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Semiquantitative Reverse Transcription Polymerase Chain Reaction (semiquantitative + reverse_transcription_polymerase_chain_reaction)
Selected AbstractsDifferent expression of P14ARF defines two groups of breast carcinomas in terms of TP73 expression and TP53 mutational statusGENES, CHROMOSOMES AND CANCER, Issue 2 2001Gemma Domínguez In 95 breast carcinomas, we investigated P14ARF and TP73 mRNA expression and their relationship to TP53 mutations, determined by an immunohistochemical method, studying several clinicopathologic features of the tumors. P14ARF and TP73 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), using ,-actin as a control. P14ARF was overexpressed in 19% of the cases and underexpressed in 24%. TP73 was overexpressed in 22% of the tumors, and normal levels were found in the remaining 78%. The analysis of TP53 showed positive immunostaining in 38% of cases. The association of P14ARF and TP73 overexpression was statistically significant, as was the association between positive TP53 staining and TP73 overexpression. P14ARF was related to TP53 only in those cases in which there was low expression of P14ARF. Concomitant overexpression of P14ARF and TP73 was statistically related to positive TP53 immunostaining. The analysis of concomitant P14ARF and TP73 overexpression and clinicopathologic parameters of the tumors showed a statistically significant difference with respect to peritumoral vessel invasion (P = 0.01), lymph node metastasis (P = 0.03), negative ERBB2 expression (P = 0.005), and more advanced pathologic stages (P = 0.03). These results suggest that overexpression of P14ARF and TP73 could be implicated in breast carcinoma tumorigenesis and, ultimately, in the phenotypic features of these lesions. © 2001 Wiley-Liss, Inc. [source] Cloning and transcriptional expression of a leucokinin-like peptide receptor from the Southern cattle tick, Boophilus microplus (Acari: Ixodidae)INSECT MOLECULAR BIOLOGY, Issue 5 2000S. P. Holmes Abstract Leucokinins are invertebrate neuropeptides that exhibit myotropic and diuretic activity. Only one leucokinin-like peptide receptor is known, the lymnokinin receptor from the mollusc Lymnaeastagnalis. A cDNA encoding a leucokinin-like peptide receptor was cloned from the Southern cattle tick, Boophilus microplus, a pest of cattle world-wide. This is the first neuropeptide receptor known from the Acari and the second known in the subfamily of leucokinin-like peptide G-protein-coupled receptors. The deduced amino acid sequence exhibits 40% identity to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative reverse transcription polymerase chain reaction. We also propose that the sequence AAF50775.1 from the Drosophila melanogaster genome (CG10626) encodes the first identified insect leucokinin receptor. [source] TLR4 monoclonal antibody blockade suppresses dextran-sulfate-sodium-induced colitis in miceJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 1 2010Yi Liu Abstract Background and Aim:, Ulcerative colitis (UC) refers to a kind of inflammatory bowel disease, of which the accurate pathogenesis is not yet well understood. Recently, the toll-like receptor 4 (TLR4) and the TLR4 signaling pathway have been proved as playing an important role in the pathogenesis of UC. The objective of this study was to evaluate the effect of TLR4 monoclonal antibody on dextran-sulfate-sodium-induced colitis in a mouse model. Methods:, We evaluated the effects of the TLR4 monoclonal antibody (TLR4mAb) on the development of dextran-sulfate-sodium-(DSS)-induced colitis. Tissue samples were evaluated by the disease activity index and histopathological score. Meanwhile, the mucosal mRNA expression of cytokines, tumor necrosis factor-,, interferon-, and interleukin-1, were analyzed by semiquantitative reverse transcription polymerase chain reaction. The mucosal protein P38-MAPK, c-jun and c-fos expressions of the TLR4-P38MAPK pathway were analyzed using Western blot. Results:, After the treatment with TLR4mAb against DSS-induced colitis, the bodyweight was significantly increased and both disease activity index and histopathological score were decreased significantly. Furthermore, the mucosal expression of messenger RNA of tumor necrosis factor-,, interferon-, and interleukin-1, were observed to be 8,15-fold more than the baseline, whereas the mucosal expressions of P38MAPK and c-jun were found to be decreased. Conclusion:, Blocking