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Quantitative RT (quantitative + rt)
Selected AbstractsQuantification of the expression and inducibility of 12 rat cytochrome P450 isoforms by quantitative RT,PCRJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Etienne Caron Abstract The administration of xenobiotics may significantly alter the expression of cytochromes P450 (CYPs), thereby leading to potentially toxic cellular, physiologic, and pharmacologic responses. Indeed, an important task in the development of new therapeutic entities is to evaluate efficiently and quantitatively their potential effects on the expression level of different CYPs. In this report, reverse transcriptase polymerase chain reaction (RT,PCR) was used to measure basal and induced mRNA of a wide range of rat CYP isoforms. Rats (n = 3 per treatment) were treated with five prototype inducers of CYP isoforms or with vehicle only. RT and PCR efficiencies were determined using appropriate RNA and DNA standards. Messenger RNA was quantified by PicoGreen standard curves and normalized to cyclophilin. Quantitative RT,PCR was used successfully to demonstrate that CYP isoforms were induced at the mRNA level following drug administration. Notably, phenobarbital resulted in significant induction of CYP2B1, CYP2B2, CYP2C6, CYP2C13, CYP2E1, CYP3A1, and CYP3A2. 3-Methylcholanthrene induced CYP1A1, CYP1A2, and CYP1B1. CYP2C11 expression was highly variable and suppressed by pyridine, whereas the expression of CYP2E1 was suppressed by dexamethasone. We demonstrated that quantitative RT,PCR can be used to evaluate efficiently the effect of compounds on the expression of a wide range of CYP isoforms. The technique is advantageous over others in that it is very sensitive, efficient and applicable to highly homologous CYP isoforms. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:368,378, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20103 [source] Role of transcription factors Ad4bp/SF-1 and DAX-1 in steroidogenesis and spermatogenesis in human testicular development and idiopathic azoospermiaINTERNATIONAL JOURNAL OF UROLOGY, Issue 6 2006YOSHIYUKI KOJIMA Background:, Ad4bp/SF-1 and DAX-1 are orphan members of the nuclear hormone receptor superfamily of transcription factors. In order to obtain better understandings of human testicular steroidogenesis and spermatogenesis, we examined the expression levels of both factors in human normal and idiopathic azoospermic testes and investigated their physical meaning. Methods:, First, we examined the expression level of Ad4bp/SF-1 and DAX-1 by quantitative reverse transcription,polymerase chain reaction (RT,PCR), immunohistochemistry and western blotting analysis using eight normal human testicular tissues from infants to adults. Second, we performed quantitative RT,PCR using testicular biopsy samples obtained from 22 idiopathic azoospermic patients to examine the expression of Ad4bp/SF-1 and DAX-1, and analysed the correlation between the expression levels of both factors and the serum hormone levels or histological evaluation to study their potential correlation with steroidogenesis and spermatogenesis on idiopathic azoospermia. Results:, The expression levels of both factors in the normal testes increased with testicular development. Ad4bp/SF-1 was abundantly expressed in Leydig cell, whereas DAX-1 was expressed in Sertoli cells. The expression level of Ad4bp/SF-1 in idiopathic azoospermic patients testes positively correlated with serum testosterone (P < 0.05). The average expression levels of DAX-1 mRNA for patients with maturation arrest (0.39 ± 0.19) and Sertoli cell-only syndrome (0.13 ± 0.08) were lower than that with hypospermatogenesis (1.60 ± 1.32) and normal spermatogenesis (1.30 ± 1.41). Conclusion:, Ad4bp/SF-1 is important for the maintenance of steroidogenesis in the human testis. DAX-1 plays a critical role in spermatogenesis in the human testis, and Sertoli cell-only syndrome and maturation arrest may result from abnormal Sertoli cell function that disrupts the normal progression of spermatogenesis. [source] Quantification of the expression and inducibility of 12 rat cytochrome P450 isoforms by quantitative RT,PCRJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Etienne Caron Abstract The administration of xenobiotics may significantly alter the expression of cytochromes P450 (CYPs), thereby leading to potentially toxic cellular, physiologic, and pharmacologic responses. Indeed, an important