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Transcript Analysis (transcript + analysis)
Selected AbstractsIn situ measurement of methane fluxes and analysis of transcribed particulate methane monooxygenase in desert soilsENVIRONMENTAL MICROBIOLOGY, Issue 10 2009Roey Angel Summary Aerated soils are a biological sink for atmospheric methane. However, the activity of desert soils and the presence of methanotrophs in these soils have hardly been studied. We studied on-site atmospheric methane consumption rates as well as the diversity and expression of the pmoA gene, coding for a subunit of the particulate methane monooxygenase, in arid and hyperarid soils in the Negev Desert, Israel. Methane uptake was only detected in undisturbed soils in the arid region (,90 mm year,1) and vertical methane profiles in soil showed the active layer to be at 0,20 cm depth. No methane uptake was detected in the hyperarid soils (,20 mm year,1) as well as in disturbed soils in the arid region (i.e. agricultural field and a mini-catchment). Molecular analysis of the methanotrophic community using terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene detected methanotrophs in the active soils, whereas the inactive ones were dominated by sequences of the homologous gene amoA, coding for a subunit of the ammonia monooxygenase. Even in the active soils, methanotrophs (as well as in situ activity) could not be detected in the soil crust, which is the biologically most important layer in desert soils. All pmoA sequences belonged to yet uncultured strains. Transcript analysis showed dominance of sequences clustering within the JR3, formerly identified in Californian grassland soils. Our results show that although active methanotrophs are prevalent in arid soils they seem to be absent or inactive in hyperarid and disturbed arid soils. Furthermore, we postulate that methanotrophs of the yet uncultured JR3 cluster are the dominant atmospheric methane oxidizers in this ecosystem. [source] Juggling multiple temporalities: the shift work story of mid-life nursesJOURNAL OF NURSING MANAGEMENT, Issue 1 2009PhD (Macq), SANDRA WEST BSc (Macq) Aim, To explore the theme of multiple temporalities revealed through a phenomenological study of the experience of mid-life shift-working nurses. Background, There are few data on the experience of mid-life women working rotating shift systems that change frequently. Concomitantly, the age profile of the current nursing workforce demands exploration of such issues. Method, This phenomenological study sought the perspectives of 13 shift-working mid-life women. Results, Sociological discussion of the temporal nature of work describes temporality as the clock time associated with an individual as determined by the constraints of their life. Transcript analysis identified the numerous temporalities surrounding a shift-working mid-life woman and a sense of disjunction between the temporalities of individuals important to them which resulted in feelings of regret and guilt. The concept of juggling is introduced to illustrate the participants' need to ,keep everything going' for important individuals in their lives. Conclusion, The personal cost of effective juggling may be high for the jugglers themselves but also for health systems that provide the mid-life shift-working nurse with no alternative than a reduction in working hours. Implications for nursing management, The development of a personal ,time map' framed within the concept of multiple temporalities is suggested for use as a staff development tool to assist with staff retention by facilitating both group and individual discussions of rostering and the complexities of managing an intergenerational work force undertaking shift work. [source] Chemokine responses in schistosomal antigen-elicited granuloma formation,PARASITE IMMUNOLOGY, Issue 6 2002Bo-Chin Chiu Summary Host immune systems have evolved specialized responses to multicellular parasites. This is well represented by the type 2 granulomatous response to Schistosoma mansoni egg antigens, which is an eosinophil-rich inflammatory response mediated by Th2-associated cytokines. Using Ag-bead models of pulmonary granuloma formation in mice, we defined characteristic chemokine (CK) profiles in the granulomatous lungs. Our findings point to a role for C-C chemokine receptor-2 (CCR2) and CCR3 agonists such as monocyte chemotactic proteins (MCPs) 1/CCL2, 3/CCL7 and 5/CCL12 as important participants that are subject to regulation by Th2 cytokines interleukin (IL)-4 and IL-13. CCR4 and CCR8 agonists are also likely contributors. Analysis of CK receptor knockout mice revealed that CCR2 ligands (e.g. MCP-1 and 5) promoted early phase granuloma macrophage accumulation, whereas anti-MCP-3 (CCL7) antibody treatment abrogated eosinophil recruitment. CCR8 knockout mice also demonstrated impaired eosinophil recruitment but this appeared to be related to impaired Th2 cell function. Transcript analysis of CD4+ T cells generated during schistosome granuloma formation failed to show biased CCR8 expression but, having a more limited receptor repertoire, these cells were likely more dependent on CCR8 ligands. Together, these studies indicate an intricate involvement of chemokines in various stages and aspects of