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排序方式: 共有158条查询结果,搜索用时 31 毫秒
91.
Alexandra Kukat Christian Kukat Jan Brocher Ingo Schfer Georg Krohne Ian A. Trounce Gaetano Villani Peter Seibel 《Nucleic acids research》2008,36(7):e44
Eukaryotic cells devoid of mitochondrial DNA (ρ0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ0 cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ0 cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK− the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK− ρ0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ0 mitochondria. 相似文献
92.
Seibel P Di Nunno C Kukat C Schäfer I Del Bo R Bordoni A Comi GP Schön A Capuano F Latorre D Villani G 《Nucleic acids research》2008,36(18):5872-5881
Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids. 相似文献
93.
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95.
Becker D Woltersdorf R Boldt W Schmitz S Braam U Schmalzing G Markwardt F 《The Journal of biological chemistry》2008,283(37):25725-25734
P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation. 相似文献
96.
E Dogru H Warzecha F Seibel S Haebel F Lottspeich J St?ckigt 《European journal of biochemistry》2000,267(5):1397-1406
The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family. 相似文献
97.
Strube CP Homann A Gamer M Jahn D Seibel J Heinz DW 《The Journal of biological chemistry》2011,286(20):17593-17600
Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases. 相似文献
98.
Metabolic temperature compensation and coevolution of locomotory performance in pteropod molluscs 总被引:1,自引:0,他引:1
Gymnosomatous pteropods are highly specialized planktonic predatorsthat feed exclusively on their thecosomatous relatives. Feedingbehavior and the morphology of gymnosome feeding structuresare diverse and have evolved in concert with the size, shape,and consistency of the thecosome shell. Here, we show that themetabolic capacity and locomotory behaviors of gymnosomes aresimilarly diverse and vary with those of their prey. Both gymnosomesand thecosomes range from gelatinous sit-and-wait forms to activepredators with high-performance locomotory muscles. We findmore than 10-fold variation in size-adjusted and temperature-adjustedmetabolic rates within both the Gymnosomata and Thecosomataand a strong correlation between the metabolic rates of predatorsand of prey. Furthermore, these characteristics are stronglyinfluenced by environmental parameters and predator and preyconverge upon similar physiological capacities under similarselection. For example, compensation of locomotory capacityin cold waters leads to elevated metabolic rates in polar species.This highly coevolved system is discussed in terms of a predator–prey"arms race" and the impending loss of both predator and preyas elevated atmospheric carbon dioxide levels threaten to dissolveprey shells via oceanic acidification. 相似文献
99.
Liselotte Wesley Andersen Ronja Dirksen Elena A. Nikulina Hans J. Baage Gunars Petersons Pter Estk Oleg L. Orlov Maria V. Orlova Florian Gloza‐Rausch Matthias Gttsche Esben Terp Fjederholt Frauke Krüger Morten Elmeros 《Ecology and evolution》2019,9(9):5292-5308
Conservation genetics is important in the management of endangered species, helping to understand their connectivity and long‐term viability, thus identifying populations of importance for conservation. The pond bat (Myotis dasycneme) is a rare species classified as “Near Threatened” with a wide but patchy Palearctic distribution. A total of 277 samples representing populations in Denmark, Germany, Latvia, Hungary, and Russia were used in the genetic analyses; 224 samples representing Denmark, Germany, and Russia were analyzed at 10 microsatellite loci; 241 samples representing all areas were analyzed using mitochondrial D‐loop and cytochrome B sequences. A Bayesian clustering approach revealed two poorly resolved clusters, one representing the Danish and German groups and the other the Russian group. However, significantly different pairwise FST and DEST estimates were observed between the Danish and German groups and between the Danish and Russian groups suggesting a recent population structure. These conflicting results might be attributed to the effect of migration or low resolution due to the number of microsatellite markers used. After concatenating the two mitochondrial sequences, analysis detected significant genetic differentiation between all populations, probably due to genetic drift combined with a founder event. The phylogenetic tree suggested a closer relationship between the Russian and Northern European populations compared to the Hungarian population, implying that the latter belongs to an older ancestral population. This was supported by the observed haplotype network and higher nucleotide diversity in this population. The genetic structuring observed in the Danish/German pond bat stresses the need for a cross‐border management between the two countries. Further, the pronounced mtDNA structuring, together with the indicated migration between nearby populations suggest philopatric female behavior but male migration, emphasizes the importance of protecting suitable habitat mosaics to maintain a continuum of patches with dense pond bat populations across the species' distribution range. 相似文献
100.
Dorota Łażewska Szczepan Mogilski Stefanie Hagenow Kamil Kuder Monika Głuch-Lutwin Agata Siwek Małgorzata Więcek Maria Kaleta Ulla Seibel Armin Buschauer Barbara Filipek Holger Stark Katarzyna Kieć-Kononowicz 《Bioorganic & medicinal chemistry》2019,27(7):1254-1262
This study focuses on the design, synthesis, molecular modeling and biological evaluation of a novel group of alkyl-1,3,5-triazinyl-methylpiperazines. New compounds were synthesized and their affinities for human histamine H4 receptor (hH4R) were evaluated. Among them, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14) exhibited hH4R affinity with a Ki of 160?nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC50?=?32?nM) and [35S]GTPγS binding assay (pKb?=?6.67). In addition, antinociceptive activity of 14 in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats). 相似文献