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Quantitative Real-time PCR (quantitative + real-time_pcr)
Terms modified by Quantitative Real-time PCR Selected AbstractsTime course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafishDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2007Sean C. Kassen Abstract Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Changes in Drosophila melanogaster midgut proteins in response to dietary Bowman,Birk inhibitorINSECT MOLECULAR BIOLOGY, Issue 5 2007H.-M. Li Abstract The midgut proteome of Drosophila melanogaster was compared in larvae fed dietary Bowman,Birk inhibitor (BBI) vs. larvae fed a control diet. By using two-dimensional gel electrophoresis, nine differentially expressed proteins were observed, which were associated with enzymes or transport functions such as sterol carrier protein X (SCPX), ubiquitin-conjugating enzyme, endopeptidase, receptor signalling protein kinase, ATP-dependent RNA helicase and ,-tocopherol transport. Quantitative real-time PCR verified differential expression of transcripts coding for six of the proteins observed from the proteomic analysis. BBI evidently affects expression of proteins associated with protein degradation, transport and fatty acid catabolism. We then tested the hypothesis that SCPX was critical for the Drosophila third instars' response to BBI treatment. Inhibition of SCPX caused the third instars to become more susceptible to dietary BBI. [source] Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål)INSECT MOLECULAR BIOLOGY, Issue 6 2000J. G. Vontas Abstract Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3,7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1,15-fold more Nl-EST1 mRNA in individual insects and 5,11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8,10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear. [source] Inhibition of 13-cis retinoic acid-induced gene expression of homeobox B7 by thalidomideINTERNATIONAL JOURNAL OF CANCER, Issue 6 2007an Milanovi Abstract Thalidomide and 13-cis retinoic acid (RA) show anticancer effects as sole agents or in combination with other drugs. However, induction of homeobox (HOX) gene expression by 13-cis RA may contribute to tumor progression thereby potentially limiting its efficacy. The purpose was to test if thalidomide can inhibit 13-cis RA-induced HOXB7 expression and whether thalidomide may enhance the antiproliferative effect of 13-cis RA in U343MG glioblastoma cells. Quantitative real-time PCR showed significant inhibition of 13-cis RA-induced HOXB7 expression by thalidomide with IC50 , 0.1,0.2 ,g/ml when given simultaneously with 13-cis RA but not when administered 18h later (p < 0.0001). 13-cis RA alone inhibited proliferation and colony formation in a concentration-dependent manner whereas growth inhibition by thalidomide alone at 5,100 ,g/ml was constant at 80,90% of controls. At 10% serum concentration, growth inhibition by a combination of the 2 drugs was additive but at 1% serum, growth inhibition was synergistic. It is concluded that thalidomide inhibits the RA-induced HOXB7 expression in glioblastoma cells and that 13-cis RA/thalidomide combinations can in principle enhance cytotoxicity. The improved cell kill induced by thalidomide is attributed to downregulation of growth stimulatory factors induced by 13-cis RA. Implications for the modus operandi of thalidomide in embryogenesis are noted. © 2007 Wiley-Liss, Inc. [source] Variation in human papillomavirus type-16 viral load within different histological grades of cervical neoplasiaJOURNAL OF MEDICAL VIROLOGY, Issue 9 2007A.N. Fiander Abstract The objective of this study was to investigate variation in human papillomavirus (HPV) type-16 load within histologically defined grades of cervical intraepithelial neoplasia. Two hundred and thirty-seven liquid based cytology samples were collected from women attending colposcopy clinics, DNA was extracted, and presence of virus determined by PCR-enzyme immunoassay. Quantitative real-time PCR was used to determine viral load for 70 HPV-16 positive single infections. Viral load was expressed as the ratio of copies of the viral L1 gene to copies of the human beta-globin gene. Measurements varied from 0.019 to 4,194 HPV genomes per cell. Our data demonstrate that in cervical neoplasia, HPV load tends to correlate with disease severity, but that the number of viral genomes/cell varies considerably within histological grades. This variation within disease grades currently limits the clinical utility of viral load measurement. [source] Altered gene