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Reverse Transcription-polymerase Chain Reaction (reverse + transcription-polymerase_chain_reaction)
Kinds of Reverse Transcription-polymerase Chain Reaction Terms modified by Reverse Transcription-polymerase Chain Reaction Selected AbstractsNucleoside transporter expression and function in cultured mouse astrocytesGLIA, Issue 1 2005Liang Peng Abstract Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755,760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1,4°C to study diffusional uptake and 1,60-min uptakes at 37°C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na+ -independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na+ -dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine. © 2005 Wiley-Liss, Inc. [source] Hepatitis C virus core protein induces malignant transformation of biliary epithelial cells by activating nuclear factor-,B pathwayJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 7 2010Zhi-Hua Li Abstract Background and Aim:, In an earlier study, we found that hepatitis C virus core protein, HCV-C, participated in the malignant transformation of HCV-C transfected normal human biliary epithelial (hBE) cells by activating telomerase. Here we further investigated the signaling of the malignant transformation. Methods:, Reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunoprecipitation were used to analyze the expression of HCV-C, human telomerase reverse transcriptase (hTERT), nuclear factor-,B (NF-,B) and NF-,B inhibitor alpha (I,B,) genes and the phosphorylation level of I,B, protein. Electrophoretic mobility shift assays (EMSA) and NF-,B-linked luciferase reporter assays were carried out to measure NF-,B activity. Results:, The expression of HCV-C and hTERT was detected only in HCV-C-transfected hBE (hBE-HCV-C) cells but not in vector-transfected or parental hBE cells. More NF-,B protein accumulated in nuclear extracts of hBE-HCV-C cells rather than in those of control cells, though total NF-,B protein level showed no difference among these cells. DNA binding activity of NF-,B and the NF-,B-linked luciferase activity were much higher in HCV-C-transfected hBE cells than those in vector- or non-transfected hBE cells. In addition, the I,B, phosphorylation level, but not the I,B, mRNA or protein levels, was increased after HCV-C transfection. Conclusions:, Hepatitis C virus core protein activates NF-,B pathway in hBE cells by increasing the phosphorylation of I,B,. The pathway may be responsible for HCV-C-induced malignant transformation of hBE cells. [source] Influence of methylated p15 INK4b and p16 INK4a genes on clinicopathological features in colorectal cancerJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 8 2006Atsushi Ishiguro Abstract Background and Aim:, Genetic silencing by promoter methylation has attracted attention in the carcinogenesis of colorectal cancer. Methylation of the p16INK4a gene has been found in primary colorectal cancer. Although the p15INK4b gene displays high homology to the p16INK4a gene in the amino acid sequence, methylation of p15INK4b has not been fully studied. We investigated p15INK4b methylation status in patients with colorectal cancer to verify the association between the methylation of p15INK4b and clinicopathological features compared with p16INK4a. Methods:, DNA samples from the tissues of primary colorectal cancer and corresponding adjacent normal colon mucosa were obtained from surgical resections of 88 patients (47 males and 41 females, aged 29,83 years). Methylation-specific polymerase chain reaction was used to analyze p15INK4b and p16INK4a methylation status after bisulfite modification. Cumulative survival rates (mean follow-up period: 53.2 months) were calculated by the Kaplan-Meier analysis. Results:, Methylations of p15INK4b and p16INK4a genes were detected in 23 (26.1%) and 20 (22.7%) colorectal cancers, respectively. Methylation of p15INK4b was not associated with any clinicopathological features. Compared with normal mucosa, the methylation of p15INK4b was more prominent in tumor tissue (P < 0.001). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that p15INK4b methylaton decreased mRNA expression. Kaplan-Meier analysis showed that patients with stage I-II had a significant difference in survival rate between those with and without methylated p15INK4b (P = 0.018). Conclusions:, Our results suggest that methylation of the p15INK4b gene contributes to the process of carcinogenesis in colorectal cancer as well as p16INK4a and is useful as a prognostic factor in the early stage. [source] Lack of susceptibility of Chacma baboons (Papio ursinus orientalis) to hepatitis C virus infectionJOURNAL OF MEDICAL VIROLOGY, Issue 4 2002N.P. Sithebe Abstract The main reason to ascertain whether baboons are susceptible to infection with hepatitis C virus (HCV) is the need to replace chimpanzees, which are endangered, as an animal model for undertaking research into the biology and host,virus interactions of HCV, and for developing a vaccine against this virus. A second reason is that baboons are a possible source of xenografts for human liver transplantation. We inoculated serum containing HCV into four Chacma baboons and monitored them for 52 weeks for evidence of infection. Serum was tested for antibody to HCV, HCV RNA, and aminotransferase concentrations at 2-week intervals for 26 weeks and thereafter at 4-week intervals. Liver tissue was examined at 28 and 52 weeks for histopathological changes and viral RNA, and at 52 weeks for viral particles using electron microscopy. Reverse transcription-polymerase chain reaction assay was used to detect HCV RNA, and the results were confirmed by Southern hybridization. Serum aminotransferase concentrations remained within the normal range and liver histology was normal during the follow-up period. Passive transmission of anti-HCV to the baboons was observed during the first 4 weeks. HCV RNA was not detectable in any serum or liver sample and electron microscopy failed to reveal viral particles in liver tissue. In conclusion, we did not find Chacma baboons to be susceptible to infection with HCV, although we cannot deny that in an immunosuppressed liver transplant recipient, infection of a baboon xenograft might occur. Another animal model for HCV infection must be sought. J. Med. Virol. 