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91.
92.
A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)
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Jérôme Morinière Lars Hendrich Michael Balke Arne J. Beermann Tobias König Monika Hess Stefan Koch Reinhard Müller Florian Leese Paul D. N. Hebert Axel Hausmann Christoph D. Schubart Gerhard Haszprunar 《Molecular ecology resources》2017,17(6):1293-1307
Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution. 相似文献
93.
Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens. 相似文献
94.
Kushnir S Marsac Y Breitling R Granovsky I Brok-Volchanskaya V Goody RS Becker CF Alexandrov K 《Bioconjugate chemistry》2006,17(3):610-617
Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease. 相似文献
95.
Telse Roggentin Reinhard G. Kleineidam Roland Schauer Peter Roggentin 《Glycoconjugate journal》1992,9(5):235-240
Three site-specific mutations were performed in two regions of a sialidase gene fromClostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed inEscherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, asK
M andV
max, as well asK
i measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies. 相似文献
96.
The Habc domain and the SNARE core complex are connected by a highly flexible linker 总被引:3,自引:0,他引:3
Syntaxin 1a is a member of the SNARE superfamily of small, mostly membrane-bound proteins that mediate membrane fusion in all eukaryotic cells. Upon membrane fusion, syntaxin 1 forms a stable complex with its partner SNAREs. Syntaxin contains a C-terminal transmembrane domain, an adjacent SNARE motif that interacts with its partner SNAREs, and an N-terminal Habc domain. The Habc domain reversibly folds back upon the SNARE motif, resulting in a "closed" conformation that is stabilized by binding to the protein munc18. The SNARE motif and the Habc domain are separated by a linker region of about 40 amino acids. When syntaxin is complexed with munc18, the linker is structured and consists of a mix of turns and small alpha-helices. When syntaxin is complexed with its partner SNAREs, the Habc domain is dissociated, but the structure of the linker region is not known. Here we used site-directed spin labeling and EPR spectroscopy to determine the structure of the linker region of syntaxin in the SNARE complex. We found that the entire linker region of syntaxin is unstructured except for three residues at the N-terminal and six residues at the C-terminal boundary whereas the structures of the flanking regions in the Habc domain and the SNARE motif correspond to the high-resolution structures of the isolated fragments. We conclude that the linker region exhibits a high degree of conformational flexibility. 相似文献
97.
Arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of Euphorbia pulcherrima 总被引:1,自引:0,他引:1
Katja Saare-Surminski Walter Preil J. Paul Knox Reinhard Lieberei 《Physiologia plantarum》2000,108(2):180-187
The arabinogalactan protein (AGP) fractions of embryogenic and non-embryogenic callus lines of Euphorbia pulcherrima Willd. ex. Klotzsch were analysed over a cultivation period of 9 weeks using the β -glucosyl Yariv reagent and an anti-AGP antibody (LM2). The amount of AGPs detected with the Yariv reagent increased in embryogenic cultures during the development of somatic embryos. The embryogenic and non-embryogenic callus contained different sets of AGPs characterized with the Yariv reagent and the LM2 monoclonal antibody. AGPs recognized by LM2 are localized primarily in the protodermal cells of globular somatic embryos. The development of somatic embryos of E. pulcherrima appears to be associated with the presence of particular AGPs. 相似文献
98.
Schliebs R 《Neurochemical research》2005,30(6-7):895-908
Alzheimer’s disease, the most common neurodegenerative disorder of senile dementia, is characterized by two major morpho-pathological
hallmarks. Deposition of extracellular neuritic, β-amyloid peptide-containing plaques (senile plaques) in cerebral cortical
regions of Alzheimer patients is accompanied by the presence of intracellular neurofibrillary tangles in cerebral pyramidal
neurons. Basal forebrain cholinergic dysfunction is also a consistent feature of Alzheimer’s disease, which has been suggested
to cause, at least partly, the cognitive deficits observed in patients with Alzheimer’s disease. Impaired cortical cholinergic
neurotransmission may also contribute to β-amyloid plaque pathology in Alzheimer’s disease by affecting expression and processing
of the β-amyloid precursor protein (APP). Vice versa, low level of soluble β-amyloid has been observed to inhibit cholinergic
synaptic function. Deposition of β-amyloid plaques in Alzheimer’s disease is also accompanied by a significant plaque-associated
glial up-regulation of interleukin-1, which has been attributed to affect expression and metabolism of APP and to interfere
with cholinergic transmission. Understanding the molecular mechanisms underlying the interrelationship between cortical cholinergic
dysfunction, β-amyloid formation and deposition, as well as local inflammatory upregulation, would allow to derive potential
treatment strategies to pharmacologically intervene in the disease-causing signaling cascade. 相似文献
99.
Marcin Moch Reinhard Windoffer Nicole Schwarz Raphaela Pohl Andreas Omenzetter Uwe Schnakenberg Fabian Herb Kraisorn Chaisaowong Dorit Merhof Lena Ramms Gloria Fabris Bernd Hoffmann Rudolf Merkel Rudolf E. Leube 《PloS one》2016,11(3)
The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells. 相似文献
100.
Heidi Borgeraas Jens Kristoffer Hertel Gard Frodahl Tveitev?g Svingen Eva Ringdal Pedersen Reinhard Seifert Ottar Nyg?rd J?ran Hjelmes?th 《PloS one》2016,11(3)