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At least 973 records · Page 54Linked to original sources
PubMed2006

Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron.

Chitin synthase (EC 2.4.1.16) is a crucial enzyme responsible for chitin biosynthesis in all chitin-containing organisms. This paper reports a complete cDNA encoding chitin synthase 1 (AqCHS1), change of AqCHS1 mRNA level in response to diflubenzuron exposure, and concentration-dependent effect of diflubenzuron on chitin synthesis in the common malaria mosquito (Anopheles quadrimaculatus). The cDNA consists of 5723 nucleotides, including an open reading frame (ORF) of 4734 nucleotides that encode 1578 amino acid residues and a non-translated region of 989 nucleotides. The deduced amino acid sequence contains all the chitin synthase signature motifs (EDR, QRRRW and SWGTR) and shows 97% identity to that of An. gambiae (AgCHS1, XM_321337). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 microg/L. As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 microg/L for 24 h. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 microg/L for 48 h resulted in decreases of chitin content by 9.0%, 43%, 58% and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased chitin synthesis may imply possible inhibition of chitin synthase, or abnormal chitin synthase translocation or chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. Further studies are necessary to elucidate the relationship between reduced chitin synthesis and increased expression of AqCHS1 in order to shed new light on trafficking and regulation of chitin biosynthesis in the mosquito affected by diflubenzuron.

Amino Acid Sequence↗
PubMed2004

Clinical applications of cell-free fetal DNA from maternal plasma.

OBJECTIVE: To describe our clinical experience with detection and analysis of cell-free fetal DNA derived from maternal plasma for prenatal sexing and fetal rhesus-D typing. METHODS: Real-time quantitative polymerase chain reactions (PCRs) of rhesus-D sequences and the SRY gene were validated and offered to patients with an enhanced risk for sex-linked fetal pathology and patients with rhesus-D antibodies. RESULTS: In the validation group, 72 samples were analyzed. Sensitivity of the rhesus-D real-time quantitative PCR in maternal plasma was 100% (95% confidence interval [CI]91.8%, 100%) and specificity was 96.6% (95% CI 82.2%, 99.9%). Sensitivity of the SRY real-time quantitative PCR was 97.2% (95% CI 85.5%, 99.9%), and specificity was 100% (95% CI 88.1%, 100%). The technique was used successfully in a clinical setting in 24 women. Overall, invasive tests were avoided in 41.7% of these patients. CONCLUSION: Detection of cell-free fetal DNA from maternal plasma is a reliable technique that can substantially reduce invasive prenatal tests.

DNA↗
PubMed2007

ERG upregulation and related ETS transcription factors in prostate cancer.

The aim of this study was to identify and validate differentially expressed genes in matched pairs of benign and malignant prostate tissue. Samples included 29 histologically verified primary tumors and 23 benign controls. Microarray analysis was initially performed using a sequence verified set of 40,000 human cDNA clones. Among the genes most consistently and highly upregulated in prostate cancer was the ETS family transcription factor ERG (ETS related gene). This finding was validated in an expanded patient series (37 tumors and 38 benign samples) using DNA oligonucleotide microarray and real-time quantitative PCR assays. ERG was 20- to more than 100-fold overexpressed in prostate cancer compared with benign prostate tissue in more than 50% of patients according to quantitative PCR. Surprisingly, ERG mRNA levels were found to be significantly higher in the endothelial cell line, HUVEC, than in the prostate cell lines PC3, DU145 and LNCaP. In situ hybridization of prostate cancer tissue revealed that ERG was abundantly expressed in both prostate cancer cells and associated endothelial cells. The consistency and magnitude of ERG overexpression in prostate cancer appeared unique, but several related ETS transcription factors were also overexpressed in matched pairs of tumor and benign samples, whereas ETS2 was significantly underexpressed. Our findings support the hypothesis that ERG overexpression and related ETS transcription factors are important for early prostate carcinogenesis.

DNA Primers↗
PubMed1998

[The experimental study on competitive PCR for quantitation of herpesviruses].

