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Primers Specific (primer + specific)
Selected AbstractsCharacterization of 6q abnormalities in childhood acute myeloid leukemia and identification of a novel t(6;11)(q24.1;p15.5) resulting in a NUP98,C6orf80 fusion in a case of acute megakaryoblastic leukemiaGENES, CHROMOSOMES AND CANCER, Issue 3 2005Sabrina Tosi Chromosome abnormalities of 6q are not frequently observed in myeloid disorders. In this article, we report the incidence of these chromosome changes in childhood myeloid leukemia as 2%,4% based on the cytogenetic database of a single institution. We applied fluorescence in situ hybridization (FISH) to characterize precisely the types of 6q abnormalities in seven patients (six with acute myeloid leukemia and one with myelodysplastic syndrome). They carried various translocations involving different breakpoints in 6q, as confirmed by FISH using a whole-chromosome-6 paint. Four cases were reported as t(6;11), although the breakpoints varied. Among these, we identified a novel translocation, t(6;11)(q24.1;p15.5), in a patient with acute megakaryoblastic leukemia. Molecular cytogenetic studies using the PAC clone RP5-1173K1 localized the genomic breakpoint on chromosome 11 to within the NUP98 gene. The breakpoint on chromosome 6 was narrowed down to a 500-kb region between BAC clones RP11-721P14 and RP11-39H10. Reverse-transcription PCR was performed using a forward primer specific for NUP98 and a reverse primer for the candidate gene in the 500-kb interval in 6q. This experiment resulted in the identification of a new fusion between NUP98 and C6orf80. Further studies will aim to fully characterize C6orf80 and will elucidate the role of this new NUP98 fusion in myeloid leukemia. © 2005 Wiley-Liss, Inc. [source] Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakesENVIRONMENTAL MICROBIOLOGY, Issue 11 2001K. Van der Gucht We characterized the bacterioplankton community and its seasonal dynamics in two neighbouring hypertrophic lakes by denaturing gradient gel electrophoresis (DGGE) analysis of short (193 bp) 16S ribosomal DNA polymerase chain reaction (PCR) products obtained with primers specific for the domain Bacteria. Lake Blankaart is turbid and has a high phytoplankton biomass and episodic cyanobacterial blooms, whereas biomanipulated Lake Visvijver is characterized by clearwater conditions and the establishment of a dense charophyte vegetation. Both lakes were dominated by bacterial groups commonly found in freshwater habitats (e.g. ACK4 cluster of Actinomycetes; ACK stands for clones isolated from the Adirondack mountain lakes). Yet, cluster analysis and principal components analysis (PCA) revealed that taxon composition of the bacterioplankton community of the two lakes differs substantially and consistently throughout the season. During the study year (1998), the bacterioplankton community of both lakes showed a distinct seasonal pattern. Lake Blankaart showed a clear differentiation between winter, spring, summer and autumn. In Lake Visvijver, summer samples differed greatly from spring, autumn and winter samples. We hypothesize that the contrasting bacterioplankton in the two neighbouring shallow lakes is determined largely by the presence or absence of macrophytes. [source] Clonally expanded CD4+CD28null T,cells in rheumatoid arthritis use distinct combinations of T cell receptor BV and BJ elementsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2003Ulf Wagner Abstract Clonally expanded, autoreactive CD4+CD28null cells can be found in the peripheral blood of patients with rheumatoid arthritis and have been shown to be associated with severeextra-articular disease manifestations. We investigated the size of the CD4+CD28null compartment and the TCR , chain repertoire of expanded CD4+ clonotypes in 94 rheumatoid arthritis patients by complementarity-determining region 3 (CDR3) length analysis (spectratyping) in the BV6 and BV14 TCR families, with primers specific for three arbitrarily chosen , chain joining elements (BJ1S2, BJ2S3 and BJ2S7). The spectratyping results showed a strong correlation of the size of the CD4+CD28null compartment with the detected number of BV14 clonotypes, whereas no association with BV6 oligoclonality was found. Only clones using the BV14,BJ1S2 and BV14,BJ2S3 combinations contributed to this correlation, however, whereas BV14,BJ2S7 clones did not. This preferential correlation implies a role for the TCR , chain in stimulating clonal outgrowth and argues against the previously suggested superantigenic stimulation of in-vivo -expanded clones. Instead, since