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TaqMan
Terms modified by TaqMan Selected AbstractsOestrogen receptor , is expressed in adult human skeletal muscle both at the mRNA and protein levelACTA PHYSIOLOGICA, Issue 4 2003A. Wiik Abstract Aim:, There are two known oestrogen receptors (ER), oestrogen receptor , (ER,) and the recently cloned oestrogen receptor , (ER,). ER, mRNA has been detected in mouse, rat, bovine and human skeletal muscle. ER, mRNA has been detected in bovine skeletal muscle. To our knowledge, no study has investigated the expression of oestrogen receptor , in human skeletal muscle. Therefore, the primary aim of the present investigation was to study ER, mRNA and protein expression in human skeletal muscle. In addition the ER, expression was also studied. Methods:, Muscle biopsies were taken from vastus lateralis in six healthy adults (three women and three men). mRNA expression was detected with real-time PCR (TaqMan) and protein localization by immunohistochemistry. Results:, A clear expression of ER, and ER, mRNA was seen in skeletal muscle in all subjects. The ER, mRNA expression was 180 fold higher compared with that of ER, mRNA. Immunohistochemistry demonstrated positive staining for ER,, but not for ER,, with localization to the nuclei of skeletal muscle fibres. On average, 70% of all nuclei were ER, -positive. Conclusion:, The present study shows for the first time ER, mRNA and protein expression in human skeletal muscle tissue in both males and females. [source] Validation of a real-time PCR method for the detection of Phytophthora ramorum1EPPO BULLETIN, Issue 2 2006A. Chandelier To validate a real-time PCR method for the detection of Phytophthora ramorum, an intra-laboratory procedure was developed. The specificity of the TaqMan probe/primer sets was determined by carrying out real-time PCR on total DNA extracted from pure culture of several Phytophthora species. The limit of detection and the potential effects of plant substrates were evaluated by conducting the test on total DNA from healthy plant materials (Rhododendron spp., Viburnum spp. and Pieris spp.) spiked with known amounts of P. ramorum genomic DNA. The PCR efficiency was estimated through the linear regression of the dilution curve. Precision of the TaqMan assay was assessed on material from a single artificially infected plant (Rhododendron spp.). Two kinds of tissues were tested: a severely infected twig and an apparently healthy leaf. Intra-assay repeatability was evaluated on 10 replicates of the same DNA sample analysed in a single assay. Inter-assay reproducibility was evaluated on the same DNA sample amplified over five separate assays while the intersample reproducibility was evaluated on separate DNA extractions of four samples from both plant tissues amplified in a single assay. [source] Real-time PCR assay for the identification of Thrips palmi,EPPO BULLETIN, Issue 1 2005L. F. F. Kox Since Thrips palmi became a regulated pest for most European countries, inspections at points of entry into Europe and monitoring in Europe have intensified not only for T. palmi but also for thrips as a whole. Morphological identification of thrips is performed on adults and to a lesser extent on second-stage larvae only, because no adequate identification keys for the separation of species based on the characteristics of eggs, first-stage larvae, pre-pupae or pupae are available. We have developed a real-time PCR assay based on TaqMan. A T. palmi -specific set of primers and probe were selected within the mitochondrial cytochrome oxidase I (COI) gene. The specificity of the assay was assessed using 15 specimens of Thrips palmi and 61 specimens of 23 other thrips species commonly occuring in Europe. All T. palmi specimens were detected, and no cross reactions with other thrips were observed. The method was tested on single larvae and adults and proved to be applicable for both those stages of T. palmi. [source] Use of multiplex real-time PCR (TaqMan) for the detection of potato viruses,EPPO BULLETIN, Issue 3-4 2000N. Boonham Certain viruses affect the quality of potato tubers for either table use or processing. Visual discrimination of these viruses is problematic because of variable symptoms, but is important if proper controls are to be implemented. Work at the Central Science Laboratory has concentrated on the detection of Potato mop-top pomovirus (PMTV), Tobacco rattle tobravirus (TRV) (both associated with the disease spraing) and the tuber necrotic strain of Potato Y potyvirus (PVYNTN), the symptoms of which can often be confused with spraing. A nucleic acid-based approach has been adopted as TRV is often found as naked RNA with no associated coat protein, and accurate discrimination of PVY strains is impossible by serology. The multiplex TaqMan assay developed in this work streamlines the testing, replacing two separate tests currently used (a TRV RT-PCR and a PMTV enzyme-linked immunosorbent assay) with a single-tube assay, which has no post-PCR manipulations. The assay has been shown to be more sensitive than either of the tests which it replaces, allowing 100- and 10000-fold increases in sensitivity for TRV and PMTV detection respectively. The test reliably detected over 40 