schistosomal egg Ag-elicited granuloma formation. [source] Modification of sunflower oil quality by seed-specific expression of a heterologous ,9-stearoyl-(acyl carrier protein) desaturase genePLANT BREEDING, Issue 2 2002P. Rousselin Abstract The coding sequence of ,9-stearoyl-(acyl carrier protein) desaturase from Ricinus communis was introduced into sunflower, under the control of seed-specific promoter and terminator sequences of the late embryogenesis abundant gene from sunflower, Hads10. Two independent primary transformants contained three and six copies of the T-DNA, as demonstrated by hybridization using nptII as a probe. The transgene proved genetically stable and was transmitted as a Mendelian trait. Transcript analysis of the heterologous ,9-stearoyl-(acyl carrier protein) desaturase under control of the Hads10 promoter verified tissue-specific expression in the developing embryos and not in the leaves. Fatty acid composition of the seed oil was followed over five generations under greenhouse and open field conditions. Some of the transgenic lines produced oil with a significantly reduced stearic acid content compared with non-transformed plants under greenhouse and field conditions. However, additional studies need to be performed to assess whether or not physiologically stable lines can be developed from these transgenic lines. [source] Proteomic analysis of rice plasma membrane reveals proteins involved in early defense response to bacterial blightPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2007Fang Chen Abstract Plant plasma membrane (PM) proteins play important roles in signal transduction during defense response to an attacking pathogen. By using an improved method of PM protein preparation and PM-bound green fluorescent protein fusion protein as a visible marker, we conducted PM proteomic analysis of the rice suspension cells expressing the disease resistance gene Xa21, to identify PM components involved in the early defense response to bacterial blight (Xanthomonas oryzae pv. oryzae). A total of 20 regulated protein spots were observed on 2-D gels of PM fractions at 12 and 24,h after pathogen inoculation, of which some were differentially regulated between the incompatible and compatible interactions mediated by Xa21, with good correlation between biological repeats. Eleven protein spots with predicted functions in plant defense were identified by MS/MS, including nine putative PM-associated proteins H+ -ATPase, protein phosphatase, hypersensitive-induced response protein (OsHIR1), prohibitin (OsPHB2), zinc finger and C2 domain protein, universal stress protein (USP), and heat shock protein. OsHIR1 was modified by the microbal challenge, leading to two differentially accumulated protein spots. Transcript analysis showed that most of the genes were also regulated at transcriptional levels. Our study would provide a starting point for functionality of PM proteins in the rice defense. [source] Selective over-expression of fibroblast growth factor receptors 1 and 4 in clinical prostate cancer,THE JOURNAL OF PATHOLOGY, Issue 1 2007K Sahadevan Abstract Fibroblast growth factor receptors (FGFRs) mediate the tumourigenic effects of FGFs in prostate cancer. These receptors are therefore potential therapeutic targets in the development of inhibitors to this pathway. To identify the most relevant targets, we simultaneously investigated FGFR1,4 expression using a prostate cancer tissue microarray (TMA) and in laser capture microdissected (LCM) prostate epithelial cells. In malignant prostates (n = 138) we observed significant FGFR1 and FGFR4 protein over-expression in comparison with benign prostates (n = 58; p < 0.0001). FGFR1 was expressed at high levels in the majority of tumours (69% of grade 3 or less, 74% of grade 4 and 70% of grade 5), while FGFR4 was strongly expressed in 83% of grade 5 cancers but in only 25% of grade 1,3 cancers (p < 0.0001). At the transcript level we observed a similar pattern, with FGFR1 and FGFR4 mRNA over-expressed in malignant epithelial cells compared to benign cells (p < 0.0005 and p < 0.05, respectively). While total FGFR2 was increased in some cancers, there was no association between expression and tumour grade or stage. Transcript analysis, however, revealed a switch in the predominant isoform expressed from FGFR2IIIb to FGFR2IIIc among malignant epithelial cells. In contrast, protein and transcript expression of FGFR3 was very similar between benign and cancer biopsies. The functional effect of targeting FGFR4 in prostate cancer cells has not previously been investigated. In in vitro experiments, suppression of FGFR4 by RNA interference effectively blocked prostate cancer cell proliferation (p < 0.0001) and invasion (p < 0.001) in response to exogenous stimulation. This effect was evident regardless of whether the cells expressed the FGFR4 Arg388 or Gly388 allele. In parallel experiments, FGFR3 suppression had no discernible effect on cancer cell behaviour. These results suggest evidence of selective over-expression of FGFR1 and FGFR4 in clinical prostate cancer and support the notion of targeted inhibition of these receptors to disrupt FGF signalling. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Qualitative Data Collection and Analysis Methods: The INSTINCT TrialACADEMIC EMERGENCY MEDICINE, Issue 11 2007William J. Meurer MD Patient care practices often lag behind current scientific evidence and professional guidelines. The failure of such knowledge translation (KT) efforts may reflect inadequate assessment and management of specific barriers confronting both physicians and patients at the point of treatment level. Effective KT in this setting may benefit from the use of qualitative methods to identify and overcome these barriers. Qualitative methodology allows in-depth exploration of the barriers involved in adopting practice change and has been infrequently used in emergency medicine research. The authors describe the methodology for qualitative analysis within the INcreasing Stroke Treatment through INteractive behavioral Change Tactics (INSTINCT) trial. This includes processes for valid data collection and reliable analysis of the textual data from focus group and interview transcripts. INSTINCT is a 24-hospital, randomized, controlled study that is designed to evaluate a system-based barrier assessment and interactive educational intervention to increase appropriate tissue plasminogen activator (tPA) use in ischemic stroke. Intervention hospitals undergo baseline barrier assessment using both qualitative as well as quantitative (survey) techniques. Investigators obtain data on local barriers to tPA use, as well as information on local attitudes, knowledge, and beliefs regarding acute stroke treatment. Targeted groups at each site include emergency physicians, emergency nurses, neurologists, radiologists, and hospital administrators. Transcript analysis using NVivo7 with a predefined barrier taxonomy is described. This will provide both qualitative insight on thrombolytic use and importance of specific barrier types for each site. The qualitative findings subsequently direct the form of professional education efforts and system interventions at treatment sites. [source] Evaluation of in silico splice tools for decision-making in molecular diagnosis,HUMAN MUTATION, Issue 7 2008Claude Houdayer Abstract It appears that all types of genomic nucleotide variations can be deleterious by affecting normal pre-mRNA splicing via disruption/creation of splice site consensus sequences. As it is neither pertinent nor realistic to perform functional testing for all of these variants, it is important to identify those that could lead to a splice defect in order to restrict transcript analyses to the most appropriate cases. Web-based tools designed to provide such predictions are available. We evaluated the performance of six of these tools (Splice Site Prediction by Neural Network [NNSplice], Splice-Site Finder [SSF], MaxEntScan [MES], Automated Splice-Site Analyses [ASSA], Exonic Splicing Enhancer [ESE] Finder, and Relative Enhancer and Silencer Classification by Unanimous Enrichment [RESCUE]-ESE) using 39 unrelated retinoblastoma patients carrying different RB1 variants (31 intronic and eight exonic). These 39 patients were screened for abnormal splicing using puromycin-treated cell lines and the results were compared to the predictions. As expected, 17 variants impacting canonical AG/GT splice sites were correctly predicted as deleterious. A total of 22 variations occurring at loosely defined positions (±60 nucleotides from an AG/GT site) led to a splice defect in 19 cases and 16 of them were classified as deleterious by at least one tool (84% sensitivity). In other words, three variants escaped in silico detection and the remaining three were correctly predicted as neutral. Overall our results suggest that a combination of complementary in silico tools is necessary to guide molecular geneticists (balance between the time and cost required by RNA analysis and the risk of missing a deleterious mutation) because the weaknesses of one in silico tool may be overcome by the results of another tool. Hum Mutat 29(7), 975,982, 2008. © 2008 Wiley-Liss, Inc. [source] The SUI-homologous translation initiation factor eIF-1 is involved in regulation of ion homeostasis in ricePLANT BIOLOGY, Issue 3 2008C. J. Diédhiou Abstract Halophytes survive high salinity by using complex adaptive mechanisms. In a search for novel molecular mechanisms involved in salt acclimation, transcript analyses revealed increased expression of a SUI-homologous translation initiation factor eIF-1 in the salt-tolerant grass species Festuca rubra ssp. littoralis but not in rice. Upon analysis of the cell specificity of eIF-1 transcription by in situ polymerase chain reaction (PCR), predominant signals were detected in rice leaf mesophyll. To further examine the role of eIF-1 in salt tolerance, transgenic rice plants were generated that over-express this factor under the control of the CaMV-35S promoter. The eIF-1 over-expressing lines showed improved growth under salt stress that was correlated with maintenance of photosynthetic activity and reduced Na+ and Cl, accumulation in leaves. The transgenic rice lines also activated expression of the vacuolar H+ -ATPase. In addition, an oxidoreductase that belongs to the aldo/keto reductase family was identified as a gene with modified expression in the eIF-1 over-expressing lines, compared with wild-type rice. Our data suggest that eIF-1 has a central