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Quantitative PCR Analysis (quantitative + pcr_analysis)
Selected AbstractsExpression patterns of the opsin 5,related genes in the developing chicken retinaDEVELOPMENTAL DYNAMICS, Issue 7 2008Sayuri Tomonari Abstract The opsin gene family encodes G protein,coupled seven-transmembrane proteins that bind to a retinaldehyde chromophore for photoreception. It has been reported that opsin 5 is expressed in mammalian neural tissue, but its function has been elusive. As a first step to understand the function for opsin 5 in the developing eye, we searched for chicken opsin 5 -related genes in the genome by a bioinformatic approach and isolated opsin 5 cDNA fragments from the embryonic retina by RT-PCR. We found that there are three opsin 5,related genes, designated cOpn5m (chicken opsin 5, mammalian type), cOpn5L1 (chicken opsin 5 - like 1), and cOpn5L2 (chicken opsin 5 - like 2), in the chicken genome. Quantitative PCR analysis has revealed that cOpn5m is the most abundant in the developing and early posthatching neural retina. In situ hybridization analysis has shown that cOpn5m is specifically expressed in subsets of differentiating ganglion cells and amacrine cells. These results suggest that the mammalian type opsin 5 may contribute to the development of these retinal cells in the chicken. Developmental Dynamics 237:1910,1922, 2008. © 2008 Wiley-Liss, Inc. [source] T-cell-receptor gene usage of Actinobacillus actinomycetemcomitans- reactive periodontal CD4+ T cells from localized juvenile periodontitis patients and human peripheral blood leukocyte-reconstituted NOD/SCID miceJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2002Xuijuan Gao We investigated the variable V, and V, gene usage of Actinobacillus actinomycetemcomitans -reactive periodontal CD4+ T cell receptors (TCR) from: (i) four A. actinomycetemcomitans-infected localized juvenile periodontitis (LJP) patients, (ii) four groups of A. actinomycetemcomitans -inoculated NOD/SCID mice engrafted with individual LJP-derived HuPBL and (iii) HuPBL samples of four LJP patients and two healthy control subjects, by quantitative PCR analyses. The results show that: (i) the majority of the TCR genes (82.5% of V, and 91.1% of V,) used by periodontal CD4+ T cells in A. actinomycetemcomitans -inoculated HuPBL-engrafted NOD/SCID mice overlap with those used by local periodontal T cells in LJP patients, (ii) although A. actinomycetemcomitans -reactive periodontal CD4+ TCR repertoire is relatively widespread, there are a few dominant genes shared by the LJP patients, suggesting a limited number of antigens or epitopes commonly recognized and (iii) A. actinomycetemcomitans likely lacks superantigenic characteristics. These results suggest A. actinomycetemcomitans -associated human CD4+ T cell repertoire established in HuPBL-NOD/SCID mice provides a useful approach to study specific aspects of immune,parasite interactions in the periodontium. [source] Sustained and stable hematopoietic donor-recipient mixed chimerism after unrelated cord blood transplantation for adult patients with severe aplastic anemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2005P. Mao Abstract:, We evaluated the engraftment of donor cells from unrelated cord blood into adult patients with severe aplastic anemia (SAA) and the outcome of allo-CBSCT (cord blood stem cell transplantation). Nine patients were conditioned with decreased dosage of immunosuppressive agents of CTX (60 mg/kg) and ALG (120 mg/kg). The prophylaxis of GVHD consisted of standard CsA and MTX. Patients have a media age of 25.3 yr (range: 15,37), and a median weight of 57.2 kg (range: 52.5,60) at the time of transplantation. Cord blood searches were all conducted at Guangzhou Cord Blood Bank. The engraftment state of the donor cells into recipients was confirmed by microsatellite DNA fingerprinting and fluorescent quantitative PCR analysis. Engrafted evidence has been found in seven patients involved by biomolecular analyses showing donor-recipient mixed chimerism post-transplant which was stable and persistent. After a median follow up of 32.2 months (range: 4,69), seven patients were alive and disease free. This study shows that durable donor-recipient stable mixed chimerism can be achieved by unrelated CBSCT in patients with SAA. Umbilical cord blood could be employed as a source of hematopoietic stem cell for adult transplantation. [source] Identification of novel alternatively spliced BRCA1-associated RING domain (BARD1) messenger RNAs in human peripheral blood lymphocytes and in sporadic breast cancer tissuesGENES, CHROMOSOMES AND CANCER, Issue 9 2007Grazia Lombardi BARD1 (BRCA1-associated RING domain) is the dominant binding partner of BRCA1 in vivo. The BARD1 gene has been reported to be mutated in a subset of breast and