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51.
The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpXH) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpXH had no activity toward NADPH or riboflavin. Coupling of TcpXH and TcpA demonstrated that TcpXH provided FADH2 for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpXH in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation. 相似文献
52.
Pentachlorophenol (PCP) is a toxic pollutant. Its biodegradation has been extensively studied in Sphingobium chlorophenolicum ATCC 39723. All enzymes required to convert PCP to a common metabolic intermediate before entering the tricarboxylic acid cycle have been characterized. One of the enzymes is tetrachloro-p-hydroquinone (TeCH) reductive dehalogenase (PcpC), which is a glutathione (GSH) S-transferase (GST). PcpC catalyzes the GSH-dependent conversion of TeCH to trichloro-p-hydroquinone (TriCH) and then to dichloro-p-hydroquinone (DiCH) in the PCP degradation pathway. PcpC is susceptible to oxidative damage, and the damaged PcpC produces glutathionyl (GS) conjugates, GS-TriCH and GS-DiCH, which cannot be further metabolized by PcpC. The fate and effect of GS-hydroquinone conjugates were unknown. A putative GST gene (pcpF) is located next to pcpC on the bacterial chromosome. The pcpF gene was cloned, and the recombinant PcpF was purified. The purified PcpF was able to convert GS-TriCH and GS-DiCH conjugates to TriCH and DiCH, respectively. The GS-hydroquinone lyase reactions catalyzed by PcpF are rather unusual for a GST. The disruption of pcpF in S. chlorophenolicum made the mutant lose the GS-hydroquinone lyase activities in the cell extracts. The mutant became more sensitive to PCP toxicity and had a significantly decreased PCP degradation rate, likely due to the accumulation of the GS-hydroquinone conjugates inside the cell. Thus, PcpF played a maintenance role in PCP degradation and converted the GS-hydroquinone conjugates back to the intermediates of the PCP degradation pathway. 相似文献
53.
Nissen MS Youn B Knowles BD Ballinger JW Jun SY Belchik SM Xun L Kang C 《The Journal of biological chemistry》2008,283(42):28710-28720
EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN. 相似文献
54.
Down-regulation of the let-7i facilitates gastric cancer invasion and metastasis by targeting COL1A1
Dear Editor,Globally,gastric cancer is the most common malignant tumor and the second highest contributor to cancer deaths after lung cancer(Murray et al.,2012).Despite improved success with treatment of early stage gastric cancer(Fuse et al.,2016),the five-year survival rate of advanced staged gastric cancer patients is still low.The aggressive growth characteristics of the tumor and metastasis are key factors responsible for poor overall survival in these patients(Ozkan et al.,2005).Therefore,investigation of the molecular mechanisms that underlie the aggressive behavior of gastric cancers,and identification of potential target genes for therapeutic interventions,is a key imperative. 相似文献
55.
反胶束中单宁酶的光学行为和稳定性 总被引:2,自引:0,他引:2
为了测定反胶束系统中单宁酶的光学行为和增溶方式,采用紫外分光光度法和荧光扫描技术对AOT水/异辛烷组成的反胶束体系中单宁酶和水相中单宁酶的光学行为进行研究,同时研究了不同反应体系中单宁酶的稳定性,并对单宁酶在反胶束体系中的增溶方式进行探讨。结果表明:反胶束体系与水相中的单宁酶,其光学行为存在很大差别。反胶束体系有利于单宁酶的稳定,脂肪醇作为反应底物,其碳链的增长有利于单宁酶在反胶束中的稳定性。单宁酶是以嵌入反胶束膜或与反胶束内膜接触的方式增溶的。 相似文献
56.
A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T alpha-synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 ( approximately 71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T alpha-synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP synthase are known to be directly involved in A53T alpha-synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, manganese-superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature-supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicated that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicated that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P alpha-synuclein Drosophila model (Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Protein expression in a Drosophila model of Parkinson's disease. J. Proteome Res. 6, 348-357; Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Lifetime proteomic profiling of an A30P alpha-synuclein Drosophila model of Parkinson's disease. J. Proteome Res. 6, 3729-3738) that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms. 相似文献
57.
微波提取天仙果多糖的工艺研究 总被引:7,自引:0,他引:7
本文通过对影响天仙果多糖提取的因素进行工艺研究 ,初步确认微波提取天仙果多糖宜在 80℃的碱性介质中结合微波前处理可获较高提取率。通过大孔树脂纯化 ,以及纸层析和硅胶G薄板层析分析表明 ,碱性介质中微波提取的天仙果多糖是复合多糖 ,可能由三个均一多糖组分组成。 相似文献
58.
59.
食虫目(Insectivora)字源来自拉丁文:insect-是昆虫;-vora是吞食。顾名思义,此类群动物,应该仅以昆虫为食;从形态上看来,其嘴甚尖,也适宜于吃虫。实际上,它们虽主要以昆虫为食,但也吃小形无脊椎动物和一些植物,是杂食性动物。从演化上来看,食虫目在真兽附纲(Eutheria)中一般公认是最原始的类群。其齿式:3/3,1/1,3/3,4/4=44,接近兽类模式;生殖器官也由泄殖腔向生殖与泌尿开孔与肛门完全分开过渡。以上种种形态,均反映出此类型比较原始,并且与现生诸目的兽类有着一定的亲缘。正由于此食虫目与其它诸目在形态上均有一定的相似之处,因此食虫目… 相似文献
60.