different isolates of TRV and PMTV obtained from a wide range of cultivars and locations, including samples where existing tests failed. A PCR-based method capable of discriminating strains of PVY was also developed. [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] Common polymorphisms in the interleukin-22 gene are not associated with chronic plaque psoriasisEXPERIMENTAL DERMATOLOGY, Issue 9 2009Wolfgang Weger Abstract Background:, Psoriasis is a chronic inflammatory skin disease. Among other cytokines, interleukin 22 (IL-22) has been implicated in the pathogenesis of chronic plaque psoriasis. The purpose of this study was to investigate a hypothesized association between common IL-22 gene polymorphisms and chronic plaque psoriasis. Methods:, Genotypes of 10 common polymorphisms of the IL-22 gene were determined by fluorogenic 5, exonuclease assays (TaqMan) in 475 patients with chronic plaque psoriasis and 252 controls. Results:, Two blocks of high linkage disequilibrium, formed by eight polymorphisms upstream of exon 5 (rs2227485, rs2227491, rs2046068, rs1179251, rs1012356, rs2227501, rs2227503, rs976748) and two polymorphisms in the 3, near gene region (rs1182844, rs1179246), were observed within the IL-22 gene. Neither single polymorphisms nor haplotypes were significantly associated with the presence or clinical features of chronic plaque psoriasis (P > 0.05). Conclusions:, Our data suggest that the investigated IL-22 gene polymorphisms are unlikely major risk factors for chronic plaque psoriasis. [source] Mitochondrial superoxide dismutase and glutathione peroxidase in idiosyncratic drug-induced liver injury,,HEPATOLOGY, Issue 1 2010M. Isabel Lucena Drug-induced liver injury (DILI) susceptibility has a potential genetic basis. We have evaluated possible associations between the risk of developing DILI and common genetic variants of the manganese superoxide dismutase (SOD2 Val16Ala) and glutathione peroxidase (GPX1 Pro200Leu) genes, which are involved in mitochondrial oxidative stress management. Genomic DNA from 185 DILI patients assessed by the Council for International Organizations of Medical Science scale and 270 sex- and age-matched controls were analyzed. The SOD2 and GPX1 genotyping was performed using polymerase chain reaction restriction fragment length polymorphism and TaqMan probed quantitative polymerase chain reaction, respectively. The statistical power to detect the effect of variant alleles with the observed odds ratio (OR) was 98.2% and 99.7% for bilateral association of SOD2 and GPX1, respectively. The SOD2 Ala/Ala genotype was associated with cholestatic/mixed damage (OR = 2.3; 95% confidence interval [CI] = 1.4-3.8; corrected P [Pc] = 0.0058), whereas the GPX1 Leu/Leu genotype was associated with cholestatic injury (OR = 5.1; 95%CI = 1.6-16.0; Pc = 0.0112). The presence of two or more combined risk alleles (SOD2 Ala and GPX1 Leu) was more frequent in DILI patients (OR = 2.1; 95%CI = 1.4-3.0; Pc = 0.0006). Patients with cholestatic/mixed injury induced by mitochondria hazardous drugs were more prone to have the SOD2 Ala/Ala genotype (OR = 3.6; 95%CI = 1.4-9.3; Pc = 0.02). This genotype was also more frequent in cholestatic/mixed DILI induced by pharmaceuticals producing quinone-like or epoxide metabolites (OR = 3.0; 95%CI = 1.7-5.5; Pc = 0.0008) and S-oxides, diazines, nitroanion radicals, or iminium ions (OR = 16.0; 95%CI = 1.8-146.1; Pc = 0.009). Conclusion: Patients homozygous for the SOD2 Ala allele and the GPX1 Leu allele are at higher risk of developing cholestatic DILI. SOD2 Ala homozygotes may be more prone to suffer DILI from drugs that are mitochondria hazardous or produce reactive intermediates. (HEPATOLOGY 2010) [source] Molecular neonatal screening for homocystinuria in the Qatari population,HUMAN MUTATION, Issue 6 2009Johannes Zschocke Abstract We report the results of molecular neonatal screening for homocystinuria (cystathionine beta-synthase deficiency) in neonates of Qatari origin, developed in conjunction with a novel biochemical screening approach. DNA was extracted from dried blood spots (DBS); the prevalent Qatari CBS gene mutation p.R336C (c.1006C>T) and a second mutation were tested with specific TaqMan assays. Over a period of 2 years we screened 12,603 neonates and identified six affected neonates homozygous for p.R336C. There were 225 heterozygous carriers for p.R336C. One additional child with homocystinuria detected through biochemical screening was homozygous for a mutation not previously identified in Qatar. Homocystinuria in the Qatari population has an incidence of 1:1,800, the highest in the world and even higher than previously estimated. Allele frequency of the mutation p.R336C is approximately 1%, displaying a significant deviation from Hardy Weinberg equilibrium. In conclusion, first-line molecular neonatal screening is technically feasible and may be developed as an option for presymptomatic identification of genetic disorders caused by specific mutations or a limited number of prevalent mutations. However, sensitivity for the diagnosis of disorders caused by various mutations is limited even in a homogeneous population such as Qatar. Hum Mutat 30:1,2, 2009. © 2009 Wiley-Liss, Inc. [source] Novel PlexorÔ SNP genotyping