task in the development of new therapeutic entities is to evaluate efficiently and quantitatively their potential effects on the expression level of different CYPs. In this report, reverse transcriptase polymerase chain reaction (RT,PCR) was used to measure basal and induced mRNA of a wide range of rat CYP isoforms. Rats (n = 3 per treatment) were treated with five prototype inducers of CYP isoforms or with vehicle only. RT and PCR efficiencies were determined using appropriate RNA and DNA standards. Messenger RNA was quantified by PicoGreen standard curves and normalized to cyclophilin. Quantitative RT,PCR was used successfully to demonstrate that CYP isoforms were induced at the mRNA level following drug administration. Notably, phenobarbital resulted in significant induction of CYP2B1, CYP2B2, CYP2C6, CYP2C13, CYP2E1, CYP3A1, and CYP3A2. 3-Methylcholanthrene induced CYP1A1, CYP1A2, and CYP1B1. CYP2C11 expression was highly variable and suppressed by pyridine, whereas the expression of CYP2E1 was suppressed by dexamethasone. We demonstrated that quantitative RT,PCR can be used to evaluate efficiently the effect of compounds on the expression of a wide range of CYP isoforms. The technique is advantageous over others in that it is very sensitive, efficient and applicable to highly homologous CYP isoforms. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:368,378, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20103 [source] Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous systemJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Steve Poirier Abstract Neural apoptosis-regulated convertase-1/proprotein convertase subtilisin-kexin like-9 (NARC-1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low-density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT,PCR analyses were performed following retinoic acid (RA)-induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC-1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element-binding protein-2, which normally up-regulates NARC-1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC-1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole-mount in situ hybridization revealed that the pattern of expression of zebrafish NARC-1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC-1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain,midbrain boundaries, leading to embryonic death at ,,96 h after fertilization. These data support a novel role for NARC-1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver. [source] Gene amplification of the transcription factor DP1 and CTNND1 in human lung cancer,THE JOURNAL OF PATHOLOGY, Issue 1 2010Sandra D. Castillo Abstract The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT,PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Frizzled-1 is down-regulated in follicular thyroid tumours and modulates growth and invasiveness,THE JOURNAL OF PATHOLOGY, Issue 1 2008A Ulivieri Abstract The mechanisms of follicular thyroid carcinoma (FTC) transformation and progression are not well understood. Previously, we detected LOH at 7q21 in all FTCs examined, indicating that loss of genetic material in that region is a common trait in these lesions. To analyse the effects of LOH on gene expression, we performed an analysis of the mRNA expression levels of six different genes, located at 7q21.1,7q21.3. A total of 23 lesions, including eight follicular hyperplasias (FHs), eight follicular adenomas (FAs), two FTCs and five papillary thyroid carcinomas (PTCs) were analysed. The Frizzled-1 (FZD-1) gene, located at 7q21.13, showed the lowest levels of mRNA expression. Down-regulation of FZD-1 expression was also confirmed in an independent series of 69 follicular neoplastic lesions compared to 25 PTCs, analysed by quantitative RT,PCR. In vitro studies showed that FZD-1 expression was also markedly reduced at both protein and mRNA levels in three FTC-derived cell lines (FRO, WRO and FTC-133), while it was normal in the three PTC-derived cell lines (Ca300, Ca301 and K1) examined. We demonstrated that over-expression of FZD-1 in 3 FTC-derived cells decreased invasiveness and proliferation rate, indicating a possible pathogenetic role. In addition, FZD-1 RNA interference in the PTC-derived cell line K1 increased invasiveness. Our data indicated that FZD-1 is involved in growth of follicular tumours and may be considered as a novel marker of this type of tumour. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] | ||||||||||