function in salt-stress adaptation in rice by regulating ion accumulation and the intracellular redox status. [source] Neural precursor cells from a fatal human motoneuron disease differentiate despite aberrant gene expressionDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2007Niklas Pakkasjärvi Abstract Precursor cells of the human central nervous system can be cultured in vitro to reveal pathogenesis of diseases or developmental disorders. Here, we have studied the biology of neural precursor cells (NPCs) from patients of lethal congenital contracture syndrome (LCCS), a severe motoneuron disease leading to prenatal death before the 32nd gestational week. LCCS fetuses are immobile because of a motoneuron defect, seen as degeneration of the anterior horn and descending tracts of the developing spinal cord. The genetic defect for the syndrome is unknown. We show that NPCs isolated postmortem from LCCS fetuses grow and are maintained in culture, but display increased cell cycle activity. Global transcript analysis of undifferentiated LCCS precursor cells present with changes in EGF-related signaling when compared with healthy age-matched human controls. Further, we show that LCCS-derived NPCs differentiate into cells of neuronal and glial lineage and that the initial differentiation is not accompanied by overt apoptosis. Cells expressing markers Islet-1 and Hb9 are also generated from the LCCS NPCs, suggesting that the pathogenic mechanism of LCCS does not directly affect the differentiation capacity or survival of the cells, but the absence of motoneurons in LCCS may be caused by a noncell autonomous mechanism. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediationENVIRONMENTAL MICROBIOLOGY, Issue 5 2008Regina A. O'Neil Summary Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments. [source] Heparin and Heparan Sulfate BiosynthesisIUBMB LIFE, Issue 4 2002Kazuyuki Sugahara Abstract Heparan sulfate is one of the most informationally rich biopolymers in Nature. Its simple sugar backbone is variously modified to different degrees depending on the cellular conditions. Thus, it matures to have an enormously complicated structure, which most likely exhibits a considerable number of unique overlapping sequences with peculiar sulfation profiles. Such sequences are recognized by specific complementary proteins, which form a huge group of "heparin-binding proteins," and the sugar sequences in turn support unique functions of the respective proteins through specific interactions. The heparan sulfate sequences are not directly encoded by genes, but are created by elaborate biosynthetic mechanisms, which ensure the generation of these indispensable sequences. In heparan sulfate biosynthesis, the tetrasaccharide sequence (GlcA-Gal-Gal-Xyl-), designated the protein linkage region, is first assembled on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparan sulfate chain is then polymerized on this fragment by alternate additions of GlcNAc and GlcA through the actions of glycosyltransferases with overlapping specificities encoded by the tumor suppressor EXT family genes. Then follow various modifications by N -deacetylation and N -sulfation of glucosamine, C5-epimerization of GlcA and multiple O -sulfations of the component sugars. Recent studies have achieved purification of several, and molecular cloning of most, of the enzymes responsible for these reactions. Some of these enzymes are bifunctional. The availability of cDNA probes has facilitated elucidation of the crystal structures for two of the biosynthetic enzymes, demonstration of their intracellular location, and their occurrence in complexes to achieve rapid and efficient synthesis of complex sugar sequences. Genomic structure and transcript analysis have shown the existence of multiple isoforms for most of the sulfotransferases. Many aspects of the heparan sulfate biosynthetic scheme are shared by the structural analog heparin, which is synthesized in mast cells and some other mammalian cells and is several-fold higher degree of polymerization and more extensive modification than heparan sulfate. [source] The bromodomain-containing protein Bdf1p acts as a phenotypic and transcriptional multicopy suppressor of YAF9 deletion in yeastMOLECULAR MICROBIOLOGY, Issue 3 2004Michele M. Bianchi Summary It was observed previously that the deletion of the open reading frame YNL107w (YAF9) was highly pleiotropic in yeast and caused defective growth phenotypes in the presence of several unrelated inhibitors, including caesium chloride. We have selected multicopy extragenic suppressor genes, revealing that this phenotype can be suppressed by overdosing the transcription factors BDF1 and GAT1 in the yaf9, strain. We focused our analysis on suppression by BDF1 and performed a genome-wide transcript analysis on a yaf9, strain, compared with the wild-type and BDF1 -suppressed strains. YAF9 deletion has a clear effect on transcription and leads to modulation of the level of expression of several genes. Transcription of a considerable portion of the underexpressed genes is restored to wild-type levels in the BDF1 -suppressed strain. We show by chromatin immunoprecipitation that both Yaf9p and Bdf1p bind to promoters of some of these genes and that the level of H3 and H4 