technology: comparisons with TaqMan® and homogenous MassEXTENDÔ MALDI-TOF mass spectrometry,HUMAN MUTATION, Issue 9 2007E.A. Tindall Abstract Analysis of SNPs for association, linkage, haplotype, and pharmacogenetic studies has led to a dramatic increase in the number and evolution of medium- to high-throughput genotyping technologies. This study introduces PlexorÔ as a new method for medium-throughput (single SNP) genotyping. We compare this fluorescent-based chemistry for call rate, accuracy, affordability, throughput, and overall efficiency against two commonly used technologies. These include fluorescent-based TaqMan® allelic discrimination for single SNP analysis (medium-throughput) and the homogenous MassEXTENDÔ (hMEÔ) chemistry using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for multiple SNP analysis (high-throughput). Analysis of 11 SNPs, including all six possible nucleotide substitutions, showed PlexorÔ to be highly comparable for both call rate (94.7%) and accuracy (99.2%) to the TaqMan® (94.6% and 99.8%, respectively) and hMEÔ (91.9% and 98.1%, respectively) chemistries. We demonstrate that this novel method is an efficient, cost-effective alternative to TaqMan® genotyping commonly used in diagnostic settings. Hum Mutat 28(9), 922,927, 2007. © 2007 Wiley-Liss, Inc. [source] Development and design of a ,ready-to-use' reaction plate for a PCR-based simultaneous detection of animal species used in foodsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2007Ines Laube Summary Different TaqManTM -polymerase chain reaction systems have been developed, which allow the detection of even minute amounts of beef, pork, lamb, goat, chicken, turkey and duck in processed foods. The species-specific systems are able to amplify DNA regions with no more than 108 bp in size (exception: duck, 212 bp) located on the single-copy genes cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase, ryanodine receptor and interleukin -2 precursor. The parallel detection of the common ingredient ,meat' produced from mammals and poultry was based on the amplification of a region of the myostatin gene. The limit of detection was determined to be ten genome copies for each system. The relative SD under repeatability condition was below 30%. In addition, a ,ready-to-use' reaction plate has been developed, which makes it possible to investigate the presence of the seven animal species in parallel after a single real-time run. [source] Real-time PCR quantification of haematopoietic chimerism after transplantation: a comparison between TaqMan and hybridization probes technologiesINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 1p1 2010J. MARTINEZ-LOPEZ Summary This study aimed to compare the sensitivity and accuracy of two methods of quantitative real-time polymerase chain reaction (qrt-PCR), in order to determine haematopoietic chimerism (CH): single nucleotide polymorphisms using TaqMan (TM) probes and insertion/deletion polymorphisms using Hybridization (Hyb) probes. A total of 106 samples from 20 patients who underwent allogenic stem cell transplantation (n = 14) or live-donor liver transplantation (n = 6) were studied. The mean level of chimerism was 8.37% for the TM method and 7.73% in the Hyb method, which was not significantly different (P = 0.69). The Pearson correlation coefficient between the two methods was r = 0.91 (P < 0.001). The estimation of the regression line, using the Passing and Balbock method was Intercept A ,0.0381 [95% confidence interval (CI) ,0.1265 to 0.0296) and Slope B: 1.04609(95% CI 0.9349,1.161). Bland,Altman data showed that the standard deviations, which differed between the two methods (%Hyb,%TM), were 0.98 and ,1.28. The accuracy and sensitivity of qrt-PCR chimerism is independent of the method used if the optimization is adequate and satisfies the criteria for adequate study. Real-time PCR, independent of the method adopted, is a very good tool for study levels of CH. [source] Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4AJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2001Kimberly B. Bleicher Abstract Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan®) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan® probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3, end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:133,142, 2001 [source] Undetectable HIV-1 RNA load with the Cobas TaqMan v1.0 in a patient diagnosed at the time of primary HIV-1 infectionJOURNAL OF MEDICAL VIROLOGY, Issue 11 2010Jade Ghosn Abstract Diagnosis of primary HIV-1 infection is challenging due to the presence of a serological window; thus, HIV-1-RNA quantitation and/or measurement of p24 antigenemia are recommended in such cases. A patient was diagnosed at the time of primary HIV-1 infection, he harbored a CFR02_AG subtype virus; quantitation of plasma HIV-1-RNA yielded an undetectable result according to one commercial assay, while HIV-1-RNA was detectable when measured with three other assays. J. Med. Virol. 