no evidence for shared antigen specificity could be detected, clonal expansion of T cells in rheumatoid arthritis might be influenced by the BJ elements because of changes in the flexibility of the protein backbone of the ,-chain. [source] Microbial community analysis at crude oil-contaminated soils targeting the 16S ribosomal RNA, xylM, C23O, and bcr genesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009Y. Higashioka Abstract Aims:, The analyses targeting multiple functional genes were performed on the samples of crude oil-contaminated soil, to investigate community structures of organisms involved in monoaromatic hydrocarbon degradation. Methods and Results:, Environmental samples were obtained from two sites that were contaminated with different components of crude oil. The analysis on 16S rRNA gene revealed that bacterial community structures were clearly different between the two sites. The cloning analyses were performed by using primers specific for the catabolic genes involved in the aerobic or anaerobic degradation of monoaromatic hydrocarbons, i.e. xylene monooxygenase (xylM), catechol 2,3-dioxygenase (C23O), and benzoyl-CoA reductase (bcr) genes. From the result of xylM gene, it was suggested that there are lineages specific to the respective sites, reflecting the differences of sampling sites. In the analysis of the C23O gene, the results obtained with two primer sets were distinct from each other. A comparison of these suggested that catabolic types of major bacteria carrying this gene were different between the two sites. As for the bcr gene, no amplicon was obtained from one sample. Phylogenetic analysis revealed that the sequences obtained from the other sample were distinct from the known sequences. Conclusions:, The differences between the two sites were demonstrated in the analyses of all tested genes. As for aerobic cleavage of the aromatic ring, it was also suggested that analysis using two primer sets provide more detailed information about microbial communities in the contaminated site. Significance and Impact of the Study:, The present study demonstrated that analysis targeting multiple functional genes as molecular markers is practical to examine microbial community in crude oil-contaminated environments. [source] The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silageJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2008R.J. Schmidt Abstract Aims:, To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods. Methods and Results:, DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation. Conclusions:, The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples. Significance and Impact of the Study:, The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri, a micro-organism used as an inoculant. [source] Diversity and abundance of Bacteria and Archaea in the Bor Khlueng Hot Spring in ThailandJOURNAL OF BASIC MICROBIOLOGY, Issue 6 2004Pattanop Kanokratana The prokaryotic diversity in the Bor Khlueng hot spring in Ratchaburi province, Thailand was investigated by a culture-independent molecular approach. This hydrothermal pool is located in the central part of Thailand and contains sulfide-rich mineral water that is believed to relieve muscle ache and pain. The water flow year-round with temperature ranging between 50,57 °C. Community DNA was extracted directly from sediment samples by coring to depth of ,20,30 cm. Small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the domains Archaea and Bacteria. The PCR products were cloned and sequenced. For the bacterial rDNA clone library, 200 clones were randomly selected for further analyses. After restriction fragment length polymorphism (RFLP) analysis of rDNA clones and exclusion of chimeric sequences 36 phylotypes were obtained. The Bor Khlueng phylotypes spanned a wide range within the domain Bacteria, occupying eleven major lineages (phyla). Almost a quarter (23%) of the clones were classifed as Acidobacteria. The other clones were grouped into the Bacteriodetes (19%), Nitrospirae (13%), Proteobacteria (12%), Deinococcus-Thermus lineage (11%), planctomycetes (6%), and Verrucomicrobia (5%). The four remaining phyla, 5% each, were assigned to Actinobacteria, Chloroflexi, Cyanobacteria, and the candidate division "OP10". For the archaeal 16S rRNA gene sequence library, 25 distinct phylotypes were obtained, 17 clones were found to be associated with Crenarahaeota and 8 clones were associated with Euryarachaeota. The findings of the molecular survey of this so far not investigated site showed that Bor Khlueng hot spring is a potential rich source of unique bacterial and archaeal species. The great majority (,80%) of the prokaryotic sequences detected in Bor Khlueng were unknown. