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Reverse-transcription PCR (reverse-transcription + pcr)
Selected AbstractsCharacterization of 6q abnormalities in childhood acute myeloid leukemia and identification of a novel t(6;11)(q24.1;p15.5) resulting in a NUP98,C6orf80 fusion in a case of acute megakaryoblastic leukemiaGENES, CHROMOSOMES AND CANCER, Issue 3 2005Sabrina Tosi Chromosome abnormalities of 6q are not frequently observed in myeloid disorders. In this article, we report the incidence of these chromosome changes in childhood myeloid leukemia as 2%,4% based on the cytogenetic database of a single institution. We applied fluorescence in situ hybridization (FISH) to characterize precisely the types of 6q abnormalities in seven patients (six with acute myeloid leukemia and one with myelodysplastic syndrome). They carried various translocations involving different breakpoints in 6q, as confirmed by FISH using a whole-chromosome-6 paint. Four cases were reported as t(6;11), although the breakpoints varied. Among these, we identified a novel translocation, t(6;11)(q24.1;p15.5), in a patient with acute megakaryoblastic leukemia. Molecular cytogenetic studies using the PAC clone RP5-1173K1 localized the genomic breakpoint on chromosome 11 to within the NUP98 gene. The breakpoint on chromosome 6 was narrowed down to a 500-kb region between BAC clones RP11-721P14 and RP11-39H10. Reverse-transcription PCR was performed using a forward primer specific for NUP98 and a reverse primer for the candidate gene in the 500-kb interval in 6q. This experiment resulted in the identification of a new fusion between NUP98 and C6orf80. Further studies will aim to fully characterize C6orf80 and will elucidate the role of this new NUP98 fusion in myeloid leukemia. © 2005 Wiley-Liss, Inc. [source] Expression of individual immunoglobulin genes occurs in an unusual system consisting of multiple independent lociEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2004Donna Abstract Humoral immunity is effected through the rearrangement of immunoglobulin (Ig) genes in individual somatic cells committed to the B,lymphocyte lineage. Haplotype or allelic exclusion restricts B,lymphocytes to the expression of a single Ig receptor that can sustain further somatic modification. In most species, a specific Ig chain is encoded at a single genetic locus. However, in cartilaginous fish, hundreds of independent Ig heavy- (IgH) and Ig light-chain (IgL) gene loci are present, many of which are joined in the germ line. Ig gene transcripts have been amplified from single peripheral blood lymphocytes isolated from the clearnose skate (Raja eglanteria) using reverse-transcription PCR, and a single productive IgH transcript was detected in the majority of cells analyzed. Similarly, only a single IgL transcript was detected in over half of the individual cells. Taken together, these findings suggest that a mechanism for haplotype exclusion arose early in the evolution of antibody diversity and is independent of a single genetic locus. [source] Alterations in Barrett's-related adenocarcinomas: A proteomic approachINTERNATIONAL JOURNAL OF CANCER, Issue 6 2008DunFa Peng Abstract In this study, we applied high-resolution, two-dimensional, gel electrophoresis and matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry analysis (MALDI TOF MS) to identify novel proteins that are involved in Barrett's tumorigenesis. We analyzed 12 primary tissue samples that included 8 Barrett's-related adenocarcinomas (BA) and 4 normal mucosae samples. Twenty-three spots were consistently altered (,2-fold) in at least half of the tumors when compared with all normal samples and thus subjected to further analysis. The MALDI TOF MS analysis demonstrated biologically interesting upregulated proteins such as ErbB3, Dr5 and Cyclin D1 as well as several members of the zinc finger proteins (Znf146, Znf212 and Znf363). Examples of downregulated proteins included Lgi1 and Klf6. We selected four proteins (ErbB3, Dr5, Znf146 and Lgi1) that are novel for BAs for validation using quantitative real-time reverse-transcription PCR on 39 BA tissue samples when compared with normal samples. We demonstrated mRNA upregulation of ERBB3 (51.3%), DR5 (41%) and ZNF146 (30.7%) and downregulation of LGI1 (100%) in BA. We have further validated the protein overexpression of ErbB3, Dr5 and Znf146, using immunohistochemical (IHC) analysis on a tissue microarray that contained 75 BAs and normal gastric and esophageal mucosae samples. BA tissue samples demonstrated overexpression of ErbB3 (42%), Dr5 (90%) and Znf146 (30%) when compared with normal tissues. In conclusion, we have identified and validated several novel proteins that are involved in Barrett's carcinogenesis. © 2007 Wiley-Liss, Inc. [source] Combined histology and molecular biology for diagnosis of early stage gastric MALT lymphomaJOURNAL OF DIGESTIVE DISEASES, Issue 1 2006Zhi Hui YI OBJECTIVE: To establish a sequential diagnostic procedure of gastric mucosa-associated lymphoid tissue (MALT) lymphoma and provide evidence for selected optimal cases to be treated in the early stage. METHODS: Thirty-one cases of gastric lymphoid hyperplasia (GLH) were selected and multiple investigations including histology, protein level, DNA and chromosome levels, combined with clinical follow-up were performed. Histological grade was according to Isaacson's criteria of GLH; CD20, UCHL-1 (CD45RO), anti-kappa (,), anti-lambda (,) and Ki-67 were used for immunohistochemical staining; semi-nested polymerase chain reaction (PCR) was used to detect IgH gene rearrangement and reverse-transcription PCR (RT-PCR) was used to detect API2-MALT1 fusion of the chromosome translocation t(11;18)(q21;q21). Twenty-nine cases underwent eradication