82:1816,1818, 2010. © 2010 Wiley-Liss, Inc. [source] Virus load dynamics of individual CMV-genotypes in lung transplant recipients with mixed-genotype infectionsJOURNAL OF MEDICAL VIROLOGY, Issue 8 2008Irene Görzer Abstract Human cytomegalovirus (CMV) is a major cause of disease and transplant dysfunction in lung transplant recipients. Simultaneous emergence of more than one CMV-genotype can occur, and appears to be disadvantageous for the patient. In this study, the dynamics of individual CMV-genotypes in blood and lung was assessed within mixed CMV-genotype populations emerging after lung transplantation. In 69 plasma and 76 bronchoalveolar lavage samples of 16 lung transplant recipients with mixed CMV-genotype infections within the first year posttransplantation each of the major glycoprotein B (gB) and glycoprotein H (gH) genotypes was selectively quantified by genotype-specific quantitative TaqMan assays. The data obtained revealed that individually different genotype dynamics occurred for the individual patients and that the relative levels of the genotypes to each other may change over time. The quantitative development was independent of the specific gB,gH-genotype. In 10 of the 16 lung recipients the patient's individual genotype composition was the same in blood and lung. Genotype development during the follow-up was influenced by antiviral treatment. These data show for the first time that the CMV load used as diagnostic tool after transplantation is not always a constant entity but reflects the sum of the individual CMV-genotype dynamics developing over time. J. Med. Virol. 80:1405,1414, 2008. © 2008 Wiley-Liss, Inc. [source] Detection of human sapovirus by real-time reverse transcription-polymerase chain reactionJOURNAL OF MEDICAL VIROLOGY, Issue 10 2006Tomoichiro Oka Abstract Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI,GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5,×,107 to 2.5,×,101 copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses. J. Med. Virol. 78:1347,1353, 2006. © 2006 Wiley-Liss, Inc. [source] Quantifying Phytophthora medicaginis in Susceptible and Resistant Alfalfa with a Real-Time Fluorescent PCR AssayJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2003G. J. Vandemark Abstract A real-time fluorescent PCR assay using a set of specific primers and a fluorochrome-labelled probe (TaqMan) was developed to quantify the amount of Phytophthora medicaginis DNA in alfalfa plants that were classified as either resistant or susceptible to the pathogen based on visual assessment of disease response. The assay clearly discriminated among three standard check alfalfa populations with different levels of resistance based on the analysis of DNA extracted from the roots of bulked plant samples. In two independent experiments, the Spearman rank correlation between pathogen DNA content and the number of susceptible plants in a bulked sample was greater than 0.89 and highly significant (P<0.0001). Significantly less pathogen DNA was detected in bulked samples of a highly resistant check population than in bulked samples from more susceptible check populations. Analysis of individual plants indicated that significantly less pathogen DNA was detected in resistant plants than in susceptible plants. Applications of the assay are considered for breeding programs and the study of microbial population dynamics in plants simultaneously infected with different pathogens. [source] OPRM1 Asn40Asp Predicts Response to Naltrexone Treatment: A Haplotype-Based ApproachALCOHOLISM, Issue 3 2009Gabor Oroszi Background:, Individualized pharmacotherapy requires identification of genetic variants predictive of treatment response. In OPRM1, Asn40Asp has been reported to be predictive of response to naltrexone treatment. Nevertheless, the in vitro function of the polymorphism remains elusive and over 300 OPRM1 sequence variants have been identified to date. Therefore we used a haplotype-based approach to capture information of other genetic variants that might predict treatment response to naltrexone in the COMBINE Study. Methods:, 5, nuclease genotyping assays (TaqMan®) were applied for 10 SNPs. Five-locus haplotypes in 2 OPRM1 haplotype blocks were assigned to Caucasian participants. The relationship of the haplotypes to medication reflected by "good clinical outcome" was analyzed in 306 Caucasians treated without Combined Behavioral Intervention and with either naltrexone or placebo. Results:, A significant haplotype by medication interaction (p = 0.03) was found in OPRM1 block 1. Naltrexone-treated alcoholics with haplotype AGCCC, the single haplotype carrying the Asp40 allele had the highest percent of good clinical outcome. When interaction of genotypes at each of the 5 loci comprising block 1 with medication was examined, only the Asn40/Asp40 and Asp40/Asp40 genotypes were found to significantly interact with naltrexone treatment. No haplotype by medication interaction was documented in OPRM1 block 2. Conclusions:, Our haplotype-based approach confirms that the single OPRM1 locus predictive of response to naltrexone treatment is Asn40Asp in exon 1. A substantial contribution of any other OPRM1 genetic variant to interindividual variations in response to naltrexone treatment (at least in terms of good clinical outcome) is not supported by our findings. [source] Association Between Alcoholism and ,-Amino Butyric Acid ,2 Receptor Subtype in a Russian PopulationALCOHOLISM, Issue 4 2005Jaakko Lappalainen