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Katharina Hiebler Zsófia Lengyel Carlos A. Castañeda Olga V. Makhlynets 《Proteins》2017,85(9):1656-1665
AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His144) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pKa value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pKa calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pKa value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pKa value of this residue in water. We determined that a short‐range favorable interaction with Glu127 contributes to the elevated pKa of His144. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pKa of its active nucleophile, His144, by 0.7 units. As a direct result of the decrease in the His144 pKa value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc. 相似文献
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In the present work, we show that we obtained nanometric structures made of water, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol (Chol), and a mixture of ethoxylated and non-ethoxylated sorbitan fatty acid esters (Tween 20, Span 20, Tween 80, and Span 80) by mixing all of them near the cloud point temperature (cp) of the ethoxylated surfactant. The influence that the constituents had on the size of the particle was determined by a pseudo-ternary phase diagram of water/Tween–Span/DPPC–Chol; the colloidal particles obtained were studied by differential scanning calorimetry, confocal fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. These studies were made for all the systems with at least 23 d of colloidal stability. The most stable system was obtained with the Tween 80–Span 80 pair, behaving as a typical suspension for 48 d; this system was made of water, Tween 80–Span 80 (80:20), DPPC–Chol (95:5) in a corresponding molar ratio of 48:37:100:10. The colloidal particles obtained were a kind of emulsion and liposome structures. The second stable system was obtained with the same mixture, but in a molar ratio of 8:6:9:0, its structure was also a kind of emulsion particles. In both systems and in other less stable ones, the “emulsion particle” was completely new, it structurally corresponds to a nucleus of mixed micelles surrounded by at least one bilayer of DPPC. 相似文献
4.
PPARγ and PGC-1α as Therapeutic Targets in Parkinson’s 总被引:1,自引:0,他引:1
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Talita Signoreti Graziano Maria Claudia Cuzzullin Gilson Cesar Franco Humberto Osvaldo Schwartz-Filho Eduardo Dias de Andrade Francisco Carlos Groppo Karina Cogo-Müller 《PloS one》2015,10(5)
Statins are important lipid-lowering agents with other pleiotropic effects. Several studies have explored a possible protective effect of statins to reduce the morbidity and mortality of many infectious diseases. Staphylococcus aureus is one of the main pathogens implicated in nosocomial infections; its ability to form biofilms makes treatment difficult. The present study observed the MIC of atorvastatin, pravastatin and simvastatin against S. aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis. Simvastatin was the only agent with activity against clinical isolates and reference strains of methicilin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Thus, the effects of simvastatin on the growth, viability and biofilm formation of S. aureus were tested. In addition, a possible synergistic effect between simvastatin and vancomycin was evaluated. Simvastatin’s MIC was 15.65 µg/mL for S. aureus 29213 and 31.25 µg/mL for the other strains of S. aureus. The effect of simvastatin was bactericidal at 4xMIC and bacteriostatic at the MIC concentration. No synergistic effect was found between simvastatin and vancomycin. However, the results obtained against S. aureus biofilms showed that, in addition to inhibiting adhesion and biofilm formation at concentrations from 1/16xMIC to 4xMIC, simvastatin was also able to act against mature biofilms, reducing cell viability and extra-polysaccharide production. In conclusion, simvastatin showed pronounced antimicrobial activity against S. aureus biofilms, reducing their formation and viability. 相似文献
7.
Adriana Fernanda K. Vizuete Matheus Mittmann Hennemann Carlos Alberto Gonçalves Diogo Losch de Oliveira 《Neurochemical research》2017,42(10):2730-2742
Epilepsy prevalence is high in infancy and in the elderly population. Lithium–pilocarpine is widely used to induce experimental animal models of epilepsy, leading to similar neurochemical and morphological alterations to those observed in temporal lobe epilepsy. As astrocytes have been implicated in epileptic disorders, we hypothesized that specific astroglial changes accompany and contribute to epileptogenesis. Herein, we evaluated time-dependent astroglial alterations in the hippocampus of young (27-day-old) rats at 1, 14 and 56 days after Li–pilocarpine-induced status epilepticus (SE), corresponding to different phases in this model of epilepsy. We determined specific markers of astroglial activation: GFAP, S100B, glutamine synthetase (GS), glutathione (GSH) content, aquaporin-4 (AQP-4) and potassium channel Kir 4.1; as well as epileptic behavioral, inflammatory and neurodegenerative changes. Phase-dependent signs of hippocampal astrogliosis were observed, as demonstrated by increments in GFAP, S100B and GS. Astrocyte dysfunction in the hippocampus was characterized, based on the decrease in GSH content, AQP-4 and Kir 4.1 channels. Degenerating neurons were identified by Fluoro-Jade C staining. We found a clear, early (at SE1) and persistent (at SE56) increase in cerebrospinal fluid (CSF) S100B levels. Additionally, serum S100B was found to decrease soon after SE induction, implicating a rapid-onset increase in the CSF/serum S100B ratio. However, serum S100B increased at SE14, possibly reflecting astroglial activation and/or long-term increase in cerebrovascular permeability. Moreover, we suggest that peripheral S100B levels may represent a useful marker for SE in young rats and for follow up during the chronic phases of this model of epilepsy. Together, results reinforce and extend the idea of astroglial involvement in epileptic disorders. 相似文献
8.
Luis Santamaría Jesús Balsa Beatriz Bidondo Angel Baltanás Carlos Montes 《Hydrobiologia》1992,246(2):89-98
Laboratory experiments and field data were used to determine salinity tolerance limits of three ostracode species (Prionocypris aragonica, Eucypris mareotica and Heterocypris barbara) from Iberian saline lakes. Salinity tolerance appeared related to ionic composition and temperature. Implications for ostracode ecology and geographical distribution are evaluated. 相似文献
9.
Miguel Angel Pujana Monica Gratacós Jordi Corral Isabel Banchs Aurora Sánchez David Genís Carlos Cervera Víctor Volpini X. Estivill 《Human genetics》1997,101(1):18-21
Genetic anticipation – increasing severity and a decrease in the age of onset with successive generations of a pedigree –
is clearly present in autosomal dominant cerebellar ataxia (ADCA). Anticipation is correlated with expansion of the CAG/CTG
repeat sequence to sizes above those in the normal range through the generations of a pedigree. Genetic heterogeneity has
been demonstrated for ADCA, with four cloned genes (SCA1, SCA2, SCA3/MJD, and SCA6) and three mapped loci (SCA4, SCA5 and
SCA7). Another related dominant ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), presents anticipation with CAG/CTG repeat
expansions. We had previously analysed ADCA patients who had not shown repeat expansions in cloned genes for CAG/CTG repeat
expansions by the repeat expansion detection method (RED) and had detected expansions of between 48 and 88 units in 17 unrelated
familial cases. We present here an analysis of 13 genes and expressed sequence tags (ESTs) containing 10 or more CAG/ CTG
repeat sequences selected from public databases in the 17 unrelated ADCA patients. Of the 13 selected genes and ESTs, 9 were
found to be polymorphic with heterozygosities ranging between 0.09 and 0.80 and 2 to 17 alleles. In ADCA patients none of
the loci showed expansions above the normal range of the CAG/CTG repeat sequences, excluding them as the mutation causing
ADCA.
Received: 28 May 1997 / Accepted: 30 June 1997 相似文献
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