66:468,471, 2002. © 2002 Wiley-Liss, Inc. [source] Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritisJOURNAL OF MEDICAL VIROLOGY, Issue 3 2002D.S. Subekti Abstract Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis. J. Med. Virol. 66:400-406, 2002. © 2002 Wiley-Liss, Inc. [source] Expression of gangliosides in an immortalized neural progenitor/stem cell lineJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2003Keiji Suetake Abstract Glycosphingolipids (GSLs) are known to play important roles in cellular growth and differentiation in the nervous system. The change in expression of gangliosides is correlated with crucial developmental events and is evolutionarily conserved among many vertebrate species. The emergence of neural progenitors represents a crucial step in neural development, but little is known about the exact composition and subcellular localization of gangliosides in neural progenitor cells. The C17.2 cell line was derived after v- myc transformation of neural progenitor cells isolated from neonatal mouse cerebellar cortex. The developmental potential of C17.2 cells is similar to that of endogenous neural progenitor/stem cells in that they are multipotential and capable of differentiating into all neural cell types. We characterized the GSL composition of C17.2 cells and found the presence of only a-series gangliosides. Subcellular localization studies revealed that GM1 and GD1a are localized mainly on the plasma membrane and partly in the cytoplasm, both as punctate clusters. Reverse transcription-polymerase chain reaction revealed the absence of ST-II transcripts in C17 cells, which most likely accounts for the lack of expression of b- and c-series complex gangliosides in this cell line. These data suggest that the divergence in ganglioside expression in C17.2 cells is regulated at the transcriptional level. © 2003 Wiley-Liss, Inc. [source] Areca nut extract represses migration and differentiation while activating matrix metalloproteinase-9 of normal gingival epithelial cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2008Y-H Tseng Background and Objective:, Areca (betel) chewing is associated with an increase in the incidence of periodontal diseases. Aberrations in matrix metalloproteinase (MMP) expression have been reported to be associated with periodontal disease. This study investigated the effects of areca nut extract on MMP activity and the phenotype of human gingival epithelial cells. Material and Methods:, Reverse transcription-polymerase chain reaction, western blotting and gelatin zymography were used to assay MMPs. Cell viability, mobility and detachment assays were performed to characterize the phenotypic impact. Confocal microscopy was employed to evaluate cell aggregation and the distribution of E-cadherin and F-actin. Results:, Treatment of gingival epithelial cells with 10 µg/mL of areca nut extract reduced its cell viability. Treatment with 5 and 10 µg/mL of areca nut extract for 24 h activated MMP-9 but not MMP-2 in gingival epithelial cells. This activation could be nuclear factor-,B dependent and was abrogated by 10 µm curcumin. Areca nut extract also reduced the migration and detachment of gingival epithelial cells. The differentiated cell,cell contact of gingival epithelial cells was markedly impaired by areca nut extract. This was accompanied by a disruption of distribution of E-cadherin and F-actin. Conclusion:, The areca nut extract-mediated activation of MMP-9 in gingival epithelial cells could signify a potential periodontal pathogenesis in areca chewers. The areca nut extract-mediated inhibition of cell viability and migration, together with the changed aggregation in gingival epithelial cells, suggests that impairment of the re-epithelization underlies the process and this, in turn, might exacerbate gingival inflammation. [source] Expression Of The Co-Stimulatory Molecule BB-1, The Ligands CTLA-4 and CD28 and Their Mrnas In Chronic Inflammatory Demyelinating PolyneuropathyJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001K Murata To examine whether the Schwann cells in patients with autoimmune neuropathies have the potential to behave as professional antigen-presenting cells, we investigated the expression of the co-stimulatory molecules BB-1, B7-1 (CD80), B7-2 (CD86) and their counter-receptors CD28 or CTLA-4 (CD152) at the protein and mRNA levels in sural nerve biopsies of patients with chronic inflammatory demyelinating polyneuropathy (CIDP), CIDP associated with human immunodeficiency virus infection (HIV-CIDP), IgM paraproteinaemic neuropathy and normal or non-immune axonal neuropathy. In single-and double-labelling experiments, we used the S-100 antigen as a pan-Schwann cell marker, myelin-associated glycoprotein as a marker for myelinating Schwann cells and the fibrillary acidic protein as a marker for unmyelinating Schwann cells. The expression of the B7 family of molecules was limited to BB-1 and was observed only on the Schwann cells. There was constitutive expression of BB-1 on unmyelinating Schwann cells in all nerves studied. However, in CIDP and HIV-CIDP, but not the other diseases, there was prominent upregulation of BB-1 on the myelinating Schwann cells. The endoneurial T cells in the proximity of BB-1-positive Schwann cells expressed the CD28 or CTLA-4 counterreceptors. Reverse transcription-polymerase chain reaction confirmed that these ligands were upregulated only in CIDP. Because the myelinating