A single pair of oligonucleotide primer selected within a highly conserved region of the DNA polymerase gene in herpesviruses was synthesized. The competitive template DNA purified from cytomegalovirus (CMV) DNA was used to carry out competitive PCR amplification with herpes simplex virus type 1 (HSV1) DNA (target sequences). And anti-HSV1 effects of acyclovir (ACV) was investigated by the method. The results showed that the efficacy of PCR amplification was equal to each other(the ratio of the quantity of competitor template with DNA to the target sequence was 1.5:1). As the concentration of ACV was increased, the quantity of HSV1, DNA was decreased. It suggests that this method is practicable and some defects of mutant template can be overcomed.

Acyclovir↗
PubMed1997

Evaluation of a quantitative plasma PCR plate assay for detecting cytomegalovirus infection in marrow transplant recipients.

A plasma PCR test, using a nonradioactive PCR plate assay, was evaluated for detection of human cytomegalovirus reactivation. This assay was compared to Southern blotting and found to perform well. As a noncompetitive method of quantitation, it was similar to a competitive method for detecting the number of genome copies per milliliter of plasma in marrow transplant recipients. This is a technically simplified assay with potential for adaptation to automation.

Base Sequence↗
PubMed1998

Clinical evaluation and applications of the Amplicor HBV Monitor test, a quantitative HBV DNA PCR assay.

Viral load has emerged recently as a reliable marker of disease progression and therapeutic efficacy in chronic infections, including AIDS and hepatitis C. The clinical management of type B hepatitis could also be improved by monitoring viremia levels in patients with chronic liver disease undergoing anti-viral treatment. To address this question we evaluated the performance of a newly developed, quantitative PCR assay (Amplicor HBV Monitor test, Roche Diagnostic Systems) in the assessment of viremia changes over time in a group of 45 patients with chronic active hepatitis (CAH) who received interferon treatment. Of the 45 patients, 14 were HBsAg and anti-HBeAg positive and 31 HBsAg, HBeAg positive. Follow-up extended up to 24 months. An average of ten samples per patient were analyzed for levels of ALT, IgM anti-HBc (Abbott Laboratories), HBV DNA by in-house dot-blot hybridization and hybridization-capture assays (HBV-DNA hybrid capture kit, Murex Diagnostics) and by Amplicor HBV Monitor. A sustained biochemical response was observed at the end of treatment in 12 HBeAg-positive and in seven anti-HBeAg positive patients. This was accompanied by the disappearance of HBeAg and of HBV DNA (hybridization assays) in all cases and by the clearance of IgM anti-HBc in 70% of the cases. Viremia (quantitative PCR assay) became undetectable only in 25-30% of cases and was associated with the loss of HBsAg. A good correlation was observed between the time course of IgM anti-HBc, quantitative PCR and dot-blot hybridization although the latter missed 33% of viremic samples. Together, these results indicate that the Amplicor HBV Monitor test is a robust and standardized assay for quantifying HBV viremia levels in the range from 10(2) to 10(7) copies/ml. Compared to other current markers, viral load may provide additional clinical information by predicting long term virologic response and HBsAg clearance in patients with normal ALT at the end of interferon therapy.

Adult↗
PubMed2004

Solanum nigrum: a model ecological expression system and its tools.

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: (i) an Agrobacterium-based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

Animals↗
PubMed1994

Direct gene quantitation by PCR reveals differential accumulation of ectopic enzyme in rat-1 cells, v-fos transformants, and revertants.

