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At least 883 records · Page 49Linked to original sources
PubMed1999

A competitive PCR assay for quantitative detection of Neospora caninum.

A quantitative-competitive PCR (QC-PCR) assay was developed for measurement of Neospora caninum levels in the tissues of infected animals. A molecule was synthesised for use in PCR as a competitor to the target Neospora-specific Nc5 genomic sequence. The assay was used to evaluate the relative level of parasites in the brain and lungs of mouse pups in a model of vertical transmission of N. caninum. Infection on day 11 of gestation resulted in similar levels of parasites in all offspring. The assay should be useful in evaluation of vaccines against Neospora infection. Incorporation of the competitor molecule in the detection assay also provides a control for PCR failure and facilitates identification of truly negative samples.

Animals↗
PubMed1998

Inhibition of human and duck hepatitis B virus by 2',3'-dideoxy-3'-fluoroguanosine in vitro.

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) has been shown to have an effect on duck hepatitis B virus (DHBV) in vivo and in vitro. In this study the inhibitory effect of FLG on DHBV and human hepatitis B virus (HBV) was evaluated in vitro. Cell lines transfected either with DHBV or HBV DNA and primary duck hepatocyte cell cultures were used. Virus production was analysed by PCR and a quantitative PCR was established for DHBV for determination of the inhibitory concentrations of the drug. 50% inhibition was achieved with an FLG concentration of 0.2 microg/ml (0.7 microM) and 90% inhibition was observed with an FLG concentration of 1.0 microg/ml (3.7 microM) using the DHBV transfected cell line. FLG showed an effect on DHBV production in primary duck hepatocyte cell cultures at concentrations down to 0.1 microg/ml (0.4 microM). However, the DHBV production returned to pre-treatment levels within a few days after cessation of treatment. HBV production in transfected cell lines was also inhibited by FLG. Both DHBV and HBV DNA-polymerases were inhibited by FLG triphosphate and 50% inhibition was observed at a concentration of 0.05 microg/ml (0.1 microM) for DHBV and 0.03 microg/ml (0.05 microM) for HBV. FLG is an efficient inhibitor of DHBV replication both in vivo and in vitro and of HBV in vitro which makes it a good candidate for treatment of HBV infections. However, it does not completely eliminate the virus since a relapse in virus production was observed when treatment was withdrawn. Therefore it would be interesting to evaluate FLG in combination with other types of anti-HBV drugs.

Animals↗
PubMed2000

Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells.

In terms of regulation of gene expression, gonadotropin-releasing hormone receptor (GnRHR) found in extrapituitary tissues has been suggested to be different from that in the pituitary. In the present study, we examined the molecular basis of this difference using the pituitary alphaT3-1 and ovarian carcinoma OVCAR-3 cells. As a first step, the different expression levels of GnRHR mRNA in the pituitary and ovarian cells were investigated using semi-quantitative RT-PCR. Quantitative analysis showed that the expression level of hGnRHR is a nine-fold higher in primary pituitary tissues than the primary culture of ovarian carcinomas (PCO). In pituitary alphaT3-1 cells, the expression level of hGnRHR was ten-fold higher than ovarian carcinoma OVCAR-3 cells. The possibility of the differential use of various cell-specific promoters in different cells was addressed by transiently transfecting cells with 3'-deletion clones of human GnRHR promoter. Sequential deletion of the 5'-flanking region of the gene revealed the use of two putative promoters, designated PR1 (-771 to -557) and PR2 (-1351 to -1022), and a negative control region (-1022 to -771), in the pituitary and ovarian cells. The same promoters appeared to be utilized for driving the basal promoter activities in both alphaT3-1 and OVCAR-3 cells, suggesting that there is no cell-specific promoter usage for the human GnRHR gene. Alternatively, the involvement of different regulatory protein factors was investigated using electrophoretic gel mobility shift assays. When end-labeled PR1 was used as a probe, two unique shifted complexes were identified in OVCAR-3 cells when compared to alphaT3-1 cells. One unique protein-DNA complex was observed in alphaT3-1 cells compared to OVCAR-3 cells when incubated with end-labeled PR2 as a probe. These DNA-protein complexes appeared to be specific, as they competed with excess amount of unlabelled competitor PR1 and PR2, respectively. In summary, it is unlikely that the use of a cell-specific promoter contributes to the different characteristics of ovarian GnRHR. Our study demonstrates that one mechanism by which cell-specific expression of human GnRHR is achieved is through the binding of distinct and/or combinations of cell-specific regulatory factors to various promoter elements in the 5'-flanking region of the gene.

