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Polymerase Chain Reaction Products (polymerase + chain_reaction_products)
Selected AbstractsEstablishment and characterization of two new cell lines derived from flounder, Paralichthys olivaceus (Temminck & Schlegel)JOURNAL OF FISH DISEASES, Issue 11-12 2003M S Kang Abstract Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets of microsatellite markers of flounder. Optimal growth temperature was 20 °C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses. [source] Characterization of porcine dentin sialoprotein (DSP) and dentin sialophosphoprotein (DSPP) cDNA clonesEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 1 2003Yasuo Yamakoshi Dentin sialophosphoprotein (DSPP) is a chimeric glycoprotein with dentin sialoprotein (DSP) on its N -terminus and dentin phosphoprotein (DPP) on its C -terminus. We have constructed and screened a unidirectional cDNA library derived from the pulp organ of developing pig teeth, and isolated cDNA clones encoding DSP-only, as well as two DSPP clones with alternative sequences in their 3, coding regions. The DSP-only transcript has an open reading frame of 386 codons, and is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region. the use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 4-encoded segment. The DSPP cDNAs contain open reading frames of 593 and 600 codons. Northern blots hybridized to radiolabeled DSP probes showed bands at 1.4, 2.5, 4.4, and 4.8 kb. Cloning and characterization of reverse transcriptase polymerase chain reaction products confirmed the existence of mRNA encoding pDSP386, pDSPP593, and pDSPP600in vivo, but also suggested that DNA sequence redundancies in the DSPP coding region make it prone to cloning artifacts. [source] B-cell diversity decreases in old age and is correlated with poor health statusAGING CELL, Issue 1 2009Kate L. Gibson Summary Older people suffer from a decline in immune system, which affects their ability to respond to infections and to raise efficient responses to vaccines. Effective and specific antibodies in responses from older individuals are decreased in favour of non-specific antibody production. We investigated the B-cell repertoire in DNA samples from peripheral blood of individuals aged 86,94 years, and a control group aged 19,54 years, using spectratype analysis of the IGHV complementarity determining region (CDR)3. We found that a proportion of older individuals had a dramatic collapse in their B-cell repertoire diversity. Sequencing of polymerase chain reaction products from a selection of samples indicated that this loss of diversity was characterized by clonal expansions of B cells in vivo. Statistical analysis of the spectratypes enabled objective comparisons and showed that loss of diversity correlated very strongly with the general health status of the individuals; a distorted spectratype can be used to predict frailty. Correlations with survival and vitamin B12 status were also seen. We conclude that B-cell diversity can decrease dramatically with age and may have important implications for the immune health of older people. B-cell immune frailty is also a marker of general frailty. [source] Association of polymorphisms of glutamate-cystein ligase and microsomal triglyceride transfer protein genes in non-alcoholic fatty liver diseaseJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2 2010Claudia Pinto Marques Souza Oliveira Abstract Background and Aims:, Although the metabolic risk factors for non-alcoholic fatty liver disease (NAFLD) progression have been recognized, the role of genetic susceptibility remains a field to be explored. The aim of this study was to examine the frequency of two polymorphisms in Brazilian patients with biopsy-proven simple steatosis or non-alcoholic steatohepatitis (NASH): ,493 G/T in the MTP gene, which codes the protein responsible for transferring triglycerides to nascent apolipoprotein B, and ,129 C/T in the GCLC gene, which codes the catalytic subunit of glutamate-cystein ligase in the formation of glutathione. Methods:, One hundred and thirty-one biopsy-proven NAFLD patients (n = 45, simple steatosis; n = 86, NASH) and 141 unrelated healthy volunteers were evaluated. Genomic DNA was extracted from peripheral blood cells, and the ,129 C/T polymorphism of the GCLC gene was determined by restriction fragment length polymorphism (RFLP). The ,493 G/T polymorphism of the MTP gene was determined by direct sequencing of the polymerase chain reaction products. Results:, The presence of at least one T allele in the ,129 C/T polymorphism of the GCLC gene was independently associated with NASH (odds ratio 12.14, 95% confidence interval 2.01,73.35; P = 0.007), whereas, the presence of at least one G allele in the ,493 G/T polymorphism of the MTP gene differed slightly between biopsy-proven NASH and simple steatosis. Conclusion:, This difference clearly warrants further investigation in larger samples. These two polymorphisms