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991.
Bergström Lind S Molin M Savitski MM Emilsson L Aström J Hedberg L Adams C Nielsen ML Engström A Elfineh L Andersson E Zubarev RA Pettersson U 《Journal of proteome research》2008,7(7):2897-2910
Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample. 相似文献
992.
Chrtien Simons Jan G.E. van Leeuwen Robert Stemmer Isabel W.C.E. Arends Thomas Maschmeyer Roger A. Sheldon Ulf Hanefeld 《Journal of Molecular Catalysis .B, Enzymatic》2008,54(3-4):67-71
To investigate the full potential of hydrolases for the removal of two amine-protecting groups, 15 different, commercially available lipases, acylases, proteases and esterases were studied for the hydrolyses of N-acetyl and N-formyl protecting groups. In addition to the well-known acylases from porcine kidney and Aspergillus melleus, this screening revealed that porcine liver esterase and the lipases from Rhizomucor miehei and Pseudomonas stutzeri are also catalysts for the hydrolysis of N-acetylalanine. The activity of lipases in this reaction was unexpected, since lipases are commonly believed not to hydrolyse amides. In addition, from these 15 enzymes, three were found to be active in the hydrolysis of N-formylalanine, i.e. porcine liver esterase and the two acylases. This is the first example where esterase is employed to deprotect N-formyl amides. 相似文献
993.
Su EJ Fredriksson L Geyer M Folestad E Cale J Andrae J Gao Y Pietras K Mann K Yepes M Strickland DK Betsholtz C Eriksson U Lawrence DA 《Nature medicine》2008,14(7):731-737
Thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is markedly limited owing to concerns about hemorrhagic complications and the requirement that tPA be administered within 3 h of symptoms. Here we report that tPA activation of latent platelet-derived growth factor-CC (PDGF-CC) may explain these limitations. Intraventricular injection of tPA or active PDGF-CC, in the absence of ischemia, leads to significant increases in cerebrovascular permeability. In contrast, co-injection of neutralizing antibodies to PDGF-CC with tPA blocks this increased permeability, indicating that PDGF-CC is a downstream substrate of tPA within the neurovascular unit. These effects are mediated through activation of PDGF-alpha receptors (PDGFR-alpha) on perivascular astrocytes, and treatment of mice with the PDGFR-alpha antagonist imatinib after ischemic stroke reduces both cerebrovascular permeability and hemorrhagic complications associated with late administration of thrombolytic tPA. These data demonstrate that PDGF signaling regulates blood-brain barrier permeability and suggest potential new strategies for stroke treatment. 相似文献
994.
Rosaura Rodicio Ulf Buchwald Hans-Peter Schmitz Jürgen J Heinisch 《Fungal genetics and biology : FG & B》2008,45(4):422-435
KlWSC1, KlWSC2/3 and KlMID2, which encode putative plasma membrane sensors for cell wall integrity signaling in Kluyveromyces lactis, were cloned and characterized. Double and triple deletion mutants show severe cell integrity defects, indicating overlapping functions. The Klwsc1 Klmid2 double deletion phenotype can be suppressed by overexpression of the downstream components KlROM2, KlPKC1 and KlBCK1. KlWsc1 sensor domain analyses showed that an amino-terminal elongation as well as an extension within the cytoplasmic domain are dispensable for function. Heterologous complementation by KlMID2 and KlWSC1 in Saccharomyces cerevisiae is only achieved upon overexpression. In contrast to ScMID2, ScWSC1 complements in K. lactis. Functional studies with chimeric Mid2 constructs indicate that species specificity is mainly conferred by the extracellular domain. Sensor-GFP fusions localize to the plasma membrane, with a cell cycle dependent distribution of KlWsc1-GFP. Both Wsc-type sensors concentrate in discrete spots within the plasma membrane. 相似文献
995.
Satou Y Mineta K Ogasawara M Sasakura Y Shoguchi E Ueno K Yamada L Matsumoto J Wasserscheid J Dewar K Wiley GB Macmil SL Roe BA Zeller RW Hastings KE Lemaire P Lindquist E Endo T Hotta K Inaba K 《Genome biology》2008,9(10):R152-11
Background
The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly.Results
We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5'-ends were precisely mapped using 5'-full-length ESTs, an important refinement even in otherwise unchanged models.Conclusion
Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever. 相似文献996.
Four nonpathogenic and nontoxigenic Epicoccum nigrum strains were evaluated for their growth, morphology and pigment producing ability in three complex and one defined liquid
media. Epicoccum nigrum IBT 41028 produced pigments in all the four media tested with a maximum pigment of 3.68 AU at 410 nm in M1 medium (unoptimized)
containing 5 g/l yeast autolysate. The color hue of the crude pigment extracts ranged from 74 to 102 exhibiting dark orange
to green-yellow color. Pelleted morphology was shown to have a positive influence on the pigment production by E. nigrum strain IBT 41028 in the liquid media, and the use of Bis-tris buffer was found to diminish or reduce the pellet formation.
Since Monascus is a well known pigment producer on rice. Pigment producing ability of E. nigrum IBT 41028 was tested on rice and compared to liquid media with Monascus ruber IBT 7904 as control. Though, both genera preferred rice but E. nigrum produced 4.6 folds higher pigment in the liquid unoptimized fermentation medium compared to M. ruber. Solid phase extraction and subsequently HPLC-DAD analysis of the crude pigment extracts showed qualitative as well as quantitative
variation in the pigment composition under solid and liquid cultivations. 相似文献
997.
The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and
diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of
associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate
with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of
longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for
a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using
an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94%
slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA
and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA
sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate
organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited
to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between
seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for
chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and
illustrate how associated microorganisms can potentially have substantial effects on host evolution.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
998.
999.
Michael C. Wimberly Michael B. Hildreth Stephen P. Boyte Erik Lindquist Lon Kightlinger 《PloS one》2008,3(12)
Background
The incidence of West Nile virus (WNv) has remained high in the northern Great Plains compared to the rest of the United States. However, the reasons for the sustained high risk of WNv transmission in this region have not been determined. To assess the environmental drivers of WNv in the northern Great Plains, we analyzed the county-level spatial pattern of human cases during the 2003 epidemic across a seven-state region.Methodology/Principal Findings
County-level data on WNv cases were examined using spatial cluster analysis, and were used to fit statistical models with weather, climate, and land use variables as predictors. In 2003 there was a single large cluster of elevated WNv risk encompassing North Dakota, South Dakota, and Nebraska along with portions of eastern Montana and Wyoming. The relative risk of WNv remained high within the boundaries of this cluster from 2004–2007. WNv incidence during the 2003 epidemic was found to have a stronger relationship with long-term climate patterns than with annual weather in either 2002 or 2003. WNv incidence increased with mean May–July temperature and had a unimodal relationship with total May–July precipitation. WNv incidence also increased with the percentage of irrigated cropland and with the percentage of the human population living in rural areas.Conclusions/Significance
The spatial pattern of WNv cases during the 2003 epidemic in the northern Great Plains was associated with both climatic gradients and land use patterns. These results were interpreted as evidence that environmental conditions across much of the northern Great Plains create a favorable ecological niche for Culex tarsalis, a particularly efficient vector of WNv. Further research is needed to determine the proximal causes of sustained WNv transmission and to enhance strategies for disease prevention. 相似文献1000.
Lundberg G Rosengren AH Håkanson U Stewénius H Jin Y Stewénius Y Påhlman S Gisselsson D 《PloS one》2008,3(8):e3099