therapy for Helicobacter pylori. Changes in histological grade, endoscopic appearance, expression of Ki-67 and IgH gene rearrangement were compared after eradication treatment. RESULTS: Of the 31 cases of GLH with predominant chronic gastritis and gastric ulcer most were histological grade 2 and 3. Only one case had , light chain restriction and 10 cases had monoclonal IgH gene rearrangement. Expression of Ki-67 and monoclonal IgH gene rearrangement were significantly increased with increased lymphoid hyperplasia (P < 0.05). Two cases had API2-MALT1 fusion. Helicobacter pylori was eradicated in 25 cases and another course of treatment had to be given in 4 cases. All cases were followed up for 1.5,37 months. Of the 27 successful eradication cases, 18 showed complete regression both histologically and endoscopically, 4 had partial regression and 7 were unchanged. CONCLUSIONS: A sequential diagnostic procedure based on histology, expression of Ki-67 combined with clonality of IgH rearrangement and API2-MALT1 fusion helps to diagnosis of early stage gastric MALT lymphoma and choose the best treatment strategy. [source] Role of ISGF3 in modulating the anti-hepatitis B virus activity of interferon-alpha in vitroJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2008Quan Zhang Abstract Background and Aim:, Although interferon-, (IFN-,) is an effective treatment for hepatitis B virus (HBV) infection, its precise mechanism of action has not been identified. In this study, we investigated the role of signal transduction pathways in the activation of anti-HBV responses mediated by IFN-,. Methods:, Using an oligo microarray, we found that four genes in the IFN-, signal pathway were markedly upregulated by IFN-, in human hepatoma cells regardless of whether they had been transfected with a plasmid containing the HBV genome: signal transducers and activators of transcription 1 (STAT1), interferon regulatory factor-9 (IRF-9, also called ISGF3, or P48), IFN-,-inducible protein 15 (IFI-15) and IFN-,-inducible protein 6,16 (IFI-6-16). We also investigated the role of IFN-stimulated gene factor3 (ISGF3) complex in IFN-,-mediated anti-HBV responses in human hepatoma cells by measuring the mRNA of the three genes within ISGF3 (STAT1, STAT2 and IRF-9) using semiquantitative reverse-transcription PCR (RT-PCR), and expression of the three proteins by western blot, and the mRNA and protein of dsRNA-dependent protein kinase (PKR). Results:, STAT1, STAT2, IRF-9 and PKR mRNA as well as protein levels were upregulated by IFN-, treatment. When cells were pretreated with genistein, STAT1, STAT2 and IRF-9 mRNA levels remained unchanged after IFN-, stimulation, but PKR mRNA levels decreased, and the expression of the STAT1, P-STAT2, IRF-9 and PKR proteins decreased. Levels of HBV DNA decreased in the supernatants of cells treated with IFN-,, while ISGF3 levels increased. The quantity of HBV DNA remained unchanged by pretreating with genistein. Conclusions:, These observations suggested that the Janus tyrosine kinase,STAT (JAK-STAT) pathway may play a major role in mediating the effects of IFN-, against HBV, and that ISGF3 might be a key factor. [source] Expression of HNFs and C/EBP, is correlated with immunocytochemical differentiation of cell lines derived from human hepatocellular carcinomas, hepatoblastomas and immortalized hepatocytesCANCER SCIENCE, Issue 9 2003Tadashi Ishiyama Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver-enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBP, using real-time reverse-transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B-17/Icrj-scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF-3,, HNF-4,, HNF-1,, and C/EBP, were specific to HCCs with well-differentiated function and morphology. Furthermore, among these four transcription factors, HNF-4, and HNF-1, expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF-4, and HNF-1, are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBP, in HCCs. [source] The effect of narrowband ultraviolet B on the expression of matrix metalloproteinase-1, transforming growth factor-,1 and type I collagen in human skin fibroblastsCLINICAL & EXPERIMENTAL DERMATOLOGY, Issue 2 2007C. P. Choi Summary Background., Ultraviolet (UV) irradiation induces chronic skin diseases, such as skin cancer and photoageing, and the mechanisms of this skin damage are associated with the upregulation of matrix metalloproteinases (MMPs) and decreased collagen synthesis. Narrowband ultraviolet B (NB-UVB) radiation is a relatively new treatment modality for vitiligo and psoriasis. However, the mechanism of NB-UVB action on photoageing is not completely understood. Aims., We investigated the effects of NB-UVB on the expression of MMP-1, transforming growth factor (TGF)-,1 and type I collagen in cultured human skin fibroblasts. Methods., Cultured human fibroblasts were irradiated with either NB-UVB (50,800 mJ/cm2) or broadband UVB (BB-UVB; 25 mJ/cm2). The expression of MMP-1, TGF-,1 and type I collagen mRNA was determined by reverse-transcription PCR. Expression of MMP-1 and TGF-,1 protein was determined by ELISA and that of type I collagen by Western blotting. Results., NB-UVB induced the expression of MMP-1 and reduced the expression of TGF-,1 and type I collagen at the mRNA and protein levels in a dose-dependent manner. The expression of type I collagen protein decreased more after irradiation with 25 mJ/cm2 of BB-UVB than 400 mJ/cm2 of NB-UVB. Conclusions., This study indicates that NB-UVB irradiation reduces type I collagen synthesis in human skin fibroblasts by inhibiting TGF-,1 expression and stimulating the release of MMP-1. It also suggested that the photoageing-related effects of NB-UVB are weaker than those of BB-UVB in vitro. [source] | ||||||||||