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Production and Stability Studies of a Neurotoxin Produced by Clostridium sp.JOURNAL OF FOOD SCIENCE, Issue 3 2006ABSTRACT: A neurotoxigenic Clostridium sp. (RKD) isolated from intestine of decaying fish produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) when tested by mouse protection bioassay. An amplicon of expected size (approximately 700 bp) was generated with primers specific for BoNT/B. Toxin was maximally released in the culture supernatant in the late stationary phase and was dependent on media composition. Growth was optimal in trypticase peptone yeast-extract glucose (TPYG) medium in a pH range of 7.5 to 8.0 and at a temperature between 35°C to 40°C while toxin production was optimum at 37°C (3 to 4 × 103 minimum lethal dose per milliliter) without any protease treatment. There was no correlation between growth and toxin production when cells were grown in media containing different concentrations of NaCl (0% to 5%). Toxin in the culture supernatant was more stable (50% reduction at 50°C in 90 min) than the partially purified fraction. Toxicity was destroyed gradually after increasing the number of freeze-thaw cycles and was almost completely inactivated after 5 cycles. It was completely inactivated by overnight treatment of 1 N NaOH while it retained 1.5% activity with a similar treatment with 1 N HCl. [source] OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE,JOURNAL OF PHYCOLOGY, Issue 2 2008Andrea Baker Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A "universal" oligonucleotide primer set, along with species and genus-specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low-light-emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples. [source] 16S rDNA Sequence Analysis of Bacterial Isolates from Die-back Affected Sissoo Trees (Dalbergia sissoo Roxb.) in BangladeshJOURNAL OF PHYTOPATHOLOGY, Issue 9 2005H. Tantau Abstract A new form of disease called ,die-back' has been established in Dalbergia sissoo trees. This disease has reached epidemic proportions in Bangladesh as well as in other countries of South Asia and is characterized by browning of the leaves, signs of wilting, and trunk lesions with gum flow. The trees die within a few months. In order to investigate the causes of this die-back disease, samples were taken for a first trial in the Rajshahi division at two sites around Sherpur. For the isolation of bacteria, surface-sterilized plant material (leaves, twigs and trunk bark) from diseased trees was transferred to LB medium and incubated. After isolation of single colonies, various bacteria species could be identified by polymerase chain reaction analysis with two primers specific for highly conserved sequence regions in the bacterial 16S rDNA and by sequencing. First indications for the presence of bacteria with phytopathogenic potential were found. [source] Screening food products for the presence of CaMV 35S promoter and NOS 3, terminatorJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2005Hanaa AS Oraby Abstract Biotechnology has enabled the modification of agricultural materials in a very precise way, thereby improving productivity and yields of economically important crops. There are a number of methods available for detecting genetically modified organisms (GMOs). In the present investigation, a qualitative PCR technique has been adopted in order to discriminate between genetically modified and non-modified food products. The qualitative PCR assay employs primers specific for genetic elements that are used to generate genetically engineered agricultural crops. Two of the most common primers used for the detection of GMOs, 35S promoter and NOS 3, terminator, have been tested over a panel of 24 food products purchased from the local market. The results indicated that, out of the 24 food products tested, three products gave positive results with the 35S promoter. The NOS 3, primers gave negative results with all tested samples. Copyright © 2005 Society of Chemical Industry [source] T-cell receptor repertoire in IgA nephropathy renal biopsiesNEPHROLOGY, Issue 2002John F Knight SUMMARY: Renal biopsies from patients with IgA nephropathy (IgAN) were studied to determine whether the presence of ,, and ,, T cells is correlated with disease progression in IgAN. The ,, and ,, T-cell receptor (TCR) repertoire was further analysed in these renal biopsies. Immunohistochemical staining using mAb (TCR, and TCR,) and molecular