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Semiquantitative Reverse Transcriptase (semiquantitative + reverse_transcriptase)

Distribution by Scientific Domains
Medical Sciences75%

Terms modified by Semiquantitative Reverse Transcriptase

  • semiquantitative reverse transcriptase polymerase chain reaction
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    Selected Abstracts

    The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells,

    MOLECULAR CARCINOGENESIS, Issue 3 2002
    Manfred Hergenhahn
    Abstract To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-, (TGF-,) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-,-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription. © 2002 Wiley-Liss, Inc. [source]

    Expression of VEGF and its receptors Flt and KDR in gastric cancer

    JOURNAL OF DIGESTIVE DISEASES, Issue 4 2001
    Dongping Liu
    OBJECTIVE: The purpose of the present study was to investigate the correlation between the expression of vascular endothelial growth factor (VEGF), the expression of its receptors, Flt and KDR, and the biological behavior of gastric cancer. METHODS: Sixty cases of gastric cancer that had been diagnosed by surgery and pathology were studied with the method of semiquantitative reverse transcriptase,polymerase chain reaction (RT-PCR), to determine the expression of VEGF, Flt and KDR in gastric cancer tissue. RESULTS: The positive rates of VEGF, Flt and KDR in gastric cancer were 73.3, 86.7 and 80.0%, respectively. In pericancerous tissue, the rates were 43.3, 33.0 and 30.0%, respectively. There were significant differences between the groups with and without lymph node metastasis (P < 0.01). For gastric cancer patients with lymphatic metastasis, the positive rates for VEGF, Flt and KDR were 90.3, 80.6 and 96.8%, respectively; for patients without lymphatic metastasis, the rates were 51.7, 41.3 and 55.1%; there were significant differences between the groups with and without lymph node metastasis (P < 0.01). The positive rates of VEGF and KDR in well-differentiated gastric cancer were 40.0 and 46.7%, markedly lower than those of the poorly differentiated and undifferentiated gastric cancer groups. The rate of positive expression of Flt in the well-differentiated gastric cancer group was 73.3%. There was no significant difference between this rate and that of the poorly differentiated and undifferentiated groups (87.5%). The positive expression of VEGF, Flt and KDR was unrelated to the depth of infiltration. CONCLUSIONS: There are some correlations between VEGF, Flt and KDR and the biological behavior of gastric cancer. Knowing where they are expressed at high rates may be of help in evaluating the prognosis of gastric cancer. [source]

    NMDA receptors mediate an early up-regulation of brain-derived neurotrophic factor expression in substantia nigra in a rat model of presymptomatic Parkinson's disease

    JOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2009
    Gonzalo Bustos
    Abstract The clinical symptoms of Parkinson's disease (PD) appear late and only when the degenerative process at the level of the nigrostriatal dopamine (DA) pathway is quite advanced. An increase in brain-derived neurotrophic factor (BDNF) expression may be one of the molecular signals associated to compensatory and plastic responses occurring in basal ganglia during presymptomatic PD. In the present study, we used in vivo microdialysis, semiquantitative reverse transcriptase,polymerase chain reaction, and immunohistochemistry to study N-methyl- D -aspartic acid (NMDA) receptor regulation of BDNF expression in substantia nigra (SN) of adult rats after partial lesioning of the nigrostriatal DA pathway with unilateral striatal injections of 6-hydroxydopamine (6-OHDA). A time-dependent partial decrease of striatal DA tissue content as well as parallel and gradual increases in extracellular glutamate and aspartate levels in SN were found 1 to 7 days after unilateral 6-OHDA intrastriatal injection. Instead, the number of tyrosine hydroxylase,immunoreactive (IR) cells in the ipsilateral SN pars compacta remained statistically unchanged after neurotoxin injection. Intrastriatal administration of 6-OHDA also produced an early and transient augmentation of pan-BDNF, exon II,BDNF, and exon III,BDNF transcripts in the ipsilateral SN. The pan-BDNF and exon II,BDNF transcript increases were completely abolished by the prior systemic administration of MK-801, a selective antagonist of NMDA receptors. MK-801 also blocked the increase in BDNF-IR cells in SN observed 7 days after unilateral 6-OHDA intrastriatal injections. Our findings suggest that a coupling between glutamate release, NMDA receptor activation, and BDNF expression may exist in the adult SN and represent an important signal in this midbrain nucleus triggered in response to partial DA loss occurring in striatal nerve endings during presymptomatic PD. © 2009 Wiley-Liss, Inc. [source]

    Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposure

    ARTHRITIS & RHEUMATISM, Issue 5 2010
    Patricia Ewert
    Objective Disorganization of acinar cell apical microvilli and the presence of stromal collagen in the acinar lumen suggest that the labial salivary gland (LSG) barrier function is impaired in patients with Sjögren's syndrome. Tight junctions define cell polarity and regulate the paracellular flow of ions and water, crucial functions of acinar cells. This study was undertaken to evaluate the expression and localization of tight junction proteins in LSGs from patients with SS and to determine in vitro the effects of tumor necrosis factor , (TNF,) and interferon-, (IFN,) on tight junction integrity of isolated acini from control subjects. Methods Twenty-two patients and 15 controls were studied. The messenger RNA and protein levels of tight junction components (claudin-1, claudin-3, claudin-4, occludin, and ZO-1) were determined by semiquantitative reverse transcriptase,polymerase chain reaction and Western blotting. Tight junction protein localization was determined by immunohistochemistry. Tight junction ultrastructure was examined by transmission electron microscopy. Isolated acini from control subjects were treated with TNF, and IFN,. Results Significant differences in tight junction protein levels were detected in patients with SS. ZO-1 and occludin were strongly down-regulated, while claudin-1 and claudin-4 were overexpressed. Tight junction proteins localized exclusively to apical domains in acini and ducts of LSGs from controls. In SS patients, the ZO-1 and occludin the apical domain presence of decreased, while claudin-3 and claudin-4 was redistributed to the basolateral plasma membrane. Exposure of isolated control acini to TNF, and IFN, reproduced these alterations in vitro. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. Conclusion Our findings indicate that local cytokine production in LSGs from SS patients may contribute to the secretory gland dysfunction observed in SS patients by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva. [source]