Background: Two recent large genetic studies in the US population have reported association between genetic variation in ,-amino butyric acid ,2 receptor subtype (GABRA2) and risk for alcohol dependence. The goal of this study was to test whether GABRA2 is associated with alcohol dependence in a sample of Russian alcohol-dependent men. Methods: A total of 113 Russian alcohol-dependent men and 100 male population control subjects were recruited in St. Petersburg and genotyped for seven GABRA2 single-nucleotide polymorphisms (SNPs) using real-time PCR (TaqMan). Six SNPs were located in a GABRA2 haplotype block previously associated with alcohol dependence (AD) in the US population. SNPs and haplotypes were tested for an association to AD using ,2 analysis and a likelihood ratio-based statistic implemented in the software COCAPHASE. Results: Significant associations between two SNPs and AD were observed (p < 0.05). In addition, a trend-level association was observed between AD and three adjacent SNPs (p < 0.1). Associated alleles were carried in a haplotype that was present at frequencies of 0.37 and 0.48 in the control and alcohol-dependent populations, respectively (p < 0.06). Tight linkage disequilibrium spanning from the central portion of the gene to the 3, end was observed in this population. Comparison of the findings to the previously published studies in the US population revealed a highly similar linkage disequilibrium pattern in this population. Conclusions: These findings suggest that genetic variants of GABRA2 increase risk for AD in the Russian population and provide additional support to the hypothesis that polymorphic variation at the GABRA2 locus plays an important role in predisposing to AD at least in European-ancestry populations. [source] Thr105Ile, a Functional Polymorphism of Histamine N-Methyltransferase, Is Associated with Alcoholism in Two Independent PopulationsALCOHOLISM, Issue 3 2005Gabor Oroszi Background: Histamine is expressed in cortical and limbic areas that are involved in emotion and cognition and modulates these behaviors. H1 receptor antagonists are sedative. Histamine N-methyltransferase (HNMT) catalyzes the N, methylation of histamine, the sole pathway for termination of the neurotransmitter action of histamine in mammalian brain. A common and functionally significant polymorphism, a C314T transition in exon 4 of the HNMT gene results in a Thr105Ile substitution of the protein encoded. The Thr105 allele is associated with ,2-fold higher enzyme activity, leading to the prediction that it might be associated with diminished histamine levels, resulting in differences in anxiety, cognition, and sedation that play important roles in alcoholism. In two ethnically distinct populations, we tested whether the Thr105Ile polymorphism was associated with alcoholism and with harm avoidance, a dimensional measure of anxious personality. Methods: A 5, exonuclease assay (TaqMan) was used to genotype Thr105Ile in psychiatrically interviewed Finnish Caucasian (n= 218) and Plains American Indian (n= 186) alcoholics, along with ethnically matched, psychiatrically interviewed, controls (Finns: n= 313, Plains Indian: n= 140). Results: Ile105 allele frequencies were significantly lower in alcoholics compared with nonalcoholics in both populations (Finns: 0.12 vs. 0.17, ,2= 6, p= 0.015; Plains Indians: 0.03 vs. 0.08, ,2= 5, p= 0.023). Genotype distributions also differed significantly. In Finns, Ile105 showed borderline significance for an association with lower harm avoidance (p= 0.070) after correcting for alcoholism diagnosis. Conclusions: Decreased levels of brain histamine consequent to the Thr105 allele may result in higher levels of anxiety and, as a consequence, vulnerability to alcoholism. [source] Allergen-induced in vitro expression of IL-18, SLAM and GATA-3 mRNA in PBMC during sublingual immunotherapyALLERGY, Issue 8 2007J. Savolainen Background:, Signalling lymphocytic activation molecule (SLAM) and interleukin (IL)-18 induce interferon (IFN)-, production from Th1 cells. The allergen-induced SLAM and IL-18 mRNA expressions are increased during subcutaneous immunotherapy (SCIT), but nothing is known about their role during sublingual immunotherapy (SLIT). Transcription factor GATA-3 is associated with Th2 cells but its role in SCIT and SLIT is yet unexplored. This study was undertaken to analyse the allergen induced in vitro mRNA expression of IL-18, SLAM and GATA-3 in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR) during SLIT. Methods:, Ten patients with AR undergoing pollen SLIT with a weekly dose of 200 000 SQ-U, 10 with 24 000 SQ-U of mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included. Peripheral blood mononuclear cell were stimulated with birch extract prior to, after 1 and 2 years of the treatment. The mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan®; Applied Biosystems, Foster City, CA, USA). Results:, The expression of IL-18 mRNA was increased in the high-dose group in comparison to the placebo group after 1 year of therapy (P = 0.028) and had an inverse correlation with the late phase skin reaction after the second study year (r = ,0.41, P = 0.041). SLAM mRNA expression increased in the high-dose group from baseline to 