studies using reverse transcription,polymerase chain reaction (RT-PCR) with primers specific for TCR families were undertaken. CDR3 length spectratyping and sequencing of TCR chains were used to analyse the diversity of the CDR3 region of these receptors. It was demonstrated that the presence of ,, T cells is associated with progressive IgAN while ,, T cells are found in both stable and progressive disease. Analysis of the TCR variable (V), repertoire showed the preferential use of V,8 with marked similarities in the CDR3 region by some renal infiltrating T cells in the kidney of some IgAN patients, although T cells infiltrating the renal interstitium of patients with IgAN express heterogeneous T cell receptors. The data from analysis of ,, T-cell repertoire showed that ,, T cells infiltrating the kidneys of IgAN patients use a restricted subset of ,, T cells with a feature of recurrent junctional amino acid motifs in V,1 T cells. The results suggest that both ,, and ,, T cells are involved in the progression of IgAN to renal failure and also that there is clonal expansion of individual ,, or ,, T cells in the kidneys of some IgAN patients. The conserved amino acid in the TCR CDR3 region of V,8 and the feature of recurrent junctional amino acid motifs in V,1 T cells may indicate antigen-driven selection. [source] T-cell receptor repertoire in IgA nephropathy renal biopsiesNEPHROLOGY, Issue 2002John F KNIGHT SUMMARY: Renal biopsies from patients with IgA nephropathy (IgAN) were studied to determine whether the presence of ,, and ,, T cells is correlated with disease progression in IgAN. the ,, and ,, T-cell receptor (TCR) repertoire was further analysed in these renal biopsies. Immunohistochemical staining using mAb (TCR, and TCR,) and molecular studies using reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for TCR families were undertaken. CDR3 length spectratyping and sequencing of TCR chains were used to analyse the diversity of the CDR3 region of these receptors. It was demonstrated that the presence of ,, T cells is associated with progressive IgAN while ,, T cells are found in both stable and progressive disease. Analysis of the TCR variable (V), repertoire showed the preferential use of V,8 with marked similarities in the CDR3 region by some renal infiltrating T cells in the kidney of some IgAN patients, although T cells infiltrating the renal interstitium of patients with IgAN express heterogeneous T cell receptors. the data from analysis of ,, T-cell repertoire showed that ,, T cells infiltrating the kidneys of IgAN patients use a restricted subset of ,, T cells with a feature of recurrent junctional amino acid motifs in V,1 T cells. the results suggest that both ,, and ,, T cells are involved in the progression of IgAN to renal failure and also that there is clonal expansion of individual ,, or ,, T cells in the kidneys of some IgAN patients. the conserved amino acid in the TCR CDR3 region of V,8 and the feature of recurrent junctional amino acid motifs in V,1 T cells may indicate antigen-driven selection. [source] Demonstration of an orexinergic central innervation of the pineal gland of the pigTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2004Chiara Fabris Abstract Orexins/hypocretins, two isoforms of the same prepropeptide, are widely distributed throughout the brain and are involved in several physiological and neuroendocrine regulatory patterns, mostly related to feeding, sleep, arousal, and cyclic sleep-wake behaviors. Orexin-A and orexin-B bind with different affinities to two G-protein-coupled transmembrane receptors, orexin-1 and orexin-2 receptors (OR-R1 and OR-R2, respectively). Because of the similarities between the human and the swine brain, we have studied the pig to investigate the orexinergic system in the diencephalon, with special emphasis on the neuroanatomical projections to the epithalamic region. By using antibodies against orexin-A and orexin-B, immunoreactive large multipolar perikarya were detected in the hypothalamic periventricular and perifornical areas at the light and electron microscopic levels. In the region of the paraventricular nucleus, the orexinergic neurons extended all the way to the lateral hypothalamic area. Immunoreactive nerve fibers, often endowed with large varicosities, were found throughout the hypothalamus and the epithalamus. Some periventricular immunoreactive nerve fibers entered the epithalamic region and continued into the pineal stalk and parenchyma to disperse among the pinealocytes. Immunoelectron microscopy confirmed the presence of orexinergic nerve fibers in the pig pineal gland. After extraction of total mRNA from the hypothalamus and pineal gland, we performed RT-PCR and nested PCR using primers specific for porcine orexin receptors. PCR products were sequenced, verifying the presence of both OR-R1 and OR-R2 in the tissues investigated. These findings, supported by previous studies on rodents, suggest a hypothalamic regulation of the pineal gland via central orexinergic nervous inputs. J. Comp. Neurol. 