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RT-PCR Technique (rt-pcr + technique)
Selected AbstractsTumoral and tissue-specific expression of the major human ,-tubulin isotypes,CYTOSKELETON, Issue 4 2010Luis J. Leandro-García Abstract The ,-tubulins are microtubule components encoded by a multigene family, which produces slightly different proteins with complex expression patterns. Several widely used anticancer drugs base their activity on ,-tubulin binding, microtubule dynamics alteration, and cell division blockage. The expression of these drug targets in tumoral and normal cells could be of crucial importance for therapy outcome, unfortunately, the complex ,-tubulin expression patterns have been poorly characterized in human. In this study, we developed a quantitative RT-PCR technique that accurately determines the mRNA expression of the eight human ,-tubulin isotypes, encoding class I, IIa, IIb, III, IVa, IVb, V, and VI and applied it to 21 nontumoral tissues and 79 tumor samples belonging to seven cancer types. In the nontumoral tissues, we found that, overall, TUBB (I), TUBB2C (IVb), and TUBB6 (V) were ubiquitous, TUBB1(VI) was hematopoietic cell-specific, and TUBB2A (IIa), TUBB2B (IIb), TUBB3 (III), and TUBB4 (IVa) had high expression in brain; however, the contribution of the different isotypes to the total ,-tubulin content varied for each tissue and had a complex pattern. In tumoral tissues, most isotypes exhibited an altered expression in specific tumor types or related to tumoral characteristics. In general, TUBB3 showed a great increase in expression while TUBB6 expression was largely decreased in most tumors. Thus, normal tissues showed a complex ,-tubulin isotype distribution, which could contribute to the toxicity profile of the microtubule-binding drugs. In addition, the specific isotypes significantly altered in tumors might represent markers for drug response. © 2010 Wiley-Liss, Inc. [source] IL-15 and IL-16 overexpression in cutaneous T-cell lymphomas: stage-dependent increase in mycosis fungoides progressionEXPERIMENTAL DERMATOLOGY, Issue 4 2000K. Asadullah Abstract: Cytokines are of major importance for the pathogenesis of cutaneous T-cell lymphomas (CTCL). Recent data suggested that IL-15 and IL-16 are survival/growth factors for the malignant T cells in these entities. To investigate the expression of IL-15 and IL-16 in mycosis fungoides (MF) and CD30+ pleomorphic T-cell lymphoma in vivo, we established a competitive RT-PCR technique. Analyzing skin biopsies from CTCL patients at different stages in comparison to psoriatic and healthy skin, we found IL-15 and IL-16 mRNA overexpression in both CTCL entities. Remarkably, there was some evidence for a stage-dependent increase during MF progression. We found only slight overexpression in early stage MF, when only few tumor cells are detectable within the infiltrates, whereas marked overexpression was found in more advanced lesions, which are characterized by a higher density of malignant cells. These results suggested that CTCL cells themselves might produce the cytokines. To further elucidate this hypothesis, two CTCL cell lines were analyzed but gave conflicting results. Therefore, the cellular origin of the IL-15 and IL-16 overexpression in CTCL remains unclear. Considering the significant overexpression of IL-15 and IL-16 and their biological capacities it is likely that these cytokines contribute to the tumor development. So, they might be involved in growth and skin homing of CTCL cells. [source] Reduction of mother-to-child transmission of HIV at Saint Camille Medical Centre in Burkina FasoJOURNAL OF MEDICAL VIROLOGY, Issue 2 2006J. Simpore Abstract One thousand three hundred and twenty-eight pregnant women with less than 32 weeks of amenorrhea received voluntary counseling and testing at Saint Camille Medical Center from May 1, 2002 to December 30, 2004. Following informed consent and pre-test counseling, HIV screening was performed in 1,202 women. According to the prevention protocol, HIV-positive women received a single dose of Nevirapine (200 mg) during their labor, while their newborn received a single dose of Nevirapine (2 mg/kg) within 72 hr from birth. HIV seroprevalence (11.2%) was higher than in the overall population. One hundred and ninty-three children were born at the end of December 2004; 53 children (27.5%) followed a short breastfeeding protocol for 4 months, while 140 (72.5%) were fed artificially. All the children underwent RT-PCR test for HIV 5,6 months after their birth: 173 (89.6%) were HIV negative whilst 20 children (10.4%) were HIV positive. Out of the 20 positive children 5/53 (9.4%) had received breast milk for 4 months, while the remaining 15/140 (10.7%) had been fed artificially (P,=,0.814). Artificially fed