1 year (P = 0.028) and correlated with IL-10 (r = 0.96, P < 0.0001) and transforming growth factor-, (r = 0.80, P = 0.0037) mRNA expression. No significant changes were seen in GATA-3 mRNA expression. Conclusions:, During SLIT, IL-18 and SLAM are upregulated, suggesting that the Th2 type inflammatory response is downregulated during SLIT by increased Th1 type response. [source] Dopamine Receptor D2 Polymorphism Moderates the Effect of Parental Education on Adolescents' School PerformanceMIND, BRAIN, AND EDUCATION, Issue 2 2008Liisa Keltikangas-Järvinen ABSTRACT, High parental socioeconomic status is known to have a positive effect on students' academic achievement. We examined whether variation in the dopamine receptor gene (DRD2 polymorphism, rs 1800497) modifies the association between parental educational level and school performance in adolescence. The participants were a randomly selected subsample of individuals participating in the Cardiovascular Risk in Young Finns study (921 girls and 742 boys) aged 12,15 years at the time school performance was assessed. The genotyping was performed using TaqMan 5,'-nuclease assay. A significant interaction was found between childhood parental educational level and students' DRD2 polymorphism on academic achievement after adjustment for age, gender, household income, parental occupation, maternal nurturance, hyperactivity, and sociability. Parental educational level was significantly positively associated with school achievement in the A2/A2 (n = 1,061) and the A1/A2 (n = 529) genotype groups, but was negative and statistically insignificant in participants carrying the A1/A1 (n = 73) genotype. It is concluded that the extent to which parental education status affects an individual's academic achievement may be dependent on the individual's genetic constitution. The findings may increase an acceptance of genetic influence in education, and, consequently, may increase accurateness of educational interventions. [source] Single nucleotide polymorphisms in the hypoxia-inducible factor-1 gene and colorectal cancer riskMOLECULAR CARCINOGENESIS, Issue 9 2010Gudrun Knechtel Abstract With an incidence of about 300,000 new cases colorectal cancer (CRC) is the second leading cause of cancer-related death in Europe and the United States. Environmental and genetic factors influence CRC risk. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric protein composed of two subunits, HIF-1 alpha and HIF-1 beta, plays a critical role in oxygen homeostasis and is involved in angiogenesis and cell proliferation. The gene for the HIF-1 alpha subunit (HIF1A) carries two common missense mutations,P582S (rs11549465) and A588T (rs11549467),which both have been related to increased trans-activation capacity of HIF1A. In our case,control study we investigated the association between these polymorphisms and CRC risk. We investigated 381 patients with histologically confirmed CRC and 2156 control subjects. HIF1A genotypes were determined by exonuclease (TaqMan) assays. For determination of microvessel density (MVD) tumor sections were stained using a mouse monoclonal antibody recognizing the pan-endothelial marker CD31. In a multivariate logistic regression analysis including age and sex neither the HIF1A 582S allele (Odds ratio: 1.204; 95% confidence interval 0.911,1.592; P,=,0.193) nor the 588T allele was significantly associated with CRC (Odds ratio: 0.851; 95% confidence interval 0.444,1.631; P,=,0.626). However, in an exploratory analysis, the HIF1A 588T allele was associated with tumor localization (P,=,0.016) and tumor size (P,=,0.003). MVD was similar in tumors of patients carrying HIF1A 588T allele and patients without this rare allele. We conclude that functional polymorphisms in the HIF1A gene do not modify CRC risk but maybe associated with clinic-pathological features of the disease. © 2010 Wiley-Liss, Inc. [source] Six diagnostic single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow troutsMOLECULAR ECOLOGY RESOURCES, Issue 3 2009AMANDA J. FINGER Abstract Ten primer pairs were screened to develop single nucleotide polymorphism (SNP) TaqMan assays that will distinguish California golden trout and some rainbow trouts (Oncorhynchus mykiss sspp., O. m. aguabonita) from the Paiute and Lahontan cutthroat trouts (Oncorhynchus clarkii seleniris, O. c. henshawi). From these 10 primer pairs, one mitochondrial and five nuclear fixed SNP differences were discovered and developed into TaqMan assays. These six assays will be useful for characterizing and monitoring hybridization between these groups. Additional Oncorhynchus clarkii sspp. and Oncorhynchus mykiss sspp. were assayed to determine if these assays are useful in closely related species. [source] Expression of FGF2 and TGF, and testis morphology during testicular hypertrophy subsequent to hemicastration in the neonatal boarMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2008R. Wells Abstract The objective was to ascertain fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), and transforming growth factor-, (TGF,) mRNA expression and testis morphology during accelerated testicular growth after hemicastration in the neonatal boar. On Day 10 after birth (Day 0), boars were assigned to control (n,=,28), no treatment; hemicastrated (n,=,28), left testis removed. The right testis in both groups (n,=,7) was removed on Days 5, 10, 15, and 20. Expression of mRNA for FGF2, EGF, and TGF, was determined by qRT-PCR using TaqMan®. Testicular morphology was determined on Day 15. On Day 10, hemicastrated boars had a greater (P,=,0.01) testis weight (6.2,±,0.8 g; mean,±,SEM) than controls (4.3,±,0.4 g) and on Day 15 testis weight in hemicastrated boars (8.8,±,0.8 g) was twice (P,<,0.01) that of control boars (4.2,±,0.3 g). Seminiferous tubule volume was approximately doubled in hemicastrated boars (P,<,0.01) and was associated with an increase (P,<,0.01) in Sertoli cell number. Interstitial compartment volume was greater (P,<,0.01) in hemicastrated boars. Leydig cell numbers were similar (P,=,0.14) but volume was greater (P,<,0.01) for hemicastrates. There were no differences (P,> 0.05) between control and hemicastrated boars in TGF, or FGF2 expression on Day 5 or Day 10, and EGF was not detected. It was concluded that upregulation of TGF, or FGF2 expression is not a pre-requisite for enhanced testicular growth and increased Sertoli cell proliferation that occurs subsequent to hemicastration in the neonatal boar. Mol. Reprod. Dev. 75: 961,966, 2008. © 2008 Wiley-Liss, Inc. [source] Parkinson's disease and LRRK2: Frequency of a common mutation in U.S. movement disorder clinicsMOVEMENT DISORDERS, Issue 4 2006Denise M. Kay PhD Abstract The G2019S mutation in the LRRK2 gene is reportedly a common cause of familial Parkinson's disease (PD) and may also have a significant role in nonfamilial PD. The objective of this study was to assess mutation carrier frequency in PD patients from movement disorder clinics in the United States, stratified by family history, age at onset, and geography; to determine carrier frequency in a large and well-characterized control population; to examine segregation of mutation in families of patients; and to correlate genotype with clinical phenotype. One thousand four hundred twenty-five unrelated PD patients from movement disorder clinics in Oregon, Washington, and New York and 1,647 unrelated controls were studied. The G2019S mutation was detected using a TaqMan assay and verified by sequencing. Eighteen of 1,425 patients and one of 1,647 controls had the mutation. Carrier frequency (± 2SE) in patients was 0.013 ± 0.006 overall, 0.030 ± 0.019 in familial PD, 0.007 ± 0.005 in nonfamilial PD, 0.016 ± 0.013 in early-onset PD, and 0.012 ± 0.007 in late-onset PD. Geographic differences were insignificant. Age at onset of mutation carriers ranged from 28 to 71 years. Mutation carriers were clinically indistinguishable from idiopathic PD. LRRK2 G2019S is the single most common pathogenic mutation linked to neurodegenerative disease to date. © 2005 Movement Disorder Society [source] Clinical outcome and IL-17, IL-23, IL-27 and FOXP3 expression in peripheral blood mononuclear cells of pollen-allergic children during sublingual immunotherapyPEDIATRIC ALLERGY AND IMMUNOLOGY, Issue 1-Part-II 2010Kaisa Nieminen Nieminen K, Valovirta E, Savolainen J. Clinical outcome and IL-17, IL-23, IL-27 and FOXP3 expression in peripheral blood mononuclear cells of pollen-allergic children during sublingual immunotherapy. Pediatr Allergy Immunol 2010: 21: e174,e184. © 2009 John Wiley & Sons A/S Induction of allergen-specific, tolerogenic, IL-10 and/or TGF-,-producing T-regulatory (Treg) cells that express transcription factor FOXP3 is considered as one of the key mechanisms of allergen-specific immunotherapy. However, little is known of the induction of FOXP3 expression in children during sublingual immunotherapy (SLIT). Recently, also, a novel subgroup of T-helper (Th) cells, the Th17 cells, secreting predominantly IL-17 (IL-17A), was identified. The expressions of IL-17 or the Th17-regulating cytokines IL-23 and IL-27 during SLIT are currently completely unexplored. This randomized, placebo-controlled dose-response study was performed to analyze the effects of SLIT on FOXP3, IL-17, IL-23, and IL-27 expressions in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis and their associations with clinical outcome. Thirty children were included: ten received SLIT with a glycerinated mixture of birch, hazel and alder with a cumulative weekly dose of 24,000 SQ-U, 10 with dose 200,000 SQ-U/wk, and ten received placebo. Cytokine and FOXP3 mRNA expressions in allergen-, purified protein derivative-stimulated and non-stimulated PBMC were determined at 0, 1 and 2 yr of SLIT by real-time RT-PCR (TaqMan®). Symptoms and medications were recorded using diary cards. Allergen-induced IL-17 mRNA expression was significantly increased in the study subjects with elevated combined Symptom Medication Score (SMS) after 2 yr. There was also a significant positive correlation between the allergen-induced IL-17 and SMS in whole study group (r = 0.38, p = 0.039) and especially the 200,000 SQ-U dose-treated group (r = 0.74, p = 0.027) at 2 yr. Allergen-induced FOXP3 mRNA expression was significantly increased in the 200,000 SQ-U dose-treated children after two study years as compared with baseline (p = 0.016) and placebo-treated children (p = 0.028). The changes in FOXP3 mRNA expression positively correlated with