BB-1-positive Schwann cells expressed HLA-DR antigen, the findings indicate that, in CIDP, Schwann cells possess the necessary markers to function as antigen-presenting cells. [source] Comparison of two methods for evaluating bone marrow metastasis of neuroblastoma: Reverse transcription-polymerase chain reaction for tyrosine hydroxylase and magnetic resonance imagingPEDIATRICS INTERNATIONAL, Issue 4 2004Chikayo Takemoto AbstractBackground:,The presence of bone marrow (BM) metastasis and circulating tumor cells in patients with neuroblastoma is a significant prognostic factor at diagnosis and might antedate detection of a relapse by other diagnostic studies. In this study, the clinical value of reverse transcription-polymerase chain reaction (RT-PCR) to amplify mRNA for tyrosine hydroxylase (TH) and magnetic resonance imaging (MRI) during the clinical course of patients with advanced neuroblastoma, was evaluated. Methods:,Four patients with Stage 1, 4 or 4S neuroblastoma, were studied. BM and peripheral blood (PB), including peripheral blood stem cell (PBSC), samples were examined for TH mRNA using RT-PCR. Concurrently, MRI detection of BM metastasis was used. Results:,In all cases, except one that had no evidence of BM invasion, TH mRNA in BM and PB at diagnosis were positive, and TH mRNA at diagnosis disappeared after chemotherapy. In two cases, although involvement in the neurocentrum BM was detected by MRI, TH mRNA in the iliac crest BM was negative. The pathological area still remained on MRI after intensive therapy. Conclusion:,RT-PCR for TH mRNA might be the most sensitive method for the detection of occult neuroblastoma cells in BM and PB. However, because invasion of the BM by neuroblastoma may have a focal distribution, sampling errors can occur. Therefore, not only RT-PCR but also MRI, need to be used to rule out marrow involvement, especially at diagnosis and BM relapse. [source] Growth Factors and Their Receptors in the Middle Ear Mucosa During Otitis Media,THE LARYNGOSCOPE, Issue 3 2002Sean D. Palacios MD Abstract Objective The hyperplastic response of the middle ear mucosa during bacterial otitis media is thought to be mediated by the actions of growth factors and their respective receptors. The purpose of the study was to explore the expression of growth factors known to stimulate epithelial cells in other systems, as well as their receptors, in the middle ear mucosa during otitis media. Study Design Expression of mRNA growth factors and receptors was measured over time after inoculation of the rat middle ear with bacteria. Methods The middle ears of 12 male Sprague-Dawley rats were injected with 105/mL Haemophilus influenzae strain 3655 (nontypeable, biotype II). Three rats were killed at 6, 24, 48, and 72 hours. Three untreated rats were also killed to serve as negative controls. The middle ear mucosa samples were surgically removed and homogenized. Reverse transcription-polymerase chain reaction was performed on each sample with primers for rat epidermal growth factor, epidermal growth factor receptor (ErbB), heparin binding epidermal-like growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, keratinocyte growth factor, betacellulin, amphiregulin, and neuregulin-,. Results Hepatocyte growth factor and epidermal growth factor receptor primers demonstrated polymerase chain reaction products of the expected size that were not displayed in the normal middle ear mucosa. Keratinocyte growth factor and hepatocyte growth factor receptor demonstrated polymerase chain reaction products at all time points tested. Betacellulin and neuregulin-, products were present at all time points except 72 hours after infection. Conclusions The results of the study support a role for growth factors in the middle ear mucosa during otitis media. These bioactive ingredients contribute to mucosal hyperplasia. [source] Identification of Dictyothrips betae as the vector of Polygonum ring spot virusANNALS OF APPLIED BIOLOGY, Issue 2 2010M. Ciuffo Dictyothrips betae (Thysanoptera: Thripidae) is the predominant thrips species on Polygonum convolvulus and Polygonum dumetorum plants infected with a recently described tospovirus species, Polygonum ring spot virus (PolRSV). Laboratory transmission experiments (leaf disk assays) with adults collected directly in the field demonstrated the competence of this thrips to transmit PolRSV, although only at a rate of 4%. However, this increased to 16% using newly emerged larvae fed on infected leaves. Frankliniella occidentalis and Thrips tabaci failed to transmit PolRSV in leaf disk assays. Reverse transcription-polymerase chain reaction (RT-PCR) with specific primers for the N protein and Western blot analysis of adult thrips to detect the N protein confirmed the presence of the virus in D. betae individuals after feeding for at least 5 days on healthy plants. For molecular identification purposes partial sequences of mitochondrial cytochrome c oxidase subunit I (COI), nuclear 28S ribosomal DNA and the elongation factor-1, (EF-1,) from D. betae were cloned. COI sequence was also used for deriving a phylogenetic tree, including D. betae. The results confirmed a relationship between this species and tospovirus-transmitting insects of the genus Thrips. [source] Gene expression profiling of the human prostate zonesBJU INTERNATIONAL, Issue 4 2006Leonie Van Der Heul-Nieuwenhuijsen OBJECTIVE To investigate differences in gene expression in different zones of the prostate by microarray analyses, to better understand why aggressive tumours predominantly occur in the peripheral zone (PZ), whereas benign prostatic hyperplasia (BPH) occurs almost exclusively in the transition zone (TZ). MATERIALS AND METHODS Expression profiling of both prostate zones was done by microarray analysis. Reverse transcription-polymerase chain reaction (RT-PCR) of the top 18 genes confirmed the microarray analyses. RT-PCR with