expression in frontal cortex and midbrain of 3,4-methylenedioxymethamphetamine (MDMA) treated mice: Differential regulation of GABA transporter subtypesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003Weiping Peng Abstract Changes in gene expression were examined in the brain of mice treated with a drug of abuse, 3,4-methylenedioxymethamphetamine (MDMA, also called Ecstasy). Frontal cortex and midbrain mRNA, analyzed by differential display polymerase chain reaction (DD-PCR) method, showed an altered expression of several cDNAs, 11 of which were isolated, cloned and sequenced. The sequence of one MDMA-induced mRNA corresponds (99.3%) to the mouse ,-amino butyric acid (GABA) transporter 1 (mGAT1). The established involvement of GABA neurotransmission in the activity of several abused drugs prompted us to focus herein on MDMA effect on the GABA transporter gene family. Semi-quantitative PCR analysis with primers selective to the reported mGAT1 sequence confirmed that MDMA treatment increased mGAT1 expression. Time-course study of the expression of the three GABA transporter subtypes showed that MDMA induced a differential temporal activation of mGAT1 and mGAT4, but had no effect on mGAT2. Quantitative real-time PCR further proved the increased expression of mGAT1 and mGAT4 upon MDMA treatment. Western immunoblotting with anti-GAT1 antibodies showed that MDMA also increased GAT1 protein levels, suggesting that neurotransmission of GABA was altered. MDMA effect was also verified in serotonin transporter knockout (,/,) mice that are insensitive behaviorally to MDMA; the drug did not increase GAT1 protein level in these mutants. In mice, tiagabine and NO-711, inhibitors of GABA transporters, restrained MDMA-induced acute toxicity and death. These results should facilitate novel approaches to prevent deleterious effects, including fatality, induced by MDMA and similar abused psychostimulants. © 2003 Wiley-Liss, Inc. [source] Identification and Regulation of Genes from a Biocontrol Strain of Fusarium oxysporumJOURNAL OF PHYTOPATHOLOGY, Issue 9 2007D. R. Fravel Abstract Differential display with three time points revealed that thiram altered expression of numerous genes in the biocontrol fungus Fusarium oxysporum CS-20. Of the 101 bands purified from the differential display gel, 86 were successfully cloned, and 64 sequenced. Based on nucleic acid sequences, homology to known products was found using BLASTn for 26 sequences and homology to hypothetical proteins was found for six sequences, also from Gibberella zeae. One band (BM1 24-1) showed homology to an ABC transporter from three different fungi. Because of its association with detoxification functions, the ABC transporter was selected for further study. Mycelia of CS-20 were exposed to 25 ,g active ingredient (a.i.) thiram in liquid culture for various times from 0 to 8 h. Quantitative real-time PCR was used to evaluate gene expression. At 30 min after treatment with thiram, the ABC transporter was upregulated 20- to 25-fold relative to the control treatment. The ABC transporter was upregulated 15-fold at 1 h after treatment and 10-fold at 2 h. At 8 h after treatment, there was no difference between treated and non-treated for expression of the ABC transporter. Transcription of the gene encoding EST BM1 24-1 is induced in response to thiram treatment and may function in providing resistance in F. oxysporum isolate CS-20 to fungicides and other toxins. Tolerance to toxins may be critical to the successful inclusion of CS-20 in disease control strategies in cropping systems. [source] Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming unitsMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2009Nina Jonsson ABSTRACT Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID50), methods that are laborious, time-consuming and take on average 3,7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID50/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID50). The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID50 and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID50 or PFU for each enterovirus serotype included was determined. [source] Changes of maternal transcripts in oocytes from persistent follicles in cattleMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2007Brandon M. Lingenfelter Abstract A high incidence of early embryonic loss is associated with prolonged dominance of follicles. The objective of the present experiment was to determine if persistence of a follicle resulted in alterations in mRNA expression of important genes in the oocyte. Cows