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91.
Roberts SJ Stewart AJ Schmid R Blindauer CA Bond SR Sadler PJ Farquharson C 《Biochimica et biophysica acta》2005,1752(1):73-82
PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate (V(max) of 633 nmol min(-1) mg(-1) and K(m) of 45.5 microM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors. 相似文献
92.
Richter S Neundorf I Loebner K Gräber M Berg T Bergmann R Steinbach J Pietzsch J Wuest F 《Bioorganic & medicinal chemistry letters》2011,21(16):4686-4689
Human polo-like kinase 1 (Plk1) is involved in cell proliferation and overexpressed in a broad variety of different cancer types. Due to its crucial role in cancerogenesis Plk1 is a potential target for diagnostic and therapeutic applications. Peptidic ligands can specifically interact with the polo-box domain (PBD) of Plk1, a C-terminal located phosphoepitope binding motif. Recently, phosphopeptide MQSpTPL has been identified as ligand with high binding affinity. However, a radiolabeled version of this peptide showed only insufficient cellular uptake. The present study investigated peptide dimers consisting of PBD-targeting phosphopeptide MQSpTPL and a cell-penetrating peptide (CPP) moiety. The new constructs demonstrate superior uptake in different cancer cell-lines compared to the phosphopeptide alone. Furthermore, we could demonstrate binding of phosphopeptide-CPP dimers to PBD of Plk1 making the compounds interesting leads for the development of molecular probes for imaging Plk1 in cancer. 相似文献
93.
94.
Naoko Mizuno Christine C. Jao Ralf Langen Alasdair C. Steven 《The Journal of biological chemistry》2010,285(30):23351-23358
Endophilin A1 is a BAR (Bin/amphiphysin/Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in presynaptic endocytic vesicles. To investigate its role in membrane remodeling, we used cryoelectron microscopy to characterize structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvature resemble those of synaptic vesicles in situ. Three-dimensional reconstructions of quasicylindrical tubes revealed arrays of BAR dimers, flanked by densities that we equate with amphipathic helices whose folding and membrane insertion were attested by EPR. We also observed the compression of bulbous coated tubes into 70-Å-wide cylindrical micelles, which appear to mimic the penultimate (hemi-fission) stage of endocytosis. Our findings suggest that the adaptability of endophilin-lipid interactions underlies dynamic changes of endocytic membranes. 相似文献
95.
Recently, applications of mass spectrometry in the field of clinical proteomics have gained tremendous visibility in the scientific and clinical community. One major objective is the search for potential biomarkers in complex body fluids like serum, plasma, urine, saliva, or cerebral spinal fluid. For this purpose, efficient visualization of large data sets derived from patient cohorts is crucial to provide clinical experts an interactive impression of the data quality. Additionally, it is necessary to apply statistical analysis and pattern matching algorithms to attain validated signal patterns that may allow for later applications in sample classification. We introduce the new ClinProTools bioinformatics software, which performs all major steps of profiling, screening, and monitoring applications in clinical proteomics. ClinProTools is the data interpretation software of the mass spectrometry-based ClinProt solutions for biomarker analysis. ClinProTools performs data pretreatment, visualization, statistics, pattern determination, pattern evaluation, and classification of spectra. This article will focus on ClinProTool's powerful and intuitive visualization options for clinical proteomics applications. 相似文献
96.
Maier HJ Marienfeld R Wirth T Baumann B 《The Journal of biological chemistry》2003,278(40):39242-39250
In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner. 相似文献
97.
Thomas Szyperski Bogdan Banecki Daniel Braun Ralf W. Glaser 《Journal of biomolecular NMR》1998,11(4):387-405
We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of 15N/13C-labeled proteins. A 3D
/
/(CO)NHN experiment is recorded either in conjunction with 3D HNN<
> or with the newly presented 3D HNN
scheme. The first combination yields sequential assignments based on the measurement of13 C chemical shifts and provides a complete 1H, 13C and 15N resonance assignment of polypeptide backbone and CHn
moieties. When employing the second combination, 13C=O chemical shifts are not measured, but the sequential assignment relies on both 13C and1 H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized15 N/13C doubly labeled proteins. 相似文献
98.
Marcelo Desimone Martina Krüger Tim Wessel Marco Wehofsky Ralf Hoffmann Edgar Wagner 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,737(1-2)
Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a Mr of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The Mr of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed. 相似文献
99.
Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6 总被引:1,自引:0,他引:1 下载免费PDF全文
Andreas Keck Jrg Rau Thorsten Reemtsma Ralf Mattes Andreas Stolz Joachim Klein 《Applied microbiology》2002,68(9):4341-4349
During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone. 相似文献
100.
Biogenesis of T Pili in Agrobacterium tumefaciens Requires Precise VirB2 Propilin Cleavage and Cyclization 下载免费PDF全文
Erh-Min Lai Ralf Eisenbrandt Markus Kalkum Erich Lanka Clarence I. Kado 《Journal of bacteriology》2002,184(1):327-330
VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens. The cleaved VirB2 protein is further cyclized to form the T pilin in A. tumefaciens but not in E. coli. Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence. No T pilus was observed in these mutants. The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed. 相似文献