common cell-type markers indicated that the differential expression between the zones was not caused by an unequal distribution of different cell types. Primary stromal and epithelial prostate cells were used to study cell type expression in the 12 highest differentially expressed zonal-specific genes. RESULTS In all, 346 genes were identified as preferentially expressed in the TZ or PZ. A few of the TZ-specific genes, including ASPA, FLJ10970 and COCH, were also stroma-specific. Comparisons with other microarray studies showed that gene expression profiles of prostate cancer and BPH correlate with the expression profiles of the PZ and TZ, respectively. CONCLUSION Gene expression differs between the PZ and TZ of the prostate, and stromal,epithelial interactions might be responsible for the distinct zonal localization of prostate diseases. [source] Acute generalized exanthematous pustulosis associated with pseudoephedrineBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2004M.A. Padial Summary Acute generalized exanthematous pustulosis (AGEP) is an uncommon skin disorder most often caused by drugs. Few adverse reactions to sympathomimetic drugs have been reported, despite their extensive use. Although the aetiology of AGEP remains uncertain, recent data have reported involvement of drug-specific T cells and interleukin (IL)-8 production. We characterized an adverse reaction to pseudoephedrine both clinically and immunologically. Histological analysis of skin biopsies confirmed the clinical entity as AGEP, while epicutaneous tests confirmed the specificity of the reaction to the drug. Moreover, immunohistochemical studies showed a mononuclear infiltrate consisting of activated memory T cells in addition to polymorphonuclear cells. Reverse transcription-polymerase chain reaction revealed an increased expression of IL-8 in AGEP-affected skin. [source] Expression and structure of interleukin 4 receptors in primary meningeal tumorsCANCER, Issue 10 2005Sachin Puri M.Sc. Abstract BACKGROUND It was reported previously that malignant human tumors, like glioma and medulloblastoma, express high-density interleukin (IL-4) receptor mRNA and protein. Because IL-4 receptors (R) are sensitive targets for targeted therapeutics, knowledge of the expression of these receptors in other central nervous system tumors is of great interest. In this study, the authors examined the expression and subunit composition of IL-4R complex in primary human meningiomas. METHODS Reverse transcription-polymerase chain reaction (RT-PCR) analysis for IL-13R,1, IL-4R, and IL-2R,c was performed on total RNA extracted from 35 meningiomas and a normal human brain tissue sample. Results were confirmed in nine randomly selected tumors by quantitative real-time PCR and in situ immunofluorescence assay. RESULTS Transcripts for the IL-4R, and IL-13R,1 chains were overexpressed in meningiomas compared with normal brain tissue. The levels of IL-4R, mRNA appeared to be higher compared with the levels of IL-13R,1 mRNA. The results also showed that tumors with higher disease grade tended to have increased mRNA expression for the IL-4R, chain. This IL-4R, mRNA overexpression appeared to be more frequent in younger patients (age < 37 years). The transcripts for IL-2R,c chain were not detected in any of the tumor samples or in normal brain tissue. Quantitative real-time PCR confirmed the results of the RT-PCR analysis. Meningiomas also demonstrated a bright immunofluorescent staining for the IL-4R, and IL-13R,1 chains but no staining for IL-2R,c. CONCLUSIONS Expression of the IL-4R, and IL-13R,1 chains and absence of IL-2,c expression established that meningiomas expressed type II IL-4Rs. These receptors may serve as a target for cytotoxin/immunotoxin therapy in patients with meningioma who are not amenable to surgical resection or for recurrent tumors. Cancer 2005. © 2005 American Cancer Society. [source] Dystrophin upregulation in pressure-overloaded cardiac hypertrophy in ratsCYTOSKELETON, Issue 1 2003Masato Maeda Abstract Dystrophin is a cytoskeletal protein localized to the sarcolemma of skeletal and cardiac muscle, and neurons. We have recently demonstrated that a significant cardiac damage including myocytes injury, inflammation, and fibrosis, was found in dystrophin-deficient myocardium during pressure overload [Kamogawa et al., 2001: Cardiovasc Res 50:509,515]. However, little is known about how the cardiac sarcolemmal cytoskeleton produces qualitative and quantitative changes in response to pressure overload. Accordingly, we investigated dystrophin gene expression and protein accumulation during cardiac hypertrophy. Cardiac hypertrophy was produced by banding of the abdominal aorta of rats. Total RNA from the left ventricle of the heart was used for a quantitative reverse transcription-polymerase chain reaction (RT-PCR). Dystrophin mRNA expression significantly increased by 33 ± 18% at 1 day (P < 0.05) and 45 ± 19% at 2 days (P < 0.01) after banding, while G3PDH mRNA showed no significant change. RT-PCR for dystrophin tissue-specific exon 1 revealed that only muscle type promoter, but not non-muscle type promoter (brain and Purkinje-cell type), was activated immediately after banding. Immunohistochemistry for dystrophin showed intense cellular membrane staining with an increase in the perimeter of the myocytes by 14% at 3 days (46.3 ,m, P < 0.01) and 19% at 7 days (51.2 ,m, P < 0.01) after banding. Western blotting also showed dystrophin protein increased by 14 ± 6% at 2 days (P < 0.05) and by 32 ± 10% at 3 days (P < 0.01) after aortic banding. In conclusion, upregulation of dystrophin mRNA expression and protein accumulation occurs in response to cardiac hypertrophy. These data and the vulnerability of dystrophin-deficient myocardium to pressure overload suggest that dystrophin could play an important role in maintaining the integrity of the sarcolemma. Cell Motil. Cytoskeleton 