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1.
Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations'' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.  相似文献   
2.
Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained.  相似文献   
3.
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by impaired degradation of very long-chain fatty acids (VLCFAs) due to mutations in the ABCD1 gene responsible for VLCFA transport into peroxisomes. Lorenzo''s oil, a 4:1 mixture of glyceryl trioleate and glyceryl trierucate, has been used to reduce the saturated VLCFA level in the plasma of X-ALD patients; however, the mechanism by which this occurs remains elusive. We report the biochemical characterization of Lorenzo''s oil activity toward elongation of very long-chain fatty acid (ELOVL) 1, the primary enzyme responsible for the synthesis of saturated and monounsaturated VLCFAs. Oleic and erucic acids inhibited ELOVL1, and, moreover, their 4:1 mixture (the FA composition of Lorenzo''s oil) exhibited the most potent inhibitory activity. The kinetics analysis revealed that this was a mixed (not a competitive) inhibition. At the cellular level, treatment with the 4:1 mixture reduced the level of SM with a saturated VLCFA accompanied by an increased level of SM with a monounsaturated VLCFA, probably due to the incorporation of erucic acid into the FA elongation cycle. These results suggest that inhibition of ELOVL1 may be an underlying mechanism by which Lorenzo''s oil exerts its action.  相似文献   
4.
Over the past several years, considerable progress has been made in the development of gene therapy as a therapeutic strategy for a variety of inherited metabolic diseases, including neuropathic lysosomal storage disorders (LSDs). The premise of gene therapy for this group of diseases is borne of findings that genetic modification of a subset of cells can provide a more global benefit by virtue of the ability of the secreted lysosomal enzymes to effect cross-correction of adjacent and distal cells. Preclinical studies in small and large animal models of these disorders support the application of either a direct in vivo approach using recombinant adeno-associated viral vectors or an ex vivo strategy using lentiviral vector-modified hematopoietic stem cells to correct the neurological component of these diseases. Early clinical studies utilizing both approaches have begun or are in late-stage planning for a small number of neuropathic LSDs. Although initial indications from these studies are encouraging, it is evident that second-generation vectors that exhibit a greater safety profile and transduction activity may be required before this optimism can be fully realized. Here, I review recent progress and the remaining challenges to treat the neurological aspects of various LSDs using this therapeutic paradigm.  相似文献   
5.
Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.  相似文献   
6.
7.
Designed retroaldolases have utilized a nucleophilic lysine to promote carbon–carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (> 10− 3 s− 1) and kcat/KM (11–25 M− 1 s− 1) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the > 105-fold rate accelerations that were achieved are within 1–3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat = 106 to 108) and an extensively evolved computational design (kcat/kuncat > 107). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.  相似文献   
8.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.  相似文献   
9.
10.
Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2Andante to form aggregates with completely blocked active sites may contribute to this phenotype.  相似文献   
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