IL-10 and TGF-, mRNAs during SLIT in whole study population. Increased allergen-induced IL-17 responses during SLIT are associated with elevated SMS. Increased tolerogenic, allergen-specific Treg responses are also observed in children during SLIT. [source] Expression of Messenger Ribonucleic Acid Encoding for Phosphodiesterase Isoenzymes in Human Female Genital TissuesTHE JOURNAL OF SEXUAL MEDICINE, Issue 6 2007Stefan Uckert PhD ABSTRACT Objectives., The use of inhibitors of phosphodiesterase 5 (PDE5) has been suggested to treat symptoms of female sexual dysfunction (FSD). Nonetheless, there has been a relatively low success rate of PDE5 inhibitors in FSD in comparison with male erectile dysfunction. The elevated expression of PDE5 in the human penile erectile tissue is considered the reason for the high clinical efficacy of PDE5 inhibitors in the pharmacotherapy of male erectile dysfunction. Aim., To evaluate by means of molecular biology the expression of messenger ribonucleic acid expression (mRNA) encoding for cyclic AMP and cyclic GMP PDE isoenzymes in female genital tissues. Main Outcome Measures., The amount of mRNA transcripts specifically encoding for cyclic AMP- and/or cyclic GMP-degrading PDE isoenzymes was determined. Methods., Human clitoral, labial, and vaginal tissue was obtained from four female cadavers (age at death: 18,42 years). The expression of mRNA specifically encoding for PDE1A, 1B, 1C, 2A, 4A, 5A, 10A, and 11A was elucidated by means of real-time polymerase chain reaction (PCR) analysis (TaqMan). Human penile erectile tissue (corpus cavernosum [HCC]) was used as a reference tissue. Results., mRNA encoding for all PDE isoforms mentioned above is expressed in the female genital tissues. Different magnitudes of mRNA expression were observed: a predominant expression of mRNA encoding for PDE1A but only insignificant amounts of PDE1B, 1C, 4A, 10, and 11A mRNA were registered. With PDE1A being the only exception, the mRNA expression was always higher in the HCC than in the female genital tissues. Especially, the expression of mRNA encoding for PDE5 was several-fold higher in the HCC. Conclusion., On the mRNA level, various PDE isoforms are expressed in the clitoris, labia, and vagina. It remains to be established as to whether the low expression of PDE5 in female genital tissue might be a negative predictor for the success of PDE5 inhibitors in the treatment of FSD. Uckert S, Ellinghaus P, Albrecht K, Jonas U, and Oelke M. Expression of messenger ribonucleic acid encoding for phosphodiesterase isoenzymes in human female genital tissues. J Sex Med 2007;4:1604,1609. [source] Establishment of a TaqMan-based real-time PCR and its application to sheep PRNP genotypingANIMAL GENETICS, Issue 4 2007Z. Lan No abstract is available for this article. [source] Influence of the leptin G-2548A polymorphism on leptin levels and anthropometric measurements in healthy Spanish adolescents,ANNALS OF HUMAN GENETICS, Issue 4 2010Pia Riestra Summary Polymorphisms in the leptin gene (LEP) have been associated with leptin levels and obesity in some studies in adults though this link has scarcely been investigated in children. In our study, we examined the relationship of the LEP G-2548A polymorphism with leptin levels, anthropometric variables and body composition in a population-based sample of pubescent children. Our study included 880 healthy schoolchildren (419 males and 461 females), 12,16 years of age. Plasma leptin levels were determined by ELISA. The LEP polymorphism was determined by allelic discrimination TaqMan® assay. Male carriers of the AA genotype had significantly lower plasma leptin levels than GA (p < 0.008) and GG (p < 0.001) carriers and significantly lower mean hip circumference (HC) values than GG carriers (p = 0.04). In girls, leptin levels were also lower in A-allele carriers than in GG carriers, and BMI and HC were significantly lower in AA carriers as compared with GG carriers. In addition, the frequency of the A allele was significantly lower (,2: 4.58, p = 0.032) in the OW-obese than in the NW group. In conclusion, the LEP G-2548A polymorphism is associated with variations in leptin levels, BMI and HC in Spanish pubertal children, and evidence suggests a link between the G allele and presence of overweight in girls. [source] MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytesARTHRITIS & RHEUMATISM, Issue 5 2010Nahid Akhtar Objective Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. Methods Chondrocytes were stimulated with interleukin-1, (IL-1,) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR),based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription,PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-,B was evaluated using specific inhibitors. Results In IL-1,,stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3,-untranslated region (3,-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3,-UTR of human MMP-13 mRNA and inhibited the IL-1,,induced expression of MMP-13 protein in chondrocytes. NF-,B and MAPK activation down-regulated the expression of miR-27b. Conclusion Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1,,induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes. [source] | |||||||||||||||||||||||||||||||