Animals↗
PubMed2004

Expression of neuropeptide Y is increased in murine endometrial epithelium during the peri-implantation period under regulation by sex steroids.

Oligopeptide hormones are involved in cell-cell interaction during embryonal implantation and neuropeptide Y (NPY) is expressed in the human placenta and decidual cells in the third trimester of pregnancy. However, there is no report regarding the intrauterine localisation and the functions of NPY during the peri-implantation period. In the present study, the spatiotemporal changes in NPY expression in the murine uterus during the peri-implantation period were investigated using reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and immunohistochemical techniques, as were the effects of sex steroids on NPY mRNA expression in primary cultured murine uterine epithelial cells. Neuropeptide Y mRNA was increased in the pregnant murine uterus, as well as in the pseudopregnant murine uterus, during the peri-implantation period. Immunohistochemical analysis revealed increases in NPY expression in luminal and glandular epithelial cells and decidualised stromal cells. Neuropeptide Y mRNA expression was strongly induced in cultured epithelial cells in response to sex steroids. The data suggest that NPY is involved in cell-cell interactions during embryonic implantation.

Animals↗
PubMed2005

Contribution of a PerR-like regulator to the oxidative-stress response and virulence of Enterococcus faecalis.

PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to H2O2 challenge (P < 0.05). Expression of eight genes with potential roles in the oxidative-stress response was determined in the wild-type and perR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.

Bacterial Proteins↗
PubMed2003

Expression of pericyte, mesangium and muscle markers in malignant rhabdoid tumor cell lines: differentiation-induction using 5-azacytidine.

Malignant rhabdoid tumor (MRT) has been considered to have multiphenotypic diversity characteristics. Some MRTs exhibit a neural phenotype. However, it is still unclear whether MRT cells can display a skeletal muscle, smooth muscle or smooth muscle-like cell phenotype, like those of pericytes and mesangial cells. To determine if MRTs exhibit skeletal muscle cell or smooth muscle-like cell phenotypes, six MRT cell lines (TM87-16, STM91-01, TTC549, TTC642, YAM-RTK1 and TTC1240) were examined for markers of skeletal muscle (MyoD, myogenin, myf-5, myf-6, acetylcholine receptor-alpha, -beta and -gamma), smooth muscle (alpha-smooth muscle actin, SM-1 and SM22), and smooth muscle-like cells, such as pericytes (angiopoietin-1 and -2) and mesangial cells (megsin), using conventional RT-PCR, semi-quantitative PCR, western blotting and immunocytochemistry before and after differentiation-induction with 5-azacytidine. alpha-Smooth muscle actin and SM22 were detected in all six MRT cell lines, while MyoD and myf-5, crucial markers for skeletal myogenic determination, were not. The TM87-16 cell line expressed SM-1 and angiopoietin-1. TTC1240 also expressed angiopoietin-1. Interestingly, STM91-01 expressed megsin, a novel marker for mesangial cells, in addition to angiopoietin-1. Our results indicated that some MRTs exhibited smooth muscle and/or smooth muscle-like cell phenotypes and some renal MRTs might be of mesangial origin. Recently, smooth muscle and also smooth muscle-like cells have been considered to be of neuroectodermal origin. MRT can thus considered to belong to the category of primitive neuroectodermal tumors (PNETs) in the broad sense.

Antimetabolites, Antineoplastic↗
PubMed2006

Multilineage involvement of the fusion gene in patients with FIP1L1/PDGFRA-positive hypereosinophilic syndrome.

Myeloproliferative hypereosinophilic syndrome (MHES) is a disorder characterised by male predominance, marked eosinophilia, splenomegaly, tissue fibrosis, elevated serum tryptase and the presence of the FIP1L1/PDGFRA fusion gene in peripheral blood mononuclear cells. The characteristic hypercellular bone marrow with dysplastic eosinophils and spindle-shaped mast cells suggest that multiple lineages may be involved in the clonal process. To determine which haematopoietic lineages are involved in MHES, we purified cells of specific lineages from patients with MHES and used nested reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR and fluorescence in situ hybridisation to analyse the purified cell populations for the presence of the fusion gene. The fusion gene was detected in eosinophils, neutrophils, mast cells, T cells, B cells and monocytes. These results suggest that the mutation arises in a pluripotential haematopoietic progenitor cell capable of giving rise to multiple lineages. The basis for the preferential expansion of eosinophils and mast cells remains unclear.