471:113,127, 2004. © 2004 Wiley-Liss, Inc. [source] A new phytoplasma detected in the South Australian native perennial shrub, Allocasuarina muellerianaANNALS OF APPLIED BIOLOGY, Issue 3 2003K S GIBB Summary Allocasuarina muelleriana shrubs growing in natural sclerophyll roadside vegetation near Willalooka in the upper south-east of South Australia have a high incidence of a yellowing disorder in either all or part of the foliage, combined in some cases with a shortening and curling of the leaf-bearing stems. Samples from symptomatic and adjacent asymptomatic plants were tested for phytoplasmas by the polymerase chain reaction (PCR) assay. All but one asymptomatic plant were negative for phytoplasmas, whereas about half of the symptomatic plants were positive. Restriction fragment polymorphism analysis of PCR products indicated that the phytoplasma was related to the buckthorn witches.-broom (BWB) and apple proliferation (AP) groups of phytoplasmas, members of which have not been previously reported in Australia. Further evidence from the sequence of the 16S rRNA gene and the use of PCR primers specific to the AP and pear decline (PD) phytoplasmas confirmed the close relationship to the BWB and AP group phytoplasmas. [source] The ,7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: A possible role for a key neurotransmitter in synovial inflammationARTHRITIS & RHEUMATISM, Issue 5 2009Marjolein A. Van Maanen Objective Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the ,7 subunit of the nicotinic acetylcholine receptor (,7nAChR) and its function in rheumatoid arthritis (RA). Methods The potential role of the ,7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An ,7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate,labeled ,-bungarotoxin, which binds specifically to the ,7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the ,7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dup,7, a variant ,7 transcript. Next, we studied the functional role of the ,7nAChR in RA FLS by examining the effects of ,7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. Results A screen using an Ad.shRNA library against 807 transcripts revealed that a specific ,7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The ,7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found ,7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dup,7 mRNA. Specific ,7nAChR agonists reduced tumor necrosis factor ,,induced IL-6 and IL-8 production by FLS. Conclusion The ,7nAChR and its dup,7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the ,7nAChR could provide a novel antiinflammatory approach to the treatment of RA. [source] Low-temperature (9°C) AMD treatment in a sulfidogenic bioreactor dominated by a mesophilic Desulfomicrobium speciesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Hannele Auvinen Abstract The possibilities for the treatment of low-temperature mine waste waters have not been widely studied. The amenability of low-temperature sulfate reduction for mine waste water treatment at 9°C was studied in a bench-scale fluidized-bed bioreactor (FBR). Formate was used as the electron and carbon source. The first influent for the FBR was acidic, synthetic waste water containing iron, nutrients, and sulfate, followed by diluted barren bioleaching solution (DBBS). The average sulfate reduction rates were 8,mmol,L,1,day,1 and 6,mmol,L,1,day,1 with synthetic waste water and DBBS, respectively. The corresponding specific activities were 2.4 and 1.6,mmol SO,g VSS,1 day,1, respectively. The composition of the microbial community and the active species of the FBR was analyzed by extracting the DNA and RNA, followed by PCR-DGGE with the universal bacterial 16S rRNA gene primers and dsrB -primers specific for sulfate-reducing bacteria. The FBR microbial community was simple and stable and the dominant and active species belonged to the genus Desulfomicrobium. In summary, long-term operation of a low-temperature bioreactor resulted in enrichment of formate-utilizing, psychrotolerant mesophilic sulfate reducing bacteria. Biotechnol. Bioeng. 2009; 104: 740,751 © 2009 Wiley Periodicals, Inc. [source] | ||||||||||||||||