acetylation at one of these promoters is significantly lowered in the yaf9 deleted strain, compared with the wild-type and the BDF1 -suppressed strains. [source] Sequence and expression analyses of , and , transcripts in patients with waldenström's macroglobulinemiaAMERICAN JOURNAL OF HEMATOLOGY, Issue 3 2001Satoshi Shiokawa Abstract Waldenström's macroglobulinemia (WM) is a malignant lymphoplasmo-proliferative disorder with monoclonal pentameric immunoglobulin (Ig)M production. The most consistent feature of clonal B cells in the bone marrow (BM) and/or lymph nodes of patients with WM is the presence of pleomorphic B-lineage cells at different stages of maturation, such as small lymphocytes, lymphoplasmacytoid cells, and plasma cells. Monoclonal lymphocytes express , chains with or without , chains. A recent DNA analysis of WM tumor clones showed WM to be derived from B cells that have been selected by antigen at a relatively late stage of differentiation. To further clarify the origin of WM tumor cells, we analyzed the variable (V) domain sequences of tumor derived , and , transcripts. The expression of , transcripts was also examined in peripheral blood (PB) and BM using the reverse transcriptase polymerase chain reaction (RT-PCR) combined with a single-strand conformation polymorphism (SSCP) analysis. The sequences were identical among the , and , transcripts in each patient and the level of somatic mutation in the VH regions expressed by tumor cells was in the same range as that of IgM-only B cells and IgM+IgD+ memory B cells. In our previous RT-PCR-SSCP analysis, a single dominant band of the , isotype was observed in BM and PB in all patients. However, common dominant bands in BM and PB were detected in only one patient in a , transcript analysis. In the rest of the patients, monoclonal , transcripts were only detected in BM. Our results suggest that a normal counterpart of WM cells is somatically mutated IgM+IgD+ and/or IgM-only B cells and the expression patterns of monoclonal , and , transcripts differ between BM and PB in some cases of WM. Am. J. Hematol. 68:139,143, 2001. © 2001 Wiley-Liss, Inc. [source] Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the BrassicaceaeTHE PLANT JOURNAL, Issue 1 2005Andreas Wachter Summary The genome of Arabidopsis thaliana reveals that in this species the enzymes of glutathione biosynthesis, GSH1 and GSH2, are encoded by single genes. In silico analysis predicts proteins with putative plastidic transit peptides (TP) for both genes, but this has not been experimentally verified. Here we report a detailed analysis of the 5,ends of GSH1 and GSH2 mRNAs and demonstrate the subcellular targeting of the proteins encoded by different transcript types. GSH1 transcript analysis revealed two mRNA populations with short and long 5,-UTRs, respectively, both including the entire TP sequence. The ratio of long/total GSH1 transcripts was subject to developmental regulation. Transient transformation experiments with reporter gene fusions, bearing long or short 5,-UTRs, indicated an exclusive targeting of GSH1 to the plastids. Corroborating these results, endogenous and ectopically expressed GSH1 proteins were always present as a single polypeptide species with the size expected for correctly processed GSH1. Finally, the plastidic GSH1 localization was confirmed by immunocytochemistry. Similar to GSH1, multiple transcript populations were found for GSH2. However, here the prevalent shorter transcripts lacked a complete TP sequence. As expected, the large (but less abundant) transcript encoded a plastidic GSH2 protein, whereas GSH2 synthesized from the shorter transcript was targeted to the cytosol. The implications of the results for the compartmentation and regulation of GSH synthesis are discussed. [source] The prevalence of, and molecular defects underlying, inherited protein S deficiency in the general populationBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Nicholas J. Beauchamp Summary The molecular basis of protein S (PS) deficiency was investigated in seven of eight donors identified with persistently low plasma PS levels from a survey of PS levels in 3788 Scottish blood donors. PROS1 gene analysis identified at least one defect in six donors. Five were heterozygous for the Heerlen polymorphism predicting a Ser460Pro substitution. Haplotype analysis revealed the possibility that this allele was inherited with the same haplotype in four of the five donors, suggesting a founder effect for the Heerlen allele in this population. One Heerlen allele carrier was also heterozygous for a 3 bp deletion 68,72 bp upstream of exon 2. Platelet PROS1 transcript analysis showed no reduction in mRNA expression from the affected allele in this donor. A T to G transversion 3 bp upstream of exon 12 was identified in one donor, which is predicted to reduce the efficiency of PS mRNA splicing. However, PROS1 transcript analysis showed no evidence of exon skipping or cryptic splicing. No PROS1 gene defect was detected in the remaining donor. This genetic information enabled us to refine our estimate of the prevalence of heritable PS deficiency in the Scottish population to between 0·16% and 0·21%, predominantly resulting from the presence of the Heerlen allele. [source] | |||||||||||||