could represent an additional factor for consideration in evaluating the risk of NAFLD progression. Further studies involving a larger population are necessary to confirm this notion. [source] Piperine inhibits eosinophil infiltration and airway hyperresponsiveness by suppressing T cell activity and Th2 cytokine production in the ovalbumin-induced asthma modelJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 3 2009Seung-Hyung Kim Abstract Objectives This study aimed to investigate the effect of piperine on airway hyper-responsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, immunoglobulin E and histamine production in a murine model of asthma. Methods Asthma was induced in Balb/c mice by ovalbumin sensitization and inhalation. Piperine (4.5 and 2.25 mg/kg) was orally administered 5 times a week for 8 weeks. At 1 day after the last ovalbumin exposure, airway hyperresponsiveness was determined and samples of bronchoalveolar lavage fluid, lung cells and serum were collected for further analysis. Key findings Piperine-treated groups had suppressed eosinophil infiltration, allergic airway inflammation and airway hyperresponsiveness, and these occurred by suppression of the production of interleukin-4, interleukin-5, immunoglobulin E and histamine. Moreover, polymerase chain reaction products for thymus and activation regulated chemokine from lung cell RNA preparations were decreased in the piperine-treated group compared with control groups, although transforming growth factor-, products were increased in the piperine-treated group. Conclusions The results suggest that the therapeutic mechanism by which piperine effectively treats asthma is based on a reduction of Th2 cytokines (interleukin-4, interleukin-5), eosinophil infiltration, and by marked reduction of thymus and activation regulated chemokine, eotaxin-2 and interleukin-13 mRNA expression (especially transcription of nuclear factor-, dependent genes) in lung tissue, as well as reduced interleukin-4, interleukin-5 and eotaxin levels in bronchoalveolar lavage fluid, and histamine and ovalbumin-specific immunoglobulin E production in serum. [source] Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskiiMOLECULAR ECOLOGY, Issue 1 2003B. A. Neilan Abstract Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic vari-ation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins. [source] Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversityMOLECULAR ECOLOGY RESOURCES, Issue 6 2009DOROTA L. PORAZINSKA Abstract Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community. [source] Isolation and characterization of microsatellite loci for storm-petrelsMOLECULAR ECOLOGY RESOURCES, Issue 3 2009ZHENGXIN SUN Abstract Primers were developed for 10 microsatellite loci for two species of Oceanodroma storm-petrels. Variability was tested in 27 O. castro and 22 O. monteiroi from the Azores, and 24 O. leucorhoa from Norway. At least six loci amplified reliably and were polymorphic in each species. The number of alleles per locus averaged 4.6, and observed heterozygosities averaged 0.41. Most primers also yielded polymerase chain reaction products in O. tethys, O. hornbyi and Pterodroma phaeopygia. These loci are being used to assay population genetic structure in storm-petrels. [source] A combination of techniques proves useful in the development of nuclear markers in the newt genus TriturusMOLECULAR ECOLOGY RESOURCES, Issue 3 2009G. ESPREGUEIRA THEMUDO Abstract To increase the number of markers available for study of phylogeny and phylogeography in the newt genus Triturus, we developed and tested 59 primer pairs using three different techniques. Primers were obtained from published sources, by designing exon-primed intron-crossing primers and from randomly cloned anonymous nuclear DNA fragments. Successful polymerase chain reaction products were cloned and sequenced. Five fragments were successfully amplified and sequenced for six species of Triturus: intron 7 of the ,-fibrinogen gene (,fibint7), third intron of the calreticulin gene (CalintC), the 11th intron of the ,-subunit of the platelet derived growth factor receptor (PDGFR,) and two anonymous markers (Cri1 and Cri4). The average percentage species divergence across all the markers is low (c. 3%), compared to what has been found in mitochondrial DNA (25,30%). [source] Polymorphic dinucleotide microsatellite loci in the clonal ascidian Diplosoma listerianum: predominance of compound and highly interrupted imperfect repeatsMOLECULAR ECOLOGY RESOURCES, Issue 2 2004Lorna Maclean Abstract We report the isolation of the first dinucleotide microsatellite loci from the clonal ascidian, Diplosoma listerianum. Most repeats were compound and highly interrupted, with flanking sequences containing many small interspersed repetitive regions. Consequently, most primers detected polymerase chain reaction products outside the expected size range, and only five out of 15 primers detected polymorphic single-locus