Antigens, CD↗
PubMed2005

Establishment and characterization of A novel Philadelphia-chromosome positive chronic myeloid leukemia cell line, TCC-S, expressing P210 and P190 BCR/ABL transcripts but missing normal ABL gene.

A novel Philadelphia-chromosome positive (Ph+) cell line, TCC-S, has been established from a patient with Ph+ chronic myeloid leukemia (CML) in the blastic crisis. TCC-S cells were shown to express both P210 and P190 BCR/ABL transcripts by reverse transcriptase-polymerase chain reaction (PCR), although quantitative-PCR revealed that TCC-S cells mainly expressed P210 BCR/ABL transcript. Karyotype analysis revealed several triploid clones which constantly harbored two der(9)del(9) (p12)t(9;22) (q34;qll)s and two del(9) (q21)s. The der(9)del(9) (p12)t(9;22) (q34;q11) is rarely found in other CML cell lines. Moreover, to the best of our knowledge, del(9) (q21) resulting in missing of a restrict region including normal ABL gene has not been found among CML cell lines previously described. Thus, TCC-S cells with only BCR/ABL gene and no normal ABL gene may be a useful tool for functional study of ABL in Ph+ CML.

Blast Crisis↗
PubMed2002

Human immunodeficiency virus cDNA metabolism: notable stability of two-long terminal repeat circles.

Early steps of retroviral replication involve reverse transcription of the viral RNA to yield a linear double-stranded cDNA copy and then integration of the viral cDNA into a chromosome of the host cell. A portion of the viral cDNA can also follow nonproductive pathways in which it becomes circularized. In one pathway, the ends of the linear cDNA become joined together by the cellular nonhomologous DNA end-joining system to form two-long terminal repeat (2-LTR) circles. It has been argued that 2-LTR circles are quickly degraded in human immunodeficiency virus (HIV)-infected cells, allowing the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. Following this idea, detection of 2-LTR circles in patients undergoing successful highly active antiretroviral therapy has led to the proposal that viral replication persists despite treatment. We have used fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cDNA early after infection. Contrary to previous work, we find that 2-LTR circles are actually quite stable in experiments where confounding variables are controlled. Thus, studies relying on the lability of 2-LTR circles are open to reinterpretation. We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation.

Cell Line↗
PubMed2003

Effects of prostaglandin analogues on human ciliary muscle and trabecular meshwork cells.

PURPOSE: To determine the effects of prostaglandin F(2alpha) analogues on gene expression of human ciliary muscle (HCM) and trabecular meshwork (HTM) cells. METHODS: Cultures of HCM and HTM cells were established from five different donors treated for 9 days with 10 microg/mL of either latanoprost (free acid) or prostaglandin F(2alpha) ethanolamide and compared with control cells. The mRNA from the cells of the five individual donors was pooled and analyzed by using gene microarrays. Gene expression changes were confirmed by either real-time PCR or relative quantitative PCR. RESULTS: Approximately 12 genes showed a twofold or greater change in expression under experimental conditions. Four of these may alter outflow. Aquaporin-1 and versican were downregulated in the HCM, whereas IGF1 and fibroleukin were upregulated in HTM. Expression levels of TNFSF10 and promelanosome-concentrating hormone also increased in the treated HTM cells. The mRNA levels for the prostaglandin FP receptor were downregulated in the ciliary muscle cells. Optineurin and alphaB-crystallin levels remained unchanged, but myocilin in the HTM cells was decreased in some samples. CONCLUSIONS: Both analogues changed gene expression similarly in either HCM or HTM cells, but the changes appeared to be cell specific, perhaps indicating that other transcription factors are influential. Outflow of aqueous humor may be increased by the prostaglandin analogues by alterations in the extracellular matrix. Other changes may influence cellular metabolism, such as the increases in IGF1, tumor necrosis factor superfamily-10 and promelanosome-concentrating hormone.

Actins↗
PubMed2005

CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use.