babies (3/140 (2.1%)) and 1/53 (1.9%) of those breast fed for 4 months deceased according to mortality rate of HIV-positive children. This shows that there is no statistically significant difference (P,=,0.648) between the mortality of artificially fed (3/140 or 2.1%) and breast-fed (1/53 or 1.9%) children. Artificially fed children (20/140 (14.3%)) and 5/53 (9.4%) of breast-fed children died within 6,10 months. This figure indicates that there is no significant difference between the mortality rate of artificially and that of breast-fed children (P,=,0.427). Although the HIV prevention program reduced significantly the vertical transmission of HIV at Saint Camille Medical Center, the mortality of artificially fed children was still high due to gastrointestinal diseases. The HIV diagnosis by RT-PCR technique was of great help in the early identification of HIV-infected children. J. Med. Virol. 78:148,152, 2006. © 2005 Wiley-Liss, Inc. [source] Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cellsJOURNAL OF NEUROCHEMISTRY, Issue 4 2008Radovan Murín Abstract The branched-chain amino acids (BCAAs) , isoleucine, leucine, and valine , belong to the limited group of substances transported through the blood,brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy. [source] Prognostic significance of CEA levels and detection of CEA mRNA in draining venous blood in patients with colorectal cancerJOURNAL OF SURGICAL ONCOLOGY, Issue 1 2006Ioannis Kanellos MD Abstract Background and Objectives: The aims of this study were to determine carcinoembryonic antigen (CEA) levels and incidence of tumor cells using the RT-PCR technique in draining venous blood of patients with colorectal cancer, correlate the results with various histopathologic factors and determine their significance as prognostic factors. Methods: From 1995 to 2000, 108 patients with adenocarcinoma of the colon or rectum, underwent curative surgery and enrolled in this prospective study. Results: The 5-year survival group had significantly lower portal CEA levels compared to the hepatic metastasis outcome group. CEA mRNA was positive in the draining venous blood from 12 (11.1%) out of 108 patients included in the study. The rate of positive tumor cell detection in portal blood was significantly higher in the hepatic metastasis outcome group than in the 5-year survival and recurrence group. The proportion of patients with portal CEA ,5 ng/ml was greater in patients with higher stage than in patients with lower stage. Conclusions: Positive CEA mRNA in draining venous blood predicted hepatic metastases and local recurrence with accuracy over 80% but with low sensitivity of 30% and 9%, respectively. Moreover, CEA level was a sensitive indicator in hepatic metastases as sensitivity was 95% and a specific indicator in predicting 5-year survival with specificity 84%. J. Surg. Oncol. 2006;94:3,8. © 2006 Wiley-Liss, Inc. [source] Identification of the M-CSF Receptor in Endometriosis by Immunohistochemistry and RT-PCRAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2004Liselotte Mettler Problem:, The aim of this paper is to provide further evidence that the dystopic proliferation of endometriotic epithelia is caused by the stimulation of peritoneal macrophages. It is essential to show that endometriotic epithelial cells express the macrophage colony-stimulating factor receptor (M-CSFR) which binds the M-CSF produced by the peritoneal macrophages. Method of study:, For the detection of M-CSFR, samples of ectopic endometrium (n = 79) and eutopic endometrium (n = 18) were compared. The specimens were gained at operative laparoscopy in the proliferative phase of the cycle. Cryostat sections were used for immunohistochemical detection. For in vitro reverse transcriptase polymerase chain reaction (RT-PCR) tests, the tissue was immediately shock frozen on paraffin sections. For the in situ RT-PCR technique the specimens were placed in a para-formaldehyde solution, embedded in paraffin and later processed. The Gene Amp 1000 in situ PCR system (Perkin Elmer) was used as the thermal cycler. Results:, M-CSF and the M-CSF receptor are present in eutopic and ectopic endometrium. Qualitatively, with both PCR techniques we found the M-CSF receptor to be present in all samples examined. Using the histochemical detection technique, the M-CSF receptor was found in nearly 70% of endometriosis patients compared with a statistically significant lower percentage in normal endometrium. Conclusions:, The in situ RT-PCR technique and immunohistochemistry elaborated the need to trace the cellular sources of the M-CSF receptor. The identification of the M-CSF receptor in endometriotic tissue and in endometrium is apt to open a new experimental field in endometriosis research. [source] | |||||||||||||