55:26,35, 2003. © 2003 Wiley-Liss, Inc. [source] Effects of metformin and oleic acid on adipocyte expression of resistinDIABETES OBESITY & METABOLISM, Issue 1 2006R Rea Aim:, The adipocyte-secreted hormone resistin has been implicated in obesity-induced insulin resistance and type 2 diabetes, but pharmacological and dietary factors that regulate resistin gene expression and the effects of resistin on cellular glucose uptake in muscle have not been clearly defined. Methods:, Expression of resistin mRNA was studied in differentiated 3T3-L1 adipocytes by using real-time semiquantitative reverse transcription-polymerase chain reaction. The effects of resistin on insulin-stimulated and insulin-independent 2-deoxyglucose uptake were evaluated in L6 muscle cells. Results:, Insulin 1 µm and rosiglitazone 10 µm markedly reduced resistin mRNA expression (relative to the control gene TF2D) by 4.7-fold (p < 0.05) and 5.3-fold (p < 0.02), respectively. Similar reductions in resistin mRNA were demonstrated with metformin 100 µm (6.2-fold reduction, p < 0.02) and oleic acid 100 µm (3.9-fold reduction, p < 0.03). Resistin 1 µm significantly reduced maximum insulin-stimulated 2-deoxyglucose uptake in L6 cells from 634 to 383% (relative to 100% for control, p < 0.001), and co-administration of rosiglitazone had no effect on resistin-induced insulin resistance. In the absence of insulin, however, resistin increased glucose uptake dose-dependently (e.g., 1.75-fold at 5 µm, p < 0.001) via a mitogen-activated protein kinase-dependent pathway. Conclusions:, These results demonstrate that various glucose-lowering therapies and oleic acid reduce resistin gene expression in isolated adipocytes, and that resistin impairs insulin-stimulated glucose uptake in skeletal muscle-derived cells. [source] Expression of caspase and apoptotic signal pathway induced by sulfur dioxideENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2010Juli Bai Abstract Sulfur dioxide (SO2) is a common air pollutant that is released in low concentrations into the atmosphere and in higher concentrations in some work places. In the present study, male Wistar rats were housed in exposure chambers and treated with 14.00 ± 1.01, 28.00 ± 1.77, and 56.00 ± 3.44 mg/m3 SO2 for 7 days (6 hr/day), while control rats were exposed to filtered air under the same conditions. The mRNA and protein levels of caspase-3, caspase-8, and caspase-9 were analyzed using a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay and an immunohistochemistry method. Activities of caspases were detected using colorimetric and fluorescent assays. Chromatin degradation and cell morphological changes were investigated by TUNEL assay and H&E staining in livers and lungs, respectively. The results showed that mRNA levels, protein levels and activities of caspase-3, caspase-8, and caspase-9 were increased in a dose-dependent manner in livers and lungs of rats after SO2 inhalation. In addition, livers were infiltrated with lymphocytes, congestion and inflammation occurred in lungs, and eosinophil cells and apoptotic cells increased in both livers and lungs after SO2 inhalation. These results suggest that SO2 exposure increases the expression and activity of both initiator and and effector caspases, and may induce apoptosis in liver and lung of rats through both death receptor and mitochondrial pathways. Environ. Mol. Mutagen. 2010. © 2009 Wiley-Liss, Inc. [source] Impact of microcystin containing diets on physiological performance of Nile tilapia (Oreochromis niloticus) concerning stress and growth,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Andrea Ziková Abstract Diets containing Microcystis with considerable amounts of the cyanotoxin microcystin-LR (MC-LR) were fed to determine their impact on the physiological performance of the omnivorous Nile tilapia (Oreochromis niloticus) with regard to stress and growth performance. Four different diets were prepared based on a commercial diet (control, MC-5% [containing 5% dried Microcystis biomass], MC-20% [containing 20% dried Microcystis biomass], and Arthrospira-20% [containing 20% dried Arthrospira sp. biomass without toxin]) and fed to female Nile tilapia. Blood and tissue samples were taken after 1, 7, and 28 d, and MC-LR was quantified in gills, muscle, and liver by using high-performance liquid chromatography (HPLC). Only in the liver were moderate concentrations of MC-LR detected. The stress hormone cortisol and glucose were analyzed from plasma, suggesting that all modified diets caused only minor to moderate stress, which was confirmed by analyses of hepatic glycogen. In addition, the effects of the different diets on growth performance were investigated by determining gene expression of hypophyseal growth hormone (GH) and hepatic insulin-like growth factor-I (IGF-I). For all diets, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) demonstrated no significant effect on gene expression of the major endocrine hormones of the growth axis, whereas classical growth data, including growth and feed conversion ratio, displayed slight inhibitory effects of all modified diets independent of their MC-LR content. However, no significant change was found in condition or hepatosomatic index among the various diets, so it seems feasible that dried cyanobacterial biomass might be even used as a component in fish diet for Nile tilapia, which requires further research in more detail. Environ. Toxicol. Chem. 2010;29:561,568. © 2009 SETAC [source] A comparison of molecular methods for the routine detection of viroids,EPPO BULLETIN, Issue 3-4 2000R. A. Mumford Viroids, such as Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd), are important plant pathogens. However, because of their unique biological properties, viroids have proved, in the past, difficult to diagnose. The use of molecular methods has now changed this and this paper reports the comparison