BACKGROUND: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. RESULTS: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1beta, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-gamma, MIF, TGF-beta1 and TNF-alpha mRNA. We showed that the beta-2 microglobulin (beta2-MG) gene was suitable for data normalisation since the level of beta2-MG transcripts in naive PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-beta1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-beta1 mRNA in response to LPS. CONCLUSION: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.

Adult↗
PubMed2003

Immunological assays for chemokine detection in in-vitro culture of CNS cells.

Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method.

Journal Article↗
PubMed2006

Bacteroides spp. as reliable marker of sewage contamination in Hawaii's environmental waters using molecular techniques.

Standard PCR (SPCR) and quantitative PCR (QPCR) assays using primers for general and for human-specific Bacteroides 16S rRNA markers were selected as the molecular tests to assess sewage contamination in recreational waters of Hawaii and these same water samples were assayed for culturable concentrations of selected faecal microbial indicators. The results of this study showed that the general primer for Bacteroides was not useful because ambient and polluted water samples were positive for this marker. However, use of human-specific primers reliably detected sewage contamination. The human-specific Bacteroides detection data supported previously reported conclusions that concentrations of alternative faecal indicators (C. perfringens, FRNA coliphages) but not traditional faecal indicators (faecal coliform, E. coli, enterococci) are reliable indicators of faecal contamination in Hawaii's environmental waters. The QPCR assay for the human-specific Bacteroides 16S rRNA marker was faster, more sensitive and more reliable than comparable SPCR assay because OPCR assay provided additional information such as melting temperatures, which confirmed that the right amplicons were being measured and Ct values, which indicated the relative level of faecal contamination.

Bacteroides↗
PubMed2000

[Molecular diagnosis and new gene engineering in hematological malignancies].

Advancement molecular diagnostics in hematological malignancy has provided us with a broad menu of new assays and techniques. By integrating the data gleaned from these techniques we can formulate a more rational broad-based diagnosis. Hematological malignancies have traditionally been classified by morphological criteria. However, molecular advances have provided new insights in the genetic backbone of chimeric genes. These malignancies have shown chromosome abnormalities of translocation with chimeric genes, and revealed the rearrangement of chimeric genes by PCR analysis and quantitative PCR system. This review summarized the recent technology for detecting chimeric genes along with concepts of laboratory performance.

Adult↗
PubMed2001

[A study of hepatitis B virus(HBV) anti-genome and its inhibitory effect on HBV replication].

OBJECTIVE: To study the combination of triplex forming oligodeoxynucleotides (TFO) with hepatitis B virus gene and its effect on replication of HBV. METHODS: An oligodeoxynucleotide (TFO20) which can form triplex with SP1 sites in HBV core promoter was synthesized and was linked with biotin. Both the liposome-TFO20 and naked TFO20 were transfected to HepG2.2.15 cells, which was detected by SABC assay and showed signals that TFO20 was combined with HBV DNA. ELISA, reverse transcript (RT)-PCR and fluorescence quantitative PCR were used to investigate the change of HBV DNA, 3.5 kb/3.4 kb RNA, 2.4 kb/2.1 kb RNA, HBsAg and HBeAg in HepG2.2.15 cells before and after treatment with TFO20. RESULTS: The ratio of liposome-TFO20 and naked TFO20 transfected to HepG2.2.15 cells were 80% and 40%, respectively. TFO20 signals mainly distributed in the nucleus and cytoplasm. After treatment with TFO20, the level of HBV DNA was reduced from 10(7.01) to 10(4.94), while 3.5 kb/3.4 kb RNA, 2.4 kb/2.1 kb RNA, HBsAg and HBeAg were also reduced by 70.2%, 31.6%, 37.0%, 78.2% respectively. CONCLUSIONS: TFO20 can be transfected into cells by packing with liposome and can be combined with HBV DNA. TFO20 can effectively inhibit replication of HBV.

DNA Viruses↗
PubMed2003

[Effect of endogenous TGF-beta1 and TNF-alpha on the As(2)O(3) inducing apoptosis of HL-60 cells].