ovarian cancer patients and BARD1 germ-line mutations have been identified in breast cancer patients negative for BRCA1 or BRCA2 gene alterations. In the present study, we show by RT-PCR and direct sequencing analysis the occurrence of seven novel and one previously identified BARD1 splicing variants in human lymphocytes and breast cancers. Two of the eight variants (BARD1, and BARD1 ,RIN) preserve a correct open reading frame and could encode BARD1 internally deleted proteins, while the remaining six variants display premature stop codons. Characterization of the relative expression of BARD1 FL, BARD1,, and BARD1 ,RIN using quantitative PCR analysis indicated that the mean expression levels of BARD1 FL, BARD1,, and BARD1 ,RIN were significantly higher in tumors than in morphologically normal tissues and lymphocytes. However, we were unable to identify either qualitatively or quantitatively tumor-specific expression patterns of the identified BARD1 splicing variants. © 2007 Wiley-Liss, Inc. [source] The occurrence of Campylobacter in river water and waterfowl within a watershed in southern Ontario, CanadaJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2010M.I. Van Dyke Abstract Aims:, Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. Methods and Results:, Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (<130 cells 100 ml,1) in 57,79% of samples taken from five locations. By comparison, a culture-based method detected Campylobacter in 0,23% of samples. Water quality parameters such as total Escherichia coli were not highly correlated with Campylobacter levels, although higher pathogen concentrations were observed at colder water temperatures (<10°C). Strains isolated from river water were primarily nalidixic acid-susceptible Campylobacter lari, and selected isolates were identified as Campylobacter lari ssp. concheus. Campylobacter from wild birds (seagulls, ducks and geese) were detected at a similar rate using PCR (32%) and culture-based (29%) methods, and although Campylobacter jejuni was isolated most frequently, C. lari ssp. concheus was also detected. Conclusions:,Campylobacter were frequently detected at low concentrations in the watershed. Higher prevalence rates using quantitative PCR was likely because of the formation of viable but nonculturable cells and low recovery of the culture method. In addition to animal and human waste, waterfowl can be an important contributor of Campylobacter in the environment. Significance and Impact of the Study:, Results of this study show that Campylobacter in surface water can be an important vector for human disease transmission and that method selection is important in determining pathogen occurrence in a water environment. [source] Real-Time quantitative PCR analysis of factor XI mRNA variants in human plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2004A. Podmore Summary., Coagulation factor XI (FXI) plays an essential role in blood coagulation. A deficiency of FXI is an unusual hemorrhagic diathesis in that the bleeding tendency can be highly variable, ranging from severe deficiencies with no symptoms to mild and moderate deficiencies requiring multiple blood transfusions for hemorrhages. This variability in bleeding has been attributed to a number of factors including the presence of a novel form of FXI associated with platelets, which ameliorates the bleeding in some cases of FXI deficiency. However, the nature of this platelet FXI molecule is controversial. Hsu et al. (J Biol Chem 1998; 273: 13787,93) suggest that it is a product of normal FXI , but lacking exon V whilst Martincic et al. (Blood 1999; 94: 3397,404) were unable to detect this alternatively spliced variant using RT-PCR. In order to resolve this controversy, we have employed the highly sensitive technique of real-time quantitative RT-PCR using RNA isolated from FXI-deficient patients. Our results indicate that the platelets of both normal and FXI deficient individuals contain FXI mRNA that is identical to the mRNA found in liver. An exon V deleted splice variant was not detected. Thus the FXI message is not alternatively spliced in platelets and therefore would not be able to produce an unusual FXI protein. [source] Molecular analysis of pyrethroid resistance conferred by target insensitivity and increased metabolic detoxification in Plutella xylostellaPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 5 2010Shoji Sonoda Abstract BACKGROUND: The pyrethroid resistance of the diamondback moth Plutella xylostella (L.) is conferred by increased gene expression of cytochrome P450 to detoxify the insecticide and/or through gene mutation of the sodium channel, which makes the individual insensitive to pyrethroids. However, no information is available about the correlation between the increased metabolic detoxification and the target