were assigned to four groups: growing follicles on day 6 (G0h) or day 8 (G48h) and persistent follicles on day 13 (P0h) or day 15 (P48h) of the estrous cycle (estrus,=,day 0). All cows were super-stimulated on day 1,4. Cows in G48h, P0h, and P48h groups received 25 mg prostaglandin (PG) F2, on day 6. Cows in P0h and P48h groups received progesterone from CIDR-B devices on day 5 through 13. Ovaries of cows in G0h, G48h, P0h, and P48h groups were removed on day 6, 8, 13, and 15, respectively. Oocytes were aspirated immediately after colpotomy and denuded of cumulus cells. Quantitative real-time PCR was used to measure the mRNA abundances of 10 selected genes important for early embryogenesis in oocytes obtained from growing and persistent follicles. Relative abundances of MSY2, PARN, and YY1 mRNA (P,<,0.05) were significantly lower in oocytes from persistent than from growing follicles. Oocytes from persistent follicles, however, had greater abundances of PAP and eIF-4E transcripts (P,<,0.05). The data indicate that persistence of a follicle leads to altered abundances of mRNA for genes important for regulation of transcription and protein translation in the oocyte, which could compromise development of early embryos in cows that ovulate a persistent follicle. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Ultraviolet-A and -B Differentially Modify the Tyrosine-Kinase Profile of Human Keratinocytes and Induce the Expression of Arg,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2008Gabriele Klosner To investigate the expression profile of protein tyrosine kinases (PTKs) in normal human epidermal keratinocytes (NHEK) in response to UVA and UVB we employed a reversed transcriptase polymerase chain reaction (PCR) approach using degenerate primers derived from the conserved catalytic domain of PTKs. Quantitative real-time PCR with specific primers was used to confirm the influence of UV on the expression of the identified PTKs. Arg (Abelson-related gene, Abl2) was the PTK with the highest prevalence (30% of all PTKs) and UVA led to a further induction of Arg expression reaching nine-fold mRNA baseline expression at 17 h after irradiation. UVB was followed by an initial downregulation and a subsequent increase in Arg mRNA reaching five-fold baseline levels after 24 h. We conclude that UVA and UVB differentially modify the expression of PTKs in NHEK, and that Arg appears to have a major role in the response of keratinocytes to UV. These results provide a basis for further studies of PTK in UV-induced signaling that regulates protective responses, cell growth and carcinogenesis in the skin. [source] Reference gene selection for real-time polymerase chain reaction in human lung cells subjected to cyclic mechanical strainRESPIROLOGY, Issue 7 2008Liao PINHU Background and objective: The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. Methods: The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. Results: The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. Conclusions: These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific. [source] Mapping and expression analyses during porcine foetal muscle development of 12 genes involved in histone modificationsANIMAL GENETICS, Issue 2 2009Y. B. Peng Summary Histone modifications (methylation and demethylation) regulate gene expression and play a role in cell proliferation and differentiation by their actions on chromatin structure. In this context, we studied the temporal expression profiles of genes acting on histone methylation and demethylation during skeletal muscle proliferation and differentiation. Quantitative real-time PCR was used to quantify the mRNA levels of CARM1, JARID1A, JMJD2A, LSD1, PRMT2, PRMT5, SMYD1, SMYD2, SMYD3, SETDB1, Suv39h2 and SUZ12 in foetal skeletal muscle. Our results showed that CARM1, JARID1A, JMJD2A, SMYD1 and SMYD2 were differentially expressed in embryonic muscles of 33 days post-conception (dpc), 65 dpc and 90 dpc. These 12 genes were mapped to porcine chromosomes (SSC) 2q21,24, 5q25, 6q35, 6q12,21, 6p15, 7q21, 3q21,27, 9q26, 10p16, 4q15,16, 10q14,16 and 12p12 respectively. Taking into account the reported QTL mapping results, gene expression analysis and radiation hybrid mapping results, these results suggest that five genes (CARM1, JARID1A, JMJD2A, SMYD1 and SMYD2) could be good candidate genes for growth and backfat thickness traits. [source] Molecular characterization, immunohistochemical localization and expression of a ribosomal protein L17 gene from Apis cerana ceranaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Fei