OBJECTIVE: To study the effect of endogenous TGF-beta(1) and TNF-alpha on As(2)O(3) inducing apoptosis of HL-60 cells. METHODS: The expressions of endogenous TGF-beta(1) and TNF-alpha in apoptotic HL-60 cells induced by As(2)O(3) were assayed by RT-PCR, quantitative RT-PCR, ELISA, DNA fragmentation and TUNEL. The effect of TGF-beta(1) and TNF-alpha antisense phosphorothioate oligodeoxynucleotides (PSODNs) on As(2)O(3) inducing apoptotic HL-60 cells was further studied. RESULTS: (1) Expressions of endogenous TGF-beta(1) and TNF-alpha were significantly up-regulated in As(2)O(3) inducing apoptotic HL-60 cells (from 13,546 +/- 124 and 497,216 +/- 187 before treatment to 23,273 +/- 229 and 674,217 +/- 189 after treatment, respectively), accompanied with down-regulated bcl-2 mRNA expression (from 10,424 +/- 274 before treatment to 3,361 +/- 89 after treatment). (2) TGF-beta(1) and TNF-alpha antisense PSODNs could rescue As(2)O(3) induced apoptosis of HL-60 cells, with a restoration of bcl-2 gene expression. CONCLUSIONS: Endogenous TGF-beta(1) and TNF-alpha played an important role in As(2)O(3) inducing HL-60 cells apoptosis through down-regulation of bcl-2 expression.

Antineoplastic Agents↗
PubMed1992

Folate-binding protein mRNA is decreased in methotrexate-resistant KB cells.

Folate-binding protein (FBP), a high-affinity folate receptor expressed in certain malignant and normal cell lines and tissues, is responsible for cellular transport of folate and structurally related antifolates such as methotrexate in human KB (nasopharyngeal carcinoma) cells. We have developed a sensitive polymerase chain reaction (PCR) assay to quantitate folate-binding protein mRNA levels. This assay was tested in KB and methotrexate (MTX)-resistant KB (KB1BT) cell lines. Based on assay results, we conclude there are 245 molecules of folate-binding protein (FBP) mRNA per KB cell, and there is an approximately 3-fold decrease in FBP mRNA levels per KB1BT cell. The data obtained by quantitative PCR correlate well with northern blot analysis of mRNA levels and membrane-associated folate-binding protein (M-FBP) levels. Moreover, when KB1BT cells were grown in the absence of MTX for 8 months, the levels of FBP and M-FBP mRNA did not change, and there was a substantial decrease in MTX uptake, compared with KB cells.

Base Sequence↗
PubMed2003

[New insight into the mechanism of p53 inducing leukemia cell apoptosis].

OBJECTIVE: To investigate the expression changes of intrinsic cytokines TGF-beta(1) and TNF-alpha, telomerase activity and bcl-2 during ongoing apoptosis of HL-60 and K562 cells induced by p53. METHODS: pN53cG (Val135), a temperature sensitive p53 mutant, which behaved like wild type p53 (wt-p53) at 32.5 degrees C, were introduced into p53-null HL-60 and K562 cells respectively by lipofectin. In the presence of G418, HL-60-pN53cG and K562 pN53cG clones expressing p53 protein were selected. The ongoing expression of intrinsic cytokines (TGF-beta(1) and TNF-alpha), bcl-2 oncogene and hTERT mRNA during the apoptosis of HL-60 and K562 cells induced by p53 and the effects of exogenous p53 gene, TGF-beta(1) and TNF-alpha antisense PS-ODNS on the apoptosis of HL-60 and K562 cells and the expression of bcl-2 were studied by RT-PCR, quantitative RT-PCR, DNA fragmentation, TdT-mediated dUTP nick end labeling (TUNEL) and flow cytometery. The levels of secreted TGF-beta(1) and telomerase activity were detected by ELISA and PCR-ELISA, respectively. RESULTS: (1) The expressions of intrinsic TGF-beta(1) and TNF-alpha mRNA were up-regulated, while that of bcl-2 and hTERT down-regulated. The levels of TGF-beta(1) in the supernatant of HL-60 and K562 cells were increased, and the level of telomerase activity decreased. (2) Antisense PS-ODNS of TGF-beta(1) and TNF-alpha could obviously inhibit the p53 inducing cell apoptosis, and restore bcl-2 mRNA and protein to pre-treated level. CONCLUSIONS: Exogenous p53 induces leukemia cell apoptosis via up-regulating the expression of intrinsic TGF-beta(1) and TNF-alpha and down-regulating the expression of hTERT and bcl-2.

Apoptosis↗