markers. Characterization of five variable loci from two UK populations revealed three to seven alleles per locus, with observed heterozygosity of 0.00,0.86 and expected heterozygosity of 0.10,0.66. Three loci showed significant heterozygote deficits either because of inbreeding, population substructure or the presence of null alleles. [source] Genetic relationships of sesame germplasm collection as revealed by inter-simple sequence repeatsPLANT BREEDING, Issue 3 2002D. H. Kim Abstract Inter-simple sequence repeats (ISSR) polymorphism was used to determine genetic relationships among 75 Sesamum indicum L. accessions of Korean and exotic sesame. Fourteen reliable ISSR primers were selected for the assessment of genetic diversity, yielding 79 amplification products. Of these polymerase chain reaction products, 33% revealed polymorphism among the 75 accessions. Genetic distances ranged from 0 to 0.255, with a mean genetic distance of 0.0687. The 75 accessions were divided into seven groups on the basis of unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis. The largest group consisted of 25 Korean cultivars, eight Korean breeding lines and 17 world-wide accessions. The other groups included 25 accessions, several of which contained useful traits. The dendrogram did not indicate any clear division among sesame accessions based on their geographical origin. However, all Korean sesame cultivars except ,Namsankkae' were clustered in the same group, indicating a narrow gene pool. Some of the Korean breeding lines were spread along the dendrogram, showing enlargement of genetic diversity. The genetic diversity data uncovered in this study can be used in future breeding programmes. [source] Matrix-assisted laser desorption/ionization detection of polymerase chain reaction products by utilizing the 5,-3, exonuclease activity of Thermus aquaticus DNA polymerase,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2003N. R. Isola The 5,-3, exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5,-3, exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo-taq assay or probe degradation assay. This method should be amenable to automation. Copyright © 2003 John Wiley & Sons, Ltd. [source] Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2001James J. Walters Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C,,,G (G,,,C on the opposite strand), were each detected by a 40.0,Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C,,,T (G,,,A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0,Da change on one strand (C,,,T) and a 16.0,Da change on the other (G,,,A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products. Copyright © 2001 John Wiley & Sons, Ltd. [source] Growth Factors and Their Receptors in the Middle Ear Mucosa During Otitis Media,THE LARYNGOSCOPE, Issue 3 2002Sean D. Palacios MD Abstract Objective The hyperplastic response of the middle ear mucosa during bacterial otitis media is thought to be mediated by the actions of growth factors and their respective receptors. The purpose of the study was to explore the expression of growth factors known to stimulate epithelial cells in other systems, as well as their receptors, in the middle ear mucosa during otitis media. Study Design Expression of mRNA growth factors and receptors was measured over time after inoculation of the rat middle ear with bacteria. Methods The middle ears of 12 male Sprague-Dawley rats were injected with 105/mL Haemophilus influenzae strain 3655 (nontypeable, biotype II). Three rats were killed at 6, 24, 48, and 72 hours. Three untreated rats were also killed to serve as negative controls. The middle ear mucosa samples were surgically removed and homogenized. Reverse transcription-polymerase chain reaction was performed on each sample with primers for rat epidermal growth factor, epidermal growth factor receptor (ErbB), heparin binding epidermal-like growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, keratinocyte growth factor, betacellulin, amphiregulin, and neuregulin-,. Results Hepatocyte growth factor and epidermal growth factor receptor primers demonstrated polymerase chain reaction products of the expected size that were not displayed in the normal middle ear mucosa. Keratinocyte growth factor and hepatocyte growth factor receptor demonstrated polymerase chain reaction products at all time points tested. Betacellulin and neuregulin-, products were present at all time points except 72 hours after infection. Conclusions The results of the study support a role for growth factors in the middle ear mucosa during otitis media. These bioactive ingredients contribute to mucosal hyperplasia. [source] Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cellsBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2006Ludovic Deriano Summary Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage-induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients' resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome. [source] | |||||||||||||