Valid comparisons of gene promoter activities between different cell lines, and within a cell line, critically depend on accurate measurements of the number of genes introduced into the nuclei of cells. We have developed a simple method that allows direct and accurate quantitation of transfected plasmid DNA in cultured cells. The transfected DNA present in nuclei is copurified with genomic DNA without using phenol/chloroform extractions. DNA is amplified by PCR, and the amount of transfected DNA is read directly from a standard curve. By using the procedures described in this report, we have studied the relative expression of the Escherichia coli beta-galactosidase gene, driven by the wild-type Mo-MuLV LTR, in Rat-1 fibroblasts, FBJ v-fos-transformed Rat-1 (1302), and a revertant of v-fos-transformant (EMS-1-19) cell lines. The relative levels of expression of the transgene at 22 hr post-transfection in these three cell lines were 1:4:1, respectively, and at 48 hr post-transfection the respective ratios were 1:10.6:4. These results have significant implications for the use of cotransfected internal control plasmids to normalize data from transient transfection experiments to study promoter activities among different cell lines.

Animals↗
PubMed2006

Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA.

BACKGROUND: Since October 1997, an international standard for hepatitis C virus (HCV) nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection. METHODS: A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits. Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture's series. RESULTS: The standard calibration curve with the series of lyophilized serum showed an excellent correlation (R(2) > 0.98), slope and intercept that were similar to those from the manufacture's series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample, lower coefficients of variation between kits from different manufactures were obtained. CONCLUSION: The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection.

Calibration↗
PubMed2002

Rapid detection of common autosomal aneuploidies by quantitative fluorescent PCR on uncultured amniocytes.

Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance for women with normal results can be performed by molecular analysis of uncultured foetal cells in less than 24 h. Today, all molecular techniques developed for a fast diagnosis of aneuploidies rely on the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. Our objective was to test a chromosome quantification method based on the analysis of fluorescent PCR products derived from non-polymorphic target genes. An easy to set up co-amplification of portions of DSCR1 (Down Syndrome Critical Region 1), DCC (Deleted in Colorectal Carcinoma), and RB1 (Retinoblastoma 1) allowed the molecular detection of aneuploidies for chromosomes 21, 18 and 13 respectively. Quantitative analysis was performed in a blind prospective study of 400 amniotic fluids. Four samples (1%) could not be analysed by PCR probably because of a low concentration of foetal DNA. Follow up karyotype analysis was done on all samples and molecular results were in agreement with the cytogenetic data with no false-positive or false-negative results. Our gene based fluorescent PCR approach is an alternative molecular method for a rapid and reliable detection of aneuploidies which can be helpful for the clinical management of high-risk pregnancies.

Amnion↗
PubMed2004

The importance of the CXCL12-CXCR4 chemokine ligand-receptor interaction in prostate cancer metastasis.

AIM: Chemokines or chemotactic cytokines are known to be important in the directional migration or chemotaxis of leucocytes in conditions of homeostasis and in inflammatory or immunological responses. However, the role of chemokines is extending beyond their involvement in mediating leucocyte trafficking with an increasing body of evidence suggesting these proteins are intimately involved in many stages of tumour development and progression. Our aim was to study the role of the CXCL12:CXCR4 chemokine ligand:receptor complex in determining the organ-specific metastasis of prostate cancer. MATERIALS AND METHODS: CXCR4 mRNA expression was determined by RT-PCR in 3 metastatic prostate cancer cell lines DU145, LNCaP and PC3, the primary prostate cancer cell line 1542 CPT3X and the normal prostate epithelial cell lines 1542 NPTX and Pre 2.8. This was followed by Taqman quantitative PCR analysis of CXCR4 mRNA in these cell lines. Flow cytometry analysis was then used to measure the expression of the CXCR4 receptor protein on the cell surface. The influence of the receptor on cell migration was studied using Transwell, Migration Assays. Finally, Taqman quantitative PCR was performed on RNA obtained from laser microdissected fresh primary prostate tumour and benign tissue samples from patients. RESULTS: In DU145, LNCaP and PC3 CXCR4 mRNA expression was approximately 1000, 400 and 21 times respectively that of 1542 NPTX, Pre 2.8 and 1542 CPT3X. In patient primary tumour samples and patient benign tissue specimens CXCR4 mRNA expression was similar to that of the metastatic cell line DU145. Flow cytometry analysis showed that significantly higher levels of the CXCR4 receptor were present on the cell surface of the 3 metastatic cell lines. Migration studies revealed that chemotaxis of the metastatic cell lines PC3 and DU145 was enhanced by CXCL12 ligand and inhibited by antibody to CXCR4. CXCL12 did not influence the migration of the normal prostate epithelial cell line 1542 NPTX. CONCLUSIONS: We have demonstrated that human prostate cell lines derived from metastases express functional CXCR4 receptor and that CXCL12 ligand enhances their migratory capabilities. Also, laser microdissected primary patient tumours and patient benign tissue specimens express CXCR4 mRNA at high levels (it is suggested that post-transcriptional modification of the CXCR4 receptor plays a major role in regulating protein expression). These results suggest prostate cancers may be influenced by the CXCL12:CXCR4 pathway during metastasis. This pathway would provide a novel target for therapeutic intervention.