Meng Abstract Ribosomal protein L17 (RPL17) is a core protein of the large (60S) ribosomal subunit and plays an important role in protein synthesis. In this report, a RPL17 gene was isolated from Apis cerana cerana, designated as AccRPL17. Alignment analysis showed that AccRPL17 exhibits high homology to other known RPL17s. Moreover, genomic sequence analysis revealed that five exons are splitted by four introns, and the position of the first intron is comparatively conservative, being localized in the 5, untranslated region. Partial putative cis -acting elements related to development were also examined. Quantitative real-time PCR showed that the highest mRNA level was detected in larvae on the fifth day. Simultaneously, immunohistochemical localization showed that AccRPL17 is primarily concentrated in muscular tissues, stigma, body wall, and the surrounding of the eye in the fifth-instar larvae. Further studies suggested that AccRPL17 might be involved in responses to abiotic stresses. This is a report attempting to analyze the expression and distribution of RPL17 in A. cerana cerana. These results indicated that AccRPL17 might play an important role in insect development, and the importance of AccRPL17 in participating in abiotic stresses is discussed. © 2010 Wiley Periodicals, Inc. [source] Hsp90 mediates insulin-like growth factor 1 and interleukin-1, signaling in an age-dependent manner in equine articular chondrocytesARTHRITIS & RHEUMATISM, Issue 7 2007Amber K. Boehm Objective Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1, (IL-1,) signaling in chondrocytes. Methods Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. The Hsp90 inhibitor geldanamycin (50 nM, 100 nM, or 500 nM) was used to assess age-related responses to Hsp90 with concurrent IGF-1 or IL-1, stimulation of chondrocytes. Quantitative real-time PCR was used to measure COL2A1 and matrix metalloproteinase 13 (MMP13) gene expression; Western blot analysis was performed to determine the phosphorylation status of p42/44 and Akt/protein kinase B. Results The effects of Hsp90 inhibition with geldanamycin were concentration dependent. Inhibition of Hsp90 with 100 nM or 500 nM geldanamycin blocked IGF-1,induced cell proliferation, Akt and p42/44 activation, and COL2A1 expression. Basal and IL-1,,induced up-regulation of MMP13 mRNA was blocked by all concentrations of geldanamycin tested. Gain-of-function assays with Hsp90 resulted in increased expression of MMP13 mRNA. Conclusion These results suggest that Hsp90 is involved in opposing signaling pathways of cartilage homeostasis, and that catabolic responses are more sensitive to Hsp90 inhibition than are anabolic responses. Further studies are needed to determine the role of Hsp90 inhibition in osteoarthritis in order to assess its potential as a therapeutic target. [source] Developmental microRNA expression profiling of murine embryonic orofacial tissueBIRTH DEFECTS RESEARCH, Issue 7 2010Partha Mukhopadhyay Abstract BACKGROUND: Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs involved in gene silencing, represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development. METHODS: To identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore microarrays from Miltenyi Biotech. Quantitative real-time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis. RESULTS: Expression of over 26% of the 588 murine miRNA genes examined was detected in murine orofacial tissues from GD-12,GD-14. Among these expressed genes, several clusters were seen to be developmentally regulated. Differential expression of miRNAs within such clusters wereshown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development. CONCLUSIONS: Using miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs. Birth Defects Research (Part A), 2010. © 2010 Wiley-Liss, Inc. [source] Effect of high-dose methylprednisolone treatment on Th17 cells in patients with multiple sclerosis in relapseACTA NEUROLOGICA SCANDINAVICA, Issue 4 2009M. Liu Objectives,,, Growing evidences have suggested that Th17 cells are involved in the pathogenic mechanisms of multiple sclerosis (MS). Treatment with high-dose intravenous methylprednisolone (IVMP) has beneficial effects on functional recovery in patients with MS during relapse. The present study was designed to analyze the influences of IVMP on Th17 cells in patients with MS after a 5-day high-dose IVMP treatment. Materials and methods,,, Th17 cell count and the production of IL-17 in peripheral blood mononuclear cells (PBMCs) were measured using flow cytometry and ELISA respectively. Quantitative real-time