of three such methods (dot-blot hybridization using DIG-labelled cRNA probes, reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan), which have been developed for routine detection of CSVd. Sensitivity comparisons show that the TaqMan assay is more sensitive than either RT-PCR (100 times) and hybridization (1000 times). RT-PCR and TaqMan assays have also been developed to detect PSTVd. In addition to the development of sensitive detection methods, considerable emphasis has been placed on making these assays amenable to mass-scale detection through the use of internal controls and the development of a rapid, reliable probe capture extraction system. [source] Evaluation of PG-M3 antibody in the diagnosis of acute promyelocytic leukaemiaEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2010Sanjeev Kumar Gupta Eur J Clin Invest 2010; 40 (10): 960,962 Abstract Background & objectives, Acute promyelocytic leukaemia (APL) is a distinct subtype of acute myeloid leukaemia (AML) characterized by a reciprocal translocation, t(15;17) and a high incidence of life-threatening coagulopathy. APL diagnosis is considered a medical emergency. As reverse transcription-polymerase chain reaction (RT-PCR) for PML-RAR, fusion oncoprotein is time consuming, there is a need for a rapid and accurate diagnostic test for APL. This study evaluates the role of PG-M3 monoclonal antibody using immunofluorescence (IF) in the early diagnosis of APL. Materials and Methods, Thirty-six new untreated APL cases diagnosed with RT-PCR for PML-RAR, as the gold standard and 38 non-APL controls (28 non-APL AMLs and 10 non-leukaemic samples) were evaluated by routine morphology and cytochemistry, RT-PCR and IF using PG-M3 monoclonal antibody. Results, Using IF, 34 of 36 (94·4%) APL cases showed a microgranular pattern suggestive of APL and two cases (5·6%) showed a speckled pattern typical of wild-type PML protein (False negative). By comparison, two of 28 (7·1%) non-APL AMLs showed microgranular pattern (false positive). Hence, IF as a diagnostic test for APL resulted in a sensitivity of 94·4%, specificity of 92·9% and positive and negative predictive values of 94·4% and 92·9% respectively. All 10 non-leukaemic samples showed a speckled pattern. Conclusions, IF using PG-M3 antibodies can be used as a rapid (takes 2 h), cheap, sensitive and specific method to identify APL. It can be a useful adjunct for diagnosis of APL especially if facilities for RT-PCR are not available, particularly in resource-limited settings. [source] Placenta growth factor stimulates the growth of Philadelphia chromosome positive acute lymphoblastic leukemia cells by both autocrine and paracrine pathwaysEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2005Toshiko Ikai Abstract:, Vascular endothelial growth factor (VEGF) and its associated molecule, placenta growth factor (PlGF) are now known to support normal hematopoiesis, and leukemia cell growth. In this study, expression of VEGF and PlGF in acute lymphoblastic leukemia (ALL) cells was examined by real time reverse transcription-polymerase chain reaction in 20 patient samples. Expression of PlGF was more intense in Philadelphia chromosome positive (Ph+) ALL than in Ph, ALL cases. On the other hand, expression level of VEGF was not different between Ph+ and Ph, cases. Then, PlGF was added to the two ALL cell lines, CRL1929 (Ph+), and Nalm6 (Ph,). The PlGF stimulated the growth of CRL1929 in time- and dose-dependent manners, although the growth of Nalm6 was not affected by PlGF. The growth stimulation of CRL1929 by PlGF was confirmed by the increase of S phase cells. And the growth promoting effect of PlGF on CRL1929 was cancelled by simultaneous addition of VEGFR1/Fc (which binds to PlGF and abrogates its function), but was not cancelled by VEGFR2/Fc (which does not bind to PlGF). Then, addition of VEGFR1/Fc to the simple culture of CRL1929 demonstrated growth inhibitory effect. These observations demonstrated that PlGF stimulates the growth of Ph+ ALL cells by both autocrine and paracrine pathways. Finally, PlGF-VEGFR1 loop might be a therapeutic target to improve the prognosis of Ph+ ALL. [source] Expression of DNA repair gene Ku80 in lymphoid neoplasmEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2005Tsai-Yun Chen Abstract:,Objectives:,Ku, a heterodimer of KU70 and Ku80 that binds to double-strand DNA breaks (DSBs) and activates the catalytic subunit (DNA-PKcs) when DNA is bound, is essential in DSB repair and V(D)J recombination. Ku80 is a putative tumor suppressor gene that might play an important role in drug resistance. Our aim was to determine the role of Ku80 in lymphoid malignancy. Patients and methods:,Competitive reverse transcription-polymerase chain reaction assays were performed and the expression levels of Ku80 were measured in normal peripheral blood mononuclear cells (n = 9) and malignant cells from 25 patients with acute lymphoblastic leukemia (ALL) (14 children, 11 adults), and chronic lymphoproliferative disorders (n = 6). The Ku80 transcripts were sequencing for the possibility of mutation. Results:,No mutation or Ku80 variant at the RNA level was seen in any patient samples or in the Raji or CCRF-CEM cell lines. In Ku80 expression, 8.8-, 1.9-, and 6.2-fold mean increases were seen in adult, pediatric ALL, and chronic lymphoid malignancies compared with the control. The Ku80 was significantly higher in adult than in pediatric ALL (P = 0.02). The amount of Ku80 expression in ALL was moderately correlated with peripheral white blood cell counts, but not with Ki67 labeling index. High Ku80 expressers (higher than the mean of all patients with ALL) tended to respond poorly to therapy: Only 22% of high Ku80 expressers achieved durable complete remission compared to 62% of low expressers. Conclusions:,Our study suggests that Ku80 might contribute to generally poor prognoses in adult ALL. [source] Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolutionEVOLUTION AND DEVELOPMENT, Issue 1 2005Shigehiro Kuraku Summary The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified. In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace. The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning. To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft-shelled turtle, Pelodiscus sinensis, using microbead-based differential cDNA analysis and real-time reverse transcription-polymerase chain reaction. We identified orthologs of Sp5, cellular retinoic acid-binding protein-I (CRABP-I), adenomatous polyposis coli down-regulated 1 (APCDD1), and lymphoid enhancer-binding factor-1 (LEF-1). Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage. In association with the expression of LEF-1, the nuclear localization of ,-catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development. These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co-option of pre-existing genes. [source] In vitro culture of skin-homing T lymphocytes from inflammatory skin diseasesEXPERIMENTAL DERMATOLOGY, Issue 5 2005Karen Bang Abstract:, We, in this study, describe how T lymphocytes in a skin biopsy can proliferate in vitro for up to 3 months by using T-cell growth factors , interleukin-2 (IL-2) and IL-4 yielding approximately 100,160 million T lymphocytes within 1 month. We established cell lines from three tuberculin skin tests, four positive patch tests, 15 of 16 biopsies from atopic dermatitis (AD), 15 of 19 biopsies from mycosis fungoides (MF), 12 of 24 biopsies from psoriasis vulgaris, which was significantly less than AD (P < 0.05), and with a reduced cumulative number of lymphocytes (P < 0.05). Omitting IL-2 and IL-4 led to immediate halt of proliferation. Blood mononuclear cells from patients and biopsies from healthy persons never gave cell lines. All cells were T lymphocytes expressing CD45RO+, HLA-DR+ and CD150. The CD7 expression was significantly increased in cell lines from AD (P < 0.05). T-cell receptor ,-chain studies by using reverse transcription-polymerase chain reaction showed that all T lymphocytes had access to the skin compartment. Single-stranded conformational analysis showed clonally expanded T cells numbering between 40 and 60 clones. After approximately 2 months of growth, the mean CD4+ : CD8+ ratio was for AD 1.20, MF 0.65 and psoriasis 0.85. Patients with AD treated with cyclosporin-A had almost no growth of CD8+ cells in vitro. Our findings indicate a changed homeostasis among skin-homing lymphocytes for in vitro culture. Our culture system of skin-homing T lymphocytes leads to a prominent cellular expansion allowing for a range of studies of in vivo activated skin T lymphocytes. [source] Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp.FEMS MICROBIOLOGY LETTERS, Issue 1 2003PCC 680 Abstract The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S -adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM. The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of ,700 mM sorbitol. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC. An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions. Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome. [source] Hepatocytes as cytotoxic effector cells can induce cell death by CD95 ligand-mediated pathway,HEPATOLOGY, Issue 6 2006Clifford S. Guy The liver plays an increasingly recognized role in the host's immune responses. The direct contribution of hepatocytes as effector cells to local immunity, pathogen containment, and liver disease is not determined. This in vitro study examined whether hepatocytes can eliminate other cells via a CD95 ligand (CD95L or FasL)/CD95 (Fas),mediated mechanism and whether this cytotoxic activity can be modulated by cytokines such as interferon gamma (IFN-,) or tumor necrosis factor alpha (TNF-,). We have found that normal woodchuck and human hepatocytes, both cultured and primary freshly isolated, as well as human HepG2 cells, intrinsically transcribe not only CD95 but also CD95L when examined by reverse transcription-polymerase chain reaction (RT-PCR) assays. The functional competence of CD95L, which was detectable in hepatocytes and HepG2 cells by Western blotting, was confirmed in bioassays by induction of apoptosis of CD95-bearing P815 and LS102.9 cell targets and validated by inhibition of the cell killing with CD95 antagonistic antibody or with a general caspase inhibitor. Furthermore, exposure of cultured hepatocytes to IFN-, or their stable transfection with IFN-, cDNA or TNF-, cDNA increased hepatocyte CD95L/CD95,mediated cell killing. In conclusion, hepatocytes express both CD95L and CD95 and they can induce death of other cells by a CD95L-dependent mechanism. IFN-, and, to a lesser extent, TNF-, can enhance hepatocyte CD95L-mediated cytotoxicity. This suggests that the local cytokine environment may modulate the hepatocyte contribution to liver immunity. (HEPATOLOGY 2006;43:1231,1240.) [source] Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated "intestinal-type" biliary cancer chemically induced in rat liverHEPATOLOGY, Issue 6 2000Guan-Hua Lai Recently, we observed that Met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), is overexpressed in epithelial cells of both early-appearing intestinal metaplastic glands in precancerous hepatic cholangiofibrotic tissue and neoplastic glands in later developed intestinal-type of cholangiocarcinoma originated from the furan rat model of cholangiocarcinogenesis when compared with normal and hyperplastic intrahepatic biliary epithelia. We now show that HGF/SF is also aberrantly expressed in a manner closely paralleling that of its receptor in the neoplastic epithelial cells of furan-induced rat cholangiocarcinomas and in a majority of metaplastic epithelial cells within earlier formed precancerous hepatic cholangiofibrotic tissue. Using in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), we