Cell Line, Tumor↗
PubMed2004

Inactivation of the ARF-MDM-2-p53 pathway in sporadic Burkitt's lymphoma in children.

Burkitt's lymphomas (BLs) are characterized by an activated MYC gene that provides a constitutive proliferative signal. However, activated myc can initiate ARF-dependent activation of p53 and apoptosis as well. Data derived from cell culture and animal models suggest that the inactivation of the ARF-MDM-2-p53 apoptotic signaling pathway may be a necessary secondary event for the development of BL. This has not been tested in freshly excised BL tissue. We investigated the ARF-MDM-2-p53 pathway in tumor specimen from 24 children with sporadic BL/B-ALL. Direct sequencing revealed a point mutation in the p53 gene in four BL. Overexpression of MDM-2 was evident in 10 of the BL samples analyzed by real-time quantitative PCR. Deletion of the CDKN2A locus that encodes ARF or reduced expression of ARF could not be detected in any BL by fluorescence in situ hybridization analysis or real-time quantitative PCR, respectively. Our results indicate that the ARF-MDM-2-p53 apoptotic pathway is disrupted in about 55% of the cases of childhood sporadic BL. We suggest that in addition to the inactivation of the ARF-MDM-2-p53 protective checkpoint function other antiapoptotic mutations may occur in a substantial part of children with sporadic BL.

Adolescent↗
PubMed2004

Change in bacterial community structure during in situ biostimulation of subsurface sediment cocontaminated with uranium and nitrate.

Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the alpha, beta, delta, and gamma subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing delta-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the delta-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.

Bacteria↗
PubMedDate not supplied

Application of real-time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped sea bream, Lithognathus mormyrus.

The striped sea bream (Lithognathus mormyrus) has been recently introduced as a bioindicator fish species in Mediterranean coastal habitats. The purpose of this study was to apply a real-time PCR assay for the determination of absolute levels of hepatic VTG transcript in this fish, identifying minimal and maximal levels and establishing the relationship between VTG RNA levels and ovarian development. A partial VTG cDNA was cloned from hepatic RNA of a striped sea bream female. Specific primers were designed based on its sequence and used for PCR and also for in vitro synthesis of partial VTG RNA standard. Hepatic VTG transcript levels were quantified by real-time PCR, using serially diluted VTG RNA standards for construction of a calibration curve and equation. VTG RNA levels were normalized to total RNA or 18S ribosomal RNA (determined by real-time PCR). VTG RNA was hardly detected in the liver of males, or females with small oocytes (diameter < 100 microm). A linear correlation was found between these two parameters at larger oocyte diameter (> 150 microm). VTG level reached a maximum of 204 fmol/pmol 18S RNA or 49 fmol/microg RNA. The results demonstrate the wide dynamic range of the established real-time PCR assay.

Animals↗
PubMed2004

Increased metallothionein in light damaged mouse retinas.