PCR was performed to analyze the mRNA expression of Th17 cell-related factors (IL-17, RORc and IL-23R) in PBMCs. Results,,, A significant reduction in IL-17 production and Th17 cells count in PBMCs was found in patients with MS after IVMP treatment. Moreover, the expression of IL-17, IL-23R and RORc mRNA decreased significantly after IVMP treatment. Conclusions,,, Treatment with methylprednisolone has a suppressive effect on Th17 cells and may be related to its clinical efficiency. [source] Expression and structure of interleukin 4 receptors in primary meningeal tumorsCANCER, Issue 10 2005Sachin Puri M.Sc. Abstract BACKGROUND It was reported previously that malignant human tumors, like glioma and medulloblastoma, express high-density interleukin (IL-4) receptor mRNA and protein. Because IL-4 receptors (R) are sensitive targets for targeted therapeutics, knowledge of the expression of these receptors in other central nervous system tumors is of great interest. In this study, the authors examined the expression and subunit composition of IL-4R complex in primary human meningiomas. METHODS Reverse transcription-polymerase chain reaction (RT-PCR) analysis for IL-13R,1, IL-4R, and IL-2R,c was performed on total RNA extracted from 35 meningiomas and a normal human brain tissue sample. Results were confirmed in nine randomly selected tumors by quantitative real-time PCR and in situ immunofluorescence assay. RESULTS Transcripts for the IL-4R, and IL-13R,1 chains were overexpressed in meningiomas compared with normal brain tissue. The levels of IL-4R, mRNA appeared to be higher compared with the levels of IL-13R,1 mRNA. The results also showed that tumors with higher disease grade tended to have increased mRNA expression for the IL-4R, chain. This IL-4R, mRNA overexpression appeared to be more frequent in younger patients (age < 37 years). The transcripts for IL-2R,c chain were not detected in any of the tumor samples or in normal brain tissue. Quantitative real-time PCR confirmed the results of the RT-PCR analysis. Meningiomas also demonstrated a bright immunofluorescent staining for the IL-4R, and IL-13R,1 chains but no staining for IL-2R,c. CONCLUSIONS Expression of the IL-4R, and IL-13R,1 chains and absence of IL-2,c expression established that meningiomas expressed type II IL-4Rs. These receptors may serve as a target for cytotoxin/immunotoxin therapy in patients with meningioma who are not amenable to surgical resection or for recurrent tumors. Cancer 2005. © 2005 American Cancer Society. [source] Molecular determinants of irinotecan efficacyINTERNATIONAL JOURNAL OF CANCER, Issue 10 2006Daniel Vallböhmer Abstract Molecular markers predicting the efficacy of CPT-11 based chemotherapies in patients with colorectal cancer (CRC) are unknown. Therefore, we investigated whether mRNA levels of drug targets (Topoisomerase I, TS), enzymes involved in 5-FU metabolism (DPD), in angiogenesis (EGFR, IL-8, VEGF) and in DNA-repair/drug detoxification (ERCC1, GST-P1) are associated with the clinical outcome of patients with CRC treated with first-line CPT-11 based chemotherapy. Thirty three patients with metastatic CRC were included in the study. Intratumoral gene expression levels were assessed from paraffin-embedded tissue samples, using laser capture microdissection and quantitative Real-Time PCR. Complete response was observed in 1 patient, partial response in 12 patients, stable disease in 13 patients and progressive disease in 6 patients. Response was inevaluable for 1 patient. Patients with complete response or partial response were classified as responders, while patients with stable disease or progressive disease were classified as nonresponders. High intratumoral mRNA levels of EGFR, ERCC1 and GSPT-P1 were each significantly associated with response to CPT-11 based chemotherapy. Recursive partitioning analysis showed that mRNA levels of EGFR and ERCC1 are primarily responsible for delineating responders from nonresponders. Also, the combination of high intratumoral gene expression levels of both EGFR and ERCC1 was significantly associated with progression-free survival. The mRNA levels of EGFR had a significant correlation with expression levels of ERCC1, GST-P1 and VEGF. This small retrospective study suggests that gene expression levels of EGFR, ERCC1 and GST-P1 may be useful in predicting the clinical outcome of patients with metastatic CRC treated with first-line CPT-11 based chemotherapy. © 2006 Wiley-Liss, Inc. [source] The putative-farnesoic acid O -methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre-imaginal life expressionARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Laura Vannini Abstract Farnesoic acid O -methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative- FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real-Time PCR in medfly pre-imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre-imaginal putative- FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well-known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control. © 2010 Wiley Periodicals, Inc. [source] Evaluation of a Non-Targeted "Omic" Approach in the Safety Assessment of Genetically Modified PlantsPLANT BIOLOGY, Issue 5 2006S. B. Metzdorff Abstract: Genetically modified plants must be approved before release in the European Union, and the approval is generally based upon a comparison of various characteristics between the transgenic plant and a conventional counterpart. As a case study, focusing on safety assessment of genetically modified plants, we here report the development and characterisation of six independently transformed Arabidopsis thaliana lines modified in the flavonoid biosynthesis. Analyses of integration events and comparative analysis for characterisation of the intended effects were performed by PCR, quantitative Real-time PCR, and High Performance Liquid Chromatography. Analysis by cDNA microarray was used as a non-targeted approach for the identification of potential unintended effects caused by the transformation. The results revealed that, although the transgenic lines possessed different types of integration events, no unintended effects were identified. However, we found that the majority of genes showing differential expression were identified as stress-related genes and that environmental conditions had a large impact on the expression of several genes, proteins, and metabolites. We suggest that the microarray approach has the potential to become a useful tool for screening of unintended effects, but state that it is crucial to have substantial information on the natural variation in traditional crops in order to be able to interpret "omics" data correctly within the framework of food safety assessment strategies of novel plant varieties, including genetically modified plant varieties. [source] Effects of short-term food deprivation on orexin-A-induced intestinal bicarbonate secretion in comparison with related secretagoguesACTA PHYSIOLOGICA, Issue 3 2010G. Flemström Abstract Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E2 (PGE2) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE2). Enterocyte intracellular calcium ([Ca2+]i) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE2. Orexin-A induces [Ca2+]i signalling in enterocytes from fed rats but no significant [Ca2+]i responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca2+]i signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status. [source] Quantitative microbial faecal source tracking with sampling guided by hydrological catchment dynamicsENVIRONMENTAL MICROBIOLOGY, Issue 10 2008G. H. Reischer Summary The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design. [source] Hexavalent uranium supports growth of Anaeromyxobacter dehalogenans and Geobacter spp. with lower than predicted biomass yieldsENVIRONMENTAL MICROBIOLOGY, Issue 11 2007Robert A. Sanford Summary The stimulation of bacteria capable of reducing soluble U(VI) to sparingly soluble U(IV) is a promising approach for containing U(VI) plumes. Anaeromyxobacter dehalogenans is capable of mediating this activity; however, its ability to couple U(VI) reduction to growth has not been established. Monitoring the increase in 16S rRNA gene copy numbers using quantitative real-time PCR (qPCR) in cultures provided with U(VI) as an electron acceptor demonstrated growth, and 7.7,8.6 × 106 cells were produced per ,mole of U(VI) reduced. This biomass yield was lower than predicted based on the theoretical free energy changes associated with U(VI)-to-U(IV) reduction. Lower than predicted growth yields with U(VI) as electron acceptor were also determined in cultures of Geobacter lovleyi and Geobacter sulfurreducens suggesting that U(VI) reduction is inefficient or imposes an additional cost to growing cells. These findings have implications for U(VI) bioremediation because Anaeromyxobacter spp. and Geobacter spp. contribute to radionuclide immobilization in contaminated subsurface environments. [source] Isolation and gene quantification of heterotrophic N2 -fixing bacterioplankton in the Baltic SeaENVIRONMENTAL MICROBIOLOGY, Issue 1 2007Kjärstin H. Boström Summary Cyanobacteria are regarded