insensitivity in pyrethroid resistance. RESULTS: Frequencies of pyrethroid-resistant alleles (L1014F, T929I and M918I) and two resistance-related mutations (A1101T and P1879S) at the sodium channel and expression levels of the cytochrome P450 gene CYP6BG1 were examined individually using laboratory and field strains of P. xylostella. Real-time quantitative PCR analysis using the laboratory strains revealed that levels of larval expression of the resistant strain, homozygous for the pyrethroid-resistant alleles other than the M918I, are significantly higher than those of the susceptible strain. In the field strains, the expression levels in insects having the same resistant alleles as those of the resistant strains varied greatly among individuals. The expression levels were not significantly higher than those in the heterozygotes. CONCLUSION: Significant correlation between the target insensitivity and the increased metabolic detoxification in pyrethroid resistance of P. xylostella was observed in the laboratory but not in the field. Copyright © 2010 Society of Chemical Industry [source] Differential distribution of haematopoietic and nonhaematopoietic progenitor cells in intralesional and extralesional keloid: do keloid scars provide a niche for nonhaematopoietic mesenchymal stem cells?BRITISH JOURNAL OF DERMATOLOGY, Issue 6 2010S.A. Iqbal Summary Background, Keloid disease is a benign, quasineoplastic disease with a high recurrence rate. Mesenchymal-like stem cells (MLSC) have previously been demonstrated in keloid scars and may be involved in keloid pathobiology. However, as these cells have only been examined by single colour fluorescence activated cell sorting (FACS) alone, they need to be more comprehensively characterized so that the key cellular contributors to keloid scars can be better understood. Objectives, To identify and characterize MLSC in intralesional and extralesional keloid, and to distinguish haematopoietic stem cells (HSC) from mesenchymal stem cells (MSC). Methods and patients, Punch biopsies from intralesional (top, middle and margin) and extralesional keloid scar sites were obtained from 17 patients with a keloid. Multicolour FACS analysis using antibodies specific for HSC markers CD34 and CD117 and MSC markers CD13, CD29, CD44 and CD90 was performed on freshly isolated keloid scar cells and on passage 0 and 1 cells. This was complemented by real-time quantitative polymerase chain reaction (PCR) and immunohistological in situ analyses. Results, Keloid scars contain distinct subpopulations of MLSCs. Cells positive for CD13, CD29, CD44 and CD90 were found to be significantly (P < 0·05) higher in the top and middle compartments of keloid scars compared with extralesional skin, where cells positive for CD34, CD90 and CD117 (representing HSCs) predominated. A unique population of CD34+ cells (cells positive for CD13, CD29, CD34, CD44 and CD90) were found in keloid scars and in extralesional skin. FACS and quantitative PCR analysis showed that many of the MSC markers were progressively downregulated and all HSC markers were lost during extended keloid fibroblast culture up to passage 1. Conclusion, We have found distinct subpopulations of haematopoietic and nonhaematopoietic MSC in keloid scars, whereby HSC accumulate extralesionally, while keloids seem to provide a niche environment for nonhaematopoietic MSC. Future therapy of keloids may have to target differentially both stem cell populations in order to deprive these tumours of their regenerative cell pools. [source] Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase I, are required for invadopodia formation in human breast cancer cellsCANCER SCIENCE, Issue 7 2010Hideki Yamaguchi Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(4,5)P2 -producing enzymes in invadopodia formation in MDA-MB-231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P2 accumulates at invadopodia on the ventral cell surface. Injection of an anti-PI(4,5)P2 antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P2 by overexpression of the phospholipase C (PLC) ,1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P2, also blocked invadopodia formation, while a mutated PLC,1-PH domain that lacks PI(4,5)P2 -binding activity had no effect. Cellular PI(4,5)P2 production is mainly mediated by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) family proteins, which include PIP5KI,, I,, and I,. Real-time quantitative PCR analysis showed that PIP5KI, is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KI, by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KI, protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KI, protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P2 by PIP5KI, is required for invadopodia formation and ECM degradation by human breast cancer cells. (Cancer Sci 2010) [source] | |||||||||||||