further showed specific expression of HGF/SF messenger RNA (mRNA) in a novel rat cholangiocarcinoma epithelial cell line overexpressing Met. This cholangiocarcinoma cell line, termed C611B, was established from tumorigenic cells isolated from a furan-induced transplantable tumor. Moreover, we detected by in situ hybridization strong expression of HGF/SF mRNA transcripts in the cancerous epithelial glands of cholangiocarcinoma developed in recipient rats after in vivo cell transplantation of C611B cells. In contrast, mRNA transcripts and protein immunoreactivity for this cytokine were not detected in hepatocytes and biliary epithelial cells in adult normal rat liver nor in rat hyperplastic intrahepatic biliary epithelium. Our results clearly show that HGF/SF becomes aberrantly expressed in cholangiocarcinoma epithelium and in putative precancerous intestinal metaplastic epithelium induced in the liver of furan-treated rats. [source] DNA demethylation of vascular endothelial growth factor-C is associated with gene expression and its possible involvement of lymphangiogenesis in gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Shunji Matsumura Abstract Previous studies have indicated that lymphangiogenesis in solid tumors is associated with lymphatic metastasis. Overexpression of Vascular endothelial growth factor (VEGF)-C plays a major role in lymphangiogenesis in cancers. In the present study, DNA methylation and expression of the VEGF-C gene was investigated in gastric cancer (GC). Four GC cell lines (MKN-45, MKN-74, HSC-39 and HSC-43) showed no expression of VEGF-C, and the VEGF-C gene was found to be methylated in these cells. In contrast, 7 GC cell lines (MKN-1, MKN-7, MKN-28, TMK-1, KATO-III, SH101-P4 and HSC-44PE) expressed VEGF-C, and the VEGF-C gene was found to be unmethylated in these cell lines. In addition, expression of VEGF-C mRNA was retrieved by treatment with a demethylating agent, Aza-2,-deoxycytidine. In GC tissue samples, bisulfite DNA sequencing analysis revealed that VEGF-C was not methylated in 9 (29.0%) of 31 GC samples, whereas demethylation was not observed in corresponding non-neoplastic mucosa samples. Overexpression of VEGF-C mRNA was observed in 16 (51.6%) of 31 GC samples by quantitative reverse transcription-polymerase chain reaction. Of the 9 GC cases with VEGF-C demethylation, 8 (88.9%) overexpressed VEGF-C. In contrast, of the 22 GC cases without VEGF-C demethylation, 8 (36.4%) overexpressed VEGF-C (p = 0.0155). Furthermore, lymphatic vessel density determined by immunostaining of podoplanin in GC tissues was associated with overexpression of VEGF-C (p < 0.0001). These results suggest that demethylation and activation of the VEGF-C gene is likely involved in lymphangiogenesis in GC. © 2007 Wiley-Liss, Inc. [source] Interleukin-10 expression significantly correlates with minor CD8+ T-cell infiltration and high microvessel density in patients with gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 8 2006Teruhisa Sakamoto Abstract We aimed to investigate the relationships between interleukin-10 (IL-10) expression and both the clinicopathological findings and prognoses in patients with gastric cancer and to compare IL-10 expression with microvessel (MV) density and CD8+ T lymphocyte infiltration to evaluate its effects on angiogenesis and immune responses in gastric cancer. IL-10 expression was determined in gastric cancer patients by reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemical procedures. Two of 7 normal gastric tissues showed IL-10 mRNA expression, while its expressions were confirmed in all advanced gastric carcinoma tissues examined (n = 11) by RT-PCR. Immunohistochemical staining demonstrated that IL-10 expression was detected in 52 (47.7%) of 109 cases. There was a close correlation between IL-10 expression and MV density. IL-10 expression inversely correlated with CD8+ T-lymphocyte infiltration. The prognoses of patients whose tumors expressed IL-10 were significantly worse than those of patients whose tumors did not express IL-10. Multivariate analysis indicated IL-10 expression was an independent prognostic factor. IL-10 might be associated with tumor progression by stimulating angiogenesis and suppressing immune responses in gastric cancer. © 2005 Wiley-Liss, Inc. [source] Adrenomedullin regulates expressions of transforming growth factor-,1 and ,1-induced matrix metalloproteinase-2 in hepatic stellate cellsINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2006Yi Wang Summary Adrenomedullin (AM), a peptide isolated from human pheochromocytoma, can be produced and secreted by various types of cells including hepatic stellate cells (HSCs), and its possible role in HSCs is not clear now. In the present study, the interactive regulation between transforming growth factor (TGF)-,1 and AM and the effect of AM on TGF-,1-induced matrix metalloproteinase (MMP)-2 expression in HSCs were investigated. TGF-,1 and AM inhibited gene transcript level mutually (real-time reverse transcription-polymerase chain reaction). AM suppressed the protein expression level of TGF-,1 (Western blot), but TGF-,1 might have no effect on AM secretion level. MMP-2 protein expression in HSCs was increased in response to TGF-,1, and upregulation of MMP-2 expression stimulated with TGF-,1 was suppressed by AM in dose-dependent manner (Western blot). AM decreased the phosphorylation level of extracellular signal-regulated kinase (ERK) in HSCs treated with TGF-,1, and TGF-,1-induced MMP-2 expression was suppressed by adding Mitogen-activated protein Kinase/ERK (MEK) inhibitor U0126 (Western blot). Our results suggest that AM may intervene the activation of HSCs by inhibiting TGF-,1 production and TGF-,1-induced MMP-2 expression; AM may suppress the upregulation of MMP-2 expression induced by TGF-,1 partially through ERK pathway. [source] | |||||||||||||||||||||||||||||||||||||