as the main N2 -fixing organisms in marine waters. However, recent clone libraries from various oceans show a wide distribution of the dinitrogenase reductase gene (nifH) originating from heterotrophic bacterioplankton. We isolated heterotrophic N2 -fixing bacteria from Baltic Sea bacterioplankton using low-nitrogen plates and semi-solid diazotroph medium (SSDM) tubes. Isolates were analysed for the nitrogenase (nifH) gene and active N2 fixation by nested polymerase chain reaction (PCR) and acetylene reduction respectively. A primer-probe set targeting the nifH gene from a , - proteobacterial isolate, 97% 16S rDNA similarity to Pseudomonas stutzeri, was designed for measuring in situ dynamics using quantitative real-time PCR. This nifH gene sequence was detected at two of 11 stations in a Baltic Proper transect at abundances of 3 × 104 and 0.8 × 103 copies per litre seawater respectively. Oxygen requirements of isolates were examined by cultivation in SSDM tubes where oxygen gradients were determined with microelectrodes. Growth, and thereby N2 fixation, was observed as horizontal bands formed at oxygen levels of 0,6% air saturation. The apparent microaerophilic or facultative anaerobic nature of the isolates explains why the SSDM approach is the most appropriate isolation method. Our study illustrates how combined isolation, functional analyses and in situ quantification yielded insights into the oxygen requirements of heterotrophic N2 -fixing bacterioplankton isolates, which were confirmed to be present in situ. [source] Isolation and characterization of Tn -Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1ENVIRONMENTAL MICROBIOLOGY, Issue 1 2005Julien Maillard Summary A new 9.9 kb catabolic transposon, Tn -Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn -Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus. [source] Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid diseaseEQUINE VETERINARY JOURNAL, Issue 4 2010R. HARALAMBUS Summary Reasons for performing study: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. Hypothesis: Given the pathogenic role of BPV-1 and BPV-2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. Methods: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid-bearing horses and one donkey. Viral load was estimated via quantitative real-time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV-1/-2 genome for one randomly selected lesion per horse and correlated with disease severity. Results: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0,556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild-type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. Conclusions: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. Potential relevance: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy. [source] Carrier frequency of SMA by quantitative analysis of the SMN1 deletion in the Iranian populationEUROPEAN JOURNAL OF NEUROLOGY, Issue 1 2010M. Hasanzad Background and purpose:, Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Carrier frequency studies of SMA have been reported for various populations. Although no large-scale population-based studies of SMA have been performed in Iran, previous estimates have indicated that the incidence of autosomal recessive disorder partly because of the high prevalence of consanguineous marriage is much higher in the Iranian population than in other populations. Methods:, In this study, we used a reliable and highly sensitive quantitative real-time PCR assay with SYBR green I dye to detect the copy number of the SMN1 gene to determine the carrier frequency of SMA in 200 healthy unrelated, non-consanguineous couples from different part of Iran. Results:, To validate the method in our samples, we determined the relative quantification (RQ) of patients with homozygous deletion (0.00) and hemyzygous carriers (0.29,0.55). The RQ in 10 of 200 normal individuals were within the carrier range of 0.31,0.57, estimating a carrier frequency of 5% in the Iranian population. Conclusions:, Our data show that the SMA carrier frequency in Iran is higher than in the European population and that further programs of population carrier detection and prenatal testing should be implemented. [source] Host plant development, water level and water parameters shape Phragmites australis -associated oomycete communities and determine reed pathogen dynamics in a large lakeFEMS MICROBIOLOGY ECOLOGY, Issue 2 2009Anna Wielgoss Abstract In a 3-year-study, we analysed the population dynamics of the reed pathogen Pythium phragmitis and other reed-associated oomycetes