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121.
Alexandra Ahlner Mats Carlsson Bengt-Harald Jonsson Patrik Lundström 《Journal of biomolecular NMR》2013,56(3):191-202
We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2. 相似文献
122.
Ulrich Weininger Annica T. Blissing Janosch Hennig Alexandra Ahlner Zhihong Liu Hans J. Vogel Mikael Akke Patrik Lundström 《Journal of biomolecular NMR》2013,57(1):47-55
Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for 1H spins in methyl 13CHD2 groups, which improves the characterization of fast exchange processes. The influence of 1H–1H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different 1H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any 1H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by 1H and 13C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results. 相似文献
123.
Alexandra Choutko Andreas P. Eichenberger Wilfred F. van Gunsteren Jožica Dolenc 《Protein science : a publication of the Protein Society》2013,22(6):809-822
The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. 相似文献
124.
Nicolas B. M. Pruvost Alexandra Hoffmann Heinz‐Ulrich Reyer 《Ecology and evolution》2013,3(9):2933-2946
The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all‐hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures. 相似文献
125.
126.
Restriction fragment length polymorphism tools is an R application which supports a complete workflow of polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), dealing with the problems which accompany analysis when PCR‐RFLP is used in diversity studies. Large numbers of different RFLP samples obtained from multiple electrophoresis runs might lead to limitations or misidentifications due to the need for band matching in most existing software applications. Due to the common problem of variation in the density of bands (i.e. distances between bands or visual intensity) in the electropherograms, it is desirable to have options for handling samples with uncertain or faint bands. As a further step in the workflow, scientists often use DNA sequencing to identify individual genotypes, so that the use of specific software to combine these tasks might be helpful. With this background, we here present an application that supports a complete workflow, starting with the analysis of single species samples by PCR‐RFLP, to PCR‐RFLP genotype identification based on a reference data set and DNA sequencing followed by similarity analysis. RFLPtools is a freely available, platform‐independent application which provides analysis functions for DNA fragment molecular weights (e.g. by RFLP analysis), including similarity calculations without the need for band matching. As it is written for the statistical software R, other statistical analyses might also be easily applied. 相似文献
127.
Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA. 相似文献
128.
Alexandra V. Stavropoulou Florentia Fostira Maroulio Pertesi Marianthi Tsitlaidou Gerassimos E. Voutsinas Olga Triantafyllidou Aristotelis Bamias Meletios A. Dimopoulos Eleni Timotheadou Dimitrios Pectasides Christos Christodoulou George Klouvas Christos Papadimitriou Thomas Makatsoris George Pentheroudakis Gerasimos Aravantinos Vassilis Karydakis Drakoulis Yannoukakos George Fountzilas Irene Konstantopoulou 《PloS one》2013,8(3)
Germline mutations in the BRCA1 and BRCA2 genes contribute to approximately 18% of hereditary ovarian cancers conferring an estimated lifetime risk from 15% to 50%. A variable incidence of mutations has been reported for these genes in ovarian cancer cases from different populations. In Greece, six mutations in BRCA1 account for 63% of all mutations detected in both BRCA1 and BRCA2 genes. This study aimed to determine the prevalence of BRCA1 mutations in a Greek cohort of 106 familial ovarian cancer patients that had strong family history or metachronous breast cancer and 592 sporadic ovarian cancer cases. All 698 patients were screened for the six recurrent Greek mutations (including founder mutations c.5266dupC, p.G1738R and the three large deletions of exon 20, exons 23–24 and exon 24). In familial cases, the BRCA1 gene was consequently screened for exons 5, 11, 12, 20, 21, 22, 23, 24. A deleterious BRCA1 mutation was found in 43/106 (40.6%) of familial cancer cases and in 27/592 (4.6%) of sporadic cases. The variant of unknown clinical significance p.V1833M was identified in 9/698 patients (1.3%). The majority of BRCA1 carriers (71.2%) presented a high-grade serous phenotype. Identifying a mutation in the BRCA1 gene among breast and/or ovarian cancer families is important, as it enables carriers to take preventive measures. All ovarian cancer patients with a serous phenotype should be considered for genetic testing. Further studies are warranted to determine the prevalence of mutations in the rest of the BRCA1 gene, in the BRCA2 gene, and other novel predisposing genes for breast and ovarian cancer. 相似文献
129.
éva Borbély Zsófia Hajna Katalin Sándor László Kereskai István Tóth Erika Pintér Péter Nagy János Szolcsányi John Quinn Andreas Zimmer James Stewart Christopher Paige Alexandra Berger Zsuzsanna Helyes 《PloS one》2013,8(4)
Objective
Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse.Methods
Complete Freund’s Adjuvant was injected intraplantarly and into the tail of Tac1−/−, Tac4−/−, Tacr1−/− (NK1 receptor deficient) and Tac1−/−/Tac4−/− mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1β concentrations of the tibiotarsal joints were performed.Results
Mechanical hyperalgesia was significantly reduced from day 11 in Tac4−/− and Tacr1−/− animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1β concentration in the joint homogenates were significantly smaller in Tac4−/− and Tac1−/−/Tac4−/− mice.Conclusions
Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1β production is suggested. 相似文献130.
Filumena Maria Gomes S. V. Subramanian Ana Maria de Ulh?a Escobar Maria Helena Valente Sandra Josefina Ferraz Ellero Grisi Alexandra Brentani Günther Fink 《PloS one》2013,8(6)