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91.
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Della Valle MC Sleat DE Zheng H Moore DF Jadot M Lobel P 《Molecular & cellular proteomics : MCP》2011,10(4):M110.006403
One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies. 相似文献
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95.
PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated
for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate
communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared
with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random
amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was
more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively.
ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints.
Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of
mixed Gram-negative bacterial strains and BIOLOG GN substrate communities.
Received: 11 September 1998 / Accepted: 29 October 1998 相似文献
96.
Helena Mata Carla Suertegaray Fontana Giovanni Nachtigall Maurício Marcos Ricardo Bornschein Marcelo Ferreira de Vasconcelos Sandro L. Bonatto 《Molecular phylogenetics and evolution》2009,53(2):450-462
Scytalopus and the recently erected Eleoscytalopus are among the Neotropical groups of birds whose taxonomy is most difficult to resolve given their very conservative morphology. We investigated the phylogeny and species limits of Eleoscytalopus and the eastern Scytalopus using two mitochondrial genes and two nuclear introns of multiple individuals from all species of these groups. The eastern Scytalopus are separated in three well defined clades also supported by morphological or vocal characteristics, although the relationships between these clades could not be resolved. We found several allopatric and very divergent lineages in these genera whose characteristics are consistent with species-level divergence, especially in S. speluncae. The great divergence between E. psychopompus and its sister species supports the former as a valid species. Our results corroborate the importance of the Bahia refuge as an avian center of endemism. 相似文献
97.
A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin 下载免费PDF全文
De Sanctis G Petrella G Ciaccio C Feis A Smulevich G Coletta M 《Biophysical journal》2007,93(6):2135-2142
The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb. 相似文献
98.
Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus 总被引:3,自引:0,他引:3 下载免费PDF全文
Gianna Palmieri Carmen Bianco Giovanna Cennamo Paola Giardina Gennaro Marino Maria Monti Giovanni Sannia 《Applied microbiology》2001,67(6):2754-2759
A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the Km value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca2+ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes. 相似文献
99.
Ballabio E Regan R Garimberti E Harbott J Bradtke J Teigler-Schlegel A Biondi A Cazzaniga G Giudici G Wainscoat JS Boultwood J Bridger JM Knight SJ Tosi S 《PloS one》2011,6(6):e20607
Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status. 相似文献
100.
Shiping Liu Eline D. Lorenzen Matteo Fumagalli Bo Li Kelley Harris Zijun Xiong Long Zhou Thorfinn Sand Korneliussen Mehmet Somel Courtney Babbitt Greg Wray Jianwen Li Weiming He Zhuo Wang Wenjing Fu Xueyan Xiang Claire C. Morgan Aoife Doherty Mary J. O’Connell James O. McInerney Erik W. Born Love Dalén Rune Dietz Ludovic Orlando Christian Sonne Guojie Zhang Rasmus Nielsen Eske Willerslev Jun Wang 《Cell》2014