colonizing fresh and dried reed leaves in the littoral zone of a large lake. Oomycete communities derived from internal transcribed spacer clone libraries were clearly differentiated according to substrate and seasonal influences. In fresh leaves, diverse communities consisting of P. phragmitis and other reed-associated pathogens were generally dominant. Pythium phragmitis populations peaked in spring with the emergence of young reed shoots, and in autumn after extreme flooding events. In summer it decreased with falling water levels, changing water chemistry and rising temperatures. Another Pythium species was also highly abundant in fresh leaves throughout the year and might represent a new, as-yet uncultured reed pathogen. In dried leaves, reed pathogens were rarely detected, whereas saprophytic species occurred abundantly during all seasons. Saprophyte communities were less diverse, less temperature sensitive and independent of reed development. In general, our results provide evidence for the occurrence of highly specialized sets of reed-associated oomycetes in a natural reed ecosystem. Quantitative analyses (clone abundances and quantitative real-time PCR) revealed that the reed pathogen P. phragmitis is particularly affected by changing water levels, water chemistry and the stage of reed development. [source] Monitoring bacterial and archaeal community shifts in a mesophilic anaerobic batch reactor treating a high-strength organic wastewaterFEMS MICROBIOLOGY ECOLOGY, Issue 3 2008Changsoo Lee Abstract Shifts in bacterial and archaeal communities, associated with changes in chemical profiles, were investigated in an anaerobic batch reactor treating dairy-processing wastewater prepared with whey permeate powder. The dynamics of bacterial and archaeal populations were monitored by quantitative real-time PCR and showed good agreement with the process data. A rapid increase in bacterial populations and a high rate of substrate fermentation were observed during the initial period. Growth and regrowth of archaeal populations occurred with biphasic production of methane, corresponding to the diauxic consumption of acetate and propionate. Bacterial community structure was examined by denaturing gel gradient electrophoresis (DGGE) targeting 16S rRNA genes. An Aeromonas -like organism was suggested to be mainly responsible for the rapid fermentation of carbohydrate during the initial period. Several band sequences closely related to the Clostridium species, capable of carbohydrate fermentation, lactate or ethanol fermentation, and/or homoacetogenesis, were also detected. Statistical analyses of the DGGE profiles showed that the bacterial community structure, as well as the process performance, varied with the incubation time. Our results demonstrated that the bacterial community shifted, reflecting the performance changes and, particularly, that a significant community shift corresponded to a considerable process event. This suggested that the diagnosis of an anaerobic digestion process could be possible by monitoring bacterial community shifts. [source] Transcription factor NF-Y is involved in regulation of the JNK pathway during Drosophila thorax developmentGENES TO CELLS, Issue 2 2008Yasuhide Yoshioka The CCAAT motif-binding factor, nuclear factor Y (NF-Y) consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in the notum compartment of wing discs by a pannir -GAL4 and UAS- dNF-YAIR mainly resulted in a thorax disclosed phenotype. Reduction of the Drosophila c-Jun N-terminal kinase (JNK) basket (bsk) gene dose enhanced the knockdown of dNF-YA-induced phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc) -LacZ enhancer trap line revealed reduction in the level of the JNK reporter, puc-LacZ signals, in dNF-YA RNAi clones. In addition, expression of wild-type Bsk effectively suppressed the phenotype induced by knockdown of dNF-YA. The bsk gene promoter contains a CCAAT motif and this motif plays a positive role in the promoter activity. We performed chromatin immunoprecipitation (ChIP) assays in S2 cells with anti-dNF-YA IgG and quantitative real-time PCR. The bsk gene promoter region containing the CCAAT boxes was effectively amplified in the immunoprecipitates by PCR. However, this region was not amplified in the immunoprecipitates from dNF-YA knockdown cells. Furthermore, the level of endogenous bsk mRNA is reduced in the dNF-YA knockdown larvae. These results suggest that dNF-Y is necessary for proper bsk expression and activity of JNK pathway during thorax development. [source] | ||||||||||||||||||||||||||||||||||