisms modulate its expression. In conclusion, we demonstrated that expression of Aurora kinases is deregulated in seminomas, suggesting that they may play a role in the progression of testicular cancers. [source] Hemoglobin regulates the metabolic and synthetic function of rat insulinoma cells cultured in a hollow fiber bioreactorBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Sharon I. Gundersen Abstract Pancreatic islet transplantation continues to benefit patients with type 1 diabetes by normalizing glucose metabolism and improving other complications of diabetes. However, islet transplantation therapy is limited by the inadequate availability of pancreatic islets. In order to address this concern, this work investigated the expansion of rat insulinoma cells (INS-1) and their ability to generate insulin in a hollow fiber bioreactor (HFB). The long-term goal of this project is to develop a bioartificial pancreas. HFBs were incubated at two different oxygenation conditions (10% and 19% O2) to determine the best scenario for O2 transport to cultured cells. Also, bovine hemoglobin (BvHb) was supplemented in the cell culture media of the HFBs in order to increase O2 transport under both oxygenation conditions. Our results show that INS-1 cells expanded under all oxygenation conditions after 2 weeks of culture, with a slightly higher cell expansion under normoxic oxygenation (19% O2) for both control HFBs and BvHb HFBs. In addition, cellular insulin production remained steady throughout the study for normoxic control HFBs and BvHb HFBs, while it increased under hypoxic oxygenation (10% O2) for both types of HFBs but to different extents. Under the two different oxygenation conditions, cellular insulin production was more uniform with time in BvHb HFBs versus control HFBs. These results, along with qRT-PCR analysis, suggest a possible dysregulation of the insulin-signaling pathway under hypoxic culture conditions. In conclusion, the HFB culture system is an environment capable of expanding insulinomas while maintaining their viability and insulin production capabilities. Biotechnol. Bioeng. 2010;107: 582,592. © 2010 Wiley Periodicals, Inc. [source] | ||||||||||