TLR4 by TLR4mAb can prevent the development of DSS-induced colitis through the TLR4-P38MAPK-c-jun pathway. [source] Profiling of differentially expressed genes induced by high linear energy transfer radiation in breast epithelial cellsMOLECULAR CARCINOGENESIS, Issue 4 2001Debasish Roy Abstract Methods to define patterns of gene expression have applications in a wide range of biological systems. Several molecular biological techniques are used to study expression patterns during the neoplastic progression of breast epithelial cells. In the present study, differential expression of human oncogenes/tumor suppressor genes in human breast epithelial cell lines irradiated with low doses of high linear energy transfer radiation and treated with estrogen was assessed with cDNA expression arrays. Transformed and tumorigenic cell lines were compared with the control cell line to identify differentially expressed genes during tumorigenic progression. Autoradiographic analysis showed that of the 190 genes analyzed, 49 genes showed a high level of altered expression, and 12 genes had minor differences in expression levels. Among these 49 genes, 17 genes were altered at all stages of transformation, 21 were altered only at the early stage, and the remaining 11 were at the late stage of transformation to the tumorigenic stage of progression. Among the 11 late stage,associated genes, seven genes were altered exclusively in the tumorigenic cell lines and in Tumor-T. Of the 17 all-stage genes, six were randomly selected, and we confirmed their altered expression by gene-specific semiquantitative reverse transcription polymerase chain reaction, followed by Northern blot analysis. The results showed that the mRNA expression patterns of all these genes were consistent with the expression pattern seen on the array. Among these six genes, five genes, including c- myc, puf, MNDA, c- yes, and Fra-1 showed upregulation, and the other gene, RBA/p48, showed downregulation in the transformed and tumorigenic cell lines compared with the control MCF-10F cell line. Investigation of these genes should help establish the molecular mechanisms of progression that are altered by radiation and estrogen treatment. A number of candidates reported here should be useful as biomarkers involved in breast carcinogenesis. © 2001 Wiley-Liss, Inc. [source] Endothelin-1 increases cholinergic nerve-mediated contraction of human bronchi via tachykinin synthesis inductionBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2001Bruno D'Agostino In some asthmatics, muscarinic receptor antagonists are effective in limiting bronchoconstrictor response, suggesting an abnormal cholinergic drive in these subjects. There is a growing body of evidences indicating that cholinergic neurotransmission is also enhanced by endothelin-1 (ET-1) in rabbit bronchi, mouse trachea and in human isolated airway preparations. We investigated the role of secondary mediators in ET-1 induced potentiation of cholinergic nerve-mediated contraction in human bronchi, in particular the possible role of neuropeptides in this phenomenon. Bronchial tissues after endothelin treatment were exposed to a standard electrical field stimulation (EFS) (30% of EFS 30Hz)-induced contraction. In addition, in some experiments, preparations were treated with a tachykinin NK2 receptor antagonist and subsequently exposed to the same protocol. HPLC and RIA were performed on organ bath fluid samples. Moreover, the human bronchi were used for the ,-PPT (preprotachykinin) mRNA extraction and semiquantitative reverse transcription polymerase chain reaction (RT , PCR), prior to and 30 , 40 min following ET-1 challenge. The selective tachykinin NK2 receptor antagonist, SR48968, was effective to reduce ET-1 potentiation of EFS mediated contraction. HPLC or RIA showed significant increased quantities of NKA in organ bath effluents after EFS stimulation in bronchi pretreated with ET-1. Finally, ,-PPT mRNA level after stimulation of bronchi with ET-1 was increased about 2 fold respect to control untreated bronchi. In conclusion, this study demonstrated that, at least in part, the ET-1 potentiation of cholinergic nerve-mediated contraction is mediated by tachykinin release, suggesting that in addition to nerves, several type of cells, such as airway smooth muscle cell, may participate to neuropeptide production. British Journal of Pharmacology (2001) 134, 1447,1454; doi:10.1038/sj.bjp.0704395 [source] | ||||||||||