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901.
The lipid phase of the photoreceptor outer segment membrane is essential to the photon capturing and signaling functions of rhodopsin. Rearrangement of phospholipids in the bilayer accompanies the formation of the active intermediates of rhodopsin following photon absorption. Furthermore, evidence for the formation of a condensation product between the photolyzed chromophore all-trans-retinal and phosphatidylethanolamine indicates that phospholipid may also participate in the movement of the retinoid in the membrane. The downside of these interactions is the formation of bisretinoid-phosphatidylethanolamine compounds that accumulate in retinal pigment epithelial cells with age and that are particularly abundant in some retinal disorders. The propensity of these compounds to negatively impact on the cells has been linked to the pathogenesis of some retinal disorders including juvenile onset recessive Stargardt disease and age-related macular degeneration. 相似文献
902.
903.
Tjakko J. van Ham Alessandro Esposito Shang-Te D. Hsu Clemens F. Kaminski Ellen A.A. Nollen Carlos W. Bertoncini 《Journal of molecular biology》2010,395(3):627-12905
Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein α-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins. 相似文献
904.
Burki Rajendar 《Journal of molecular biology》2010,399(5):665-347
The removal of damaged or unneeded proteins by ATP-dependent proteases is crucial for cell survival in all organisms. Integral components of ATP-dependent proteases are motor proteins that unfold stably folded proteins that have been targeted for removal. These protein unfoldases/polypeptide translocases use ATP to unfold the target proteins and translocate them into a proteolytic component. Despite the central role of these motor proteins in cell homeostasis, a number of important questions regarding the molecular mechanisms of enzyme catalyzed protein unfolding and translocation remain unanswered. Here, we demonstrate that Escherichia coli ClpA, in the absence of the proteolytic component ClpP, processively and directionally steps along the polypeptide backbone with a kinetic step size of ∼ 14 amino acids, independent of the concentration of ATP with a rate of ∼ 19 amino acids s−1 at saturating concentrations of ATP. In contrast to earlier studies by others, we have developed single-turnover fluorescence stopped-flow methods that allow us to quantitatively examine the molecular mechanism of the motor component ClpA decoupled from the proteolytic component ClpP. For the first time, we reveal that in the absence of ClpP ClpA translocates polypeptides directionally, processively and in discrete steps similar to other motor proteins that translocate vectorially on a linear lattice, such as nucleic acid helicases and kinesin. We believe that the methods employed here will be generally applicable to the examination of other AAA?+ protein translocases involved in a variety of important biological functions where the substrate is not covalently modified; for example, membrane fusion, membrane transport, protein disaggregation, and protein refolding. 相似文献
905.
Chun-Liang Lai Kyle E. Landgraf Gregory A. Voth Joseph J. Falke 《Journal of molecular biology》2010,402(2):301-1572
Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca2+-regulated membrane targeting element. Upon activation by Ca2+, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca2+ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca2+ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP2) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP2 simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP2 headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP2-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP2:C2 stoichiometry approaching 2:1 at a high PIP2 mole density. Direct binding measurements titrating the C2 domain with PIP2 in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP2 mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP2 stoichiometries are discussed. 相似文献
906.
Zhanjia Hou 《Journal of molecular biology》2010,402(1):210-35050
To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 Å. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle. 相似文献
907.
The potentials of a series of one-electron oxidation and reduction reactions have been determined for manganese group half-sandwich complexes of the tricarbadecaboranyl ligand PhC3B7H9 and the penta-organo fullerene ligand C60Bn2PhH2 (Bn = benzyl). The anodic processes were studied in CH2Cl2 and the cathodic processes were studied in both CH2Cl2 and THF, the supporting electrolyte being [NBu4][B(C6F5)4]. The manganese complex Mn(CO)2(PMe3)(PhC3B7H9) (1) is a member of a three-electron transfer series which includes oxidation to 1+ (0.51 V versus ferrocene) and successive reductions to 1− (−1.66 V) and 12− (−1.77 V). Both the oxidation and reduction of the closely-related complex Mn(CO)2(PPh3)(PhC3B7H9) (2) are chemically irreversible under slow-scan cyclic voltammetry conditions. The rhenium complex Re(CO)2(PPh3)(PhC3B7H9) (3) oxidizes (E1/2 = 0.82 V versus ferrocene) to a radical cation which, unlike its cyclopentadienyl analogue, shows no evidence of dimerization. Oxidation of the fullerene-based complex Re(CO)3(C60Bn2PhH2) is more facile than that of its cyclopentadienyl analogue, in contrast to previous findings in this class of metal-fullerene derivatives. An electrochemical ligand factor, EL, of 0.63 is calculated for the PhC3B7H9 ligand in manganese group half-sandwich complexes. 相似文献
908.
Zhi Xie Don Kulasiri Sandhya Samarasinghe Jiang Qian 《Biotechnology and bioengineering》2010,105(2):250-259
To assess the importance of model parameters in kinetic models, sensitivity analysis is generally employed to provide key measures. However, it is quite often that no information is available for a significant number of parameters in biochemical models. Therefore, the results of sensitivity analysis that heavily rely on the accuracy of parameters are largely ambiguous. In this study, we propose a computational approach to determine the relative importance of parameters controlling the performance of the circadian clock in Drosophila. While previous attempts to sensitivity analysis largely depend on the knowledge of model parameters which are generally unknown, our study depicts a consistent picture of sensitivity assessment for a large number of parameters, even when the values of these parameters are not available in vivo. The resulting parametric sensitivity analysis suggests that PER/TIM negative loop is critical to maintain the stable periodicity of the circadian clock, which is consistent to the previously experimental and computational findings. Furthermore, our analysis generates a rich hypothesis of important parameters in the circadian clock that can be further tested experimentally. This approach can also be extended to assess the sensitivity of parameters in any biochemical system where a large number of parameters have unknown values. Biotechnol. Bioeng. 2010; 105: 250–259. © 2009 Wiley Periodicals, Inc. 相似文献
909.
Many studies have examined consensus sequences required for protein‐glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the α‐helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein‐heparin binding interactions when the charged residues are aligned linearly. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 290–298, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
910.