Oxidative stress plays a role in human age-related macular degeneration and in the light damage model of retinal degeneration. Metallothionein (MT), an antioxidant, has been reported to protect retinal pigment epithelial cells against apoptosis and oxidative stress. The purpose of this study was to evaluate changes in MT expression level and retinal localization following light damage. To accomplish this, Balb/c mice were exposed to cool white fluorescent light (10,000 lx) for 7 hr. In three independent experiments, at several intervals after the light injury, retinal MTs were studied at the protein level by immunohistochemistry (IHC) and Western analysis, and at the mRNA level by quantitative PCR with isoform-specific primers. Western analysis and IHC indicated an increase in metallothionein protein following light damage. MT localized to the retinal pigment epithelium and several layers of neural retina. Quantitative PCR identified the expression of MT I-III isoforms, not the MT IV isoform in the mouse retina, and, following light damage, showed increased expression of retinal MT-I and MT-II mRNAs by 8- and 22-fold, respectively. Increased expression of the antioxidant MT in the light damaged mouse retina suggests that upregulation of MT is an important acute retinal response to photo-oxidative stress.

Animals↗
PubMed2006

Overexpression of cyclase-associated protein 2 in multistage hepatocarcinogenesis.

PURPOSE: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC. EXPERIMENTAL DESIGN: We measured the level of CAP2 mRNA by real-time quantitative PCR. We raised a polyclonal antibody against CAP2 and we confirmed the expression of CAP2 by immunoblotting and immunohistochemistry in HCC cell lines and HCC tissues. RESULTS: According to real-time quantitative PCR, the level of CAP2 mRNA was up-regulated in early HCC when compared with noncancerous liver tissue, and it was further up-regulated in progressed HCC. We raised a polyclonal antibody against CAP2, which showed a single 53-kDa band of strong intensity in the human HCC cell lines and HCC tissues but only a weak band in the noncancerous liver tissues in Western blot analysis. Immunohistochemical examination of CAP2 revealed its significant overexpression in early HCC when compared with noncancerous and precancerous lesions and in progressed HCC when compared with early HCC. CONCLUSION: Our findings show that CAP2 is up-regulated in HCC when compared with noncancerous and precancerous lesions. This is the first report that proves that CAP2 is up-regulated in human cancers and that this is possibly related to multistage hepatocarcinogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

[Identification of genes related to cell phenotypic transition by differential display analysis].

To identify the genes that are differentially expressed during the phenotypic transition from vascular adventitial fibroblasts to myofibroblasts, the adventitial fibroblasts were cultured from rat thoracic aorta, and myofibroblasts were obtained by treatment of fibroblasts with TGF-beta1. Differential display PCR (DD-PCR) was used to screen for differentially expressed genes by comparison of mRNA extracted from the two cell populations. Bands upregulated or downregulated on DD gels were excised, reamplified, cloned and sequenced. DD results were verified by quantitative PCR and Northern blot analysis.Antisense oligonucleotide was transfected to study the effect of osteopontin on migration of AF. Differential display showed a significant difference in gene expression profile between the two cell types. A transcript that was downregulated in myofibroblasts showed high DNA sequence homology to part of the gene for NADH dehydrogenase subunit 5. An upregulated transcript showed significant sequence homology to osteopontin gene. Quantitative PCR and Northern blot analysis confirmed the DD results. Among the other differential bands detected, 4 candidate sequences showed no homology to the known genes. The AF numbers of migration were significantly decreased by use of OPN antisense oligonucleotide. This study suggests that the downregulation of gene encoding NADH dehydrogenase subunit 5 and upregulation of osteopontin gene and several other unknown genes may be involved in the phenotypic transition of adventitial fibroblasts to myofibroblats. Inhibition of the expression of OPN may play an important role in the process of vascular remodeling.

Animals↗
PubMed2000

Quantitative immunocapture PCR assay for detection of Campylobacter jejuni in foods.

The rapid detection of food-borne bacterial pathogens as part of a quality control program is necessary for the maintenance of a safe food supply. In this report, we present our findings for an immunocapture PCR method for the detection of Campylobacter jejuni in foods. The method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. Assay results are quantitative, and one cell in a milliliter sample can be detected. Application of the method to spiked milk samples and chicken skin washes did not affect the sensitivity of the assay.

Animals↗