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1.
Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt2/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.  相似文献   
2.
The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.  相似文献   
3.
镉对茶条槭和五角槭光合作用和叶绿素荧光特性的影响   总被引:4,自引:0,他引:4  
以北方阔叶树种茶条槭(Acer ginnala)和五角槭(Acer mono)2年生苗木为材料,采用土壤和风化砂混合物作为盆栽基质,设置0(CK)、10、50、100、200 mg·kg-1 5种土壤镉浓度,研究了土壤镉胁迫对苗木叶片光合作用和叶绿素荧光特性的影响.结果表明:随着土壤镉胁迫浓度增加,茶条槭净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)先升后降,胞间二氧化碳浓度(Ci)则先降后升,且均在10 mg·kg-1 Cd2+时达到峰值或谷值;同时五角槭的Pn逐渐降低,Ci持续升高,Gs和Tr则先升后降且均在10 mg·kg-1 Cd2+时达到峰值.随土壤镉处理浓度的增加,茶条槭和五角槭的Fv/Fm、Fv/F0和qN均呈先升后降趋势,ΦPSⅡ则均持续下降;qP在茶条槭表现为先升后降,而在五角槭则逐渐下降.所有指标(除Ci外)在随镉浓度的变化过程中,其上升幅度均为茶条槭大于五角槭,而下降幅度则为五角槭大于茶条槭.研究发现,重金属镉通过破坏或抑制光合作用过程来影响茶条槭和五角槭的生长,高浓度镉导致的Pn下降是由非气孔限制因素所致;茶条槭对土壤镉的耐性大于五角槭,100和50 mg·kg-1 Cd2+可能分别是茶条槭和五角槭对土壤镉污染的耐受极限.  相似文献   
4.
We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca2+ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca2+]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca2+ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca2+ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.  相似文献   
5.
Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.  相似文献   
6.
A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2′-furil to form fluorescent difurylimidazole derivatives that were separated on a C18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, respectively. The method showed good linearity over the concentration ranges of 0.100–5.00, 0.200–10.0, 0.200–40.0, and 0.400–10.0 nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions.  相似文献   
7.
瑞香狼毒是分布在青海省高寒草甸的主要毒害草之一,近年来其迅速蔓延对当地畜牧业危害严重并使草地生态系统日趋退化.在海北州祁连县选取狼毒分布的典型退化草甸,采用2012—2014年狼毒盛花期获取的实测光谱数据,分析狼毒与牧草的光谱差异性.结果表明: 在350~900 nm的可见光 近红外波段,狼毒顶花的光谱反射特征明显异于狼毒叶片和同期牧草等绿色背景,顶花与绿色背景的光谱反射率差异主要体现在红谷和蓝谷.随着盖度的增加,狼毒群落光谱反射率整体升高,在近红外反射峰处狼毒群落与牧草群落光谱反射率具有最大差值,且不同盖度狼毒群落之间的差异性最明显.顶花与绿色背景以及狼毒群落与牧草群落的一阶导数光谱差异均体现在黄边幅值和蓝边幅值.狼毒群落盖度与光谱特征参量的线性回归分析表明,红谷与狼毒群落盖度的相关性最好(R2=0.94),反演狼毒群落盖度的精度最高.盛花期区分狼毒与牧草的主要光谱特征参量为红谷、蓝谷与近红外反射峰,其对应的红、蓝及近红外波段的组合可用于构建狼毒提取的敏感指数.  相似文献   
8.
9.
The accessory light-harvesting polypeptides associated with photosystem I (LHCI) in Porphyridium cruentum bind chlorophyll a, zeaxanthin and -carotene. A cDNA library of P. cruentum was screened with an antiserum specific to the LHCI polypeptides, and an 0.9 kb fragment was identified as coding for an LHCI polypeptide. This cDNA, which we named LhcaR1, has an open reading frame encoding 222 amino acid residues including a putative transit peptide of 28 amino acids. Hydropathy analysis suggests that there are three transmembrane helices in the mature polypeptide. Each of the amino acid residues that bind chlorophyll (six residues) and serve in stabilizing the helices in higher-plant LHCs are conserved in helices 1 and 3 of P. cruentum LhcaR1. The N-terminal flanking regions of these two helices also show high sequence conservation with other LHCs. Helix 2 contains a seventh putative chlorophyll-binding site, but resembles helix 2 of higher-plant LHCs to a lesser degree. A sequence motif of 11 residues found near the N-terminus and in each of the three helices suggests the possibility that the red algal LhcaR1 derives from a gene duplication. Polypeptides of the expected molecular weight in six other red algae (Achrochaetium, Bangia, Callithamnion, Cyanidium, Polysiphonia, Spermothamnion) were recognized by the antiserum to P. cruentum LHCI, indicating a wide distribution of LHCI in rhodophytes.  相似文献   
10.
Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease that is characterized by synovial inflammation and bone erosion. We have investigated the mechanism(s) by which essential trace metals may initiate and propagate inflammatory phenotypes in synovial fibroblasts. We used HIG-82, rabbit fibroblast-like synovial cells (FLS), as a model system for potentially initiating RA through oxidative stress. We used potassium peroxychromate (PPC, Cr+5), ferrous chloride (FeCl2, Fe+2), and cuprous chloride (CuCl, Cu+) trace metal agents as exogenous pro-oxidants. Intracellular ROS was quantified by fluorescence microscopy and confirmed by flow cytometry (FC). Protein expression levels were measured by western blot and FC, while ELISA was used to quantify the levels of cytokines. Trace metal agents in different valence states acted as exogenous pro-oxidants that generate reactive oxygen species (ROS), which signal through TLR4 stimulation. ROS/TLR4- coupled activation resulted in the release of HMGB1, TNF-α, IL-1β, and IL-10 in conjunction with upregulation of myeloid-related protein (MRP8/14) inflammatory markers that may contribute to the RA pathophysiology. Our results indicate that oxidant-induced TLR4 activation can release HMGB1 in combination with other inflammatory cytokines to mediate pro-inflammatory actions that contribute to RA pathogenesis. The pathway by which inflammatory and tissue erosive changes may occur in this model system possibly underlies the need for functioning anti-HMGB1-releasing agents and antioxidants that possess both dual trace metal chelating and oxidant scavenging properties in a directed combinatorial therapy for RA.  相似文献   
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