首页 | 本学科首页   官方微博 | 高级检索  
文章检索
  按 检索   检索词:      
出版年份:   被引次数:   他引次数: 提示:输入*表示无穷大
  收费全文   804508篇
  免费   90330篇
  国内免费   598篇
  2018年   7047篇
  2017年   6762篇
  2016年   9956篇
  2015年   14641篇
  2014年   16747篇
  2013年   23231篇
  2012年   27065篇
  2011年   27554篇
  2010年   18390篇
  2009年   16846篇
  2008年   24206篇
  2007年   25081篇
  2006年   23163篇
  2005年   22467篇
  2004年   22302篇
  2003年   20980篇
  2002年   20302篇
  2001年   34853篇
  2000年   34985篇
  1999年   27825篇
  1998年   10216篇
  1997年   10410篇
  1996年   9936篇
  1995年   9624篇
  1994年   9466篇
  1993年   9349篇
  1992年   23050篇
  1991年   22377篇
  1990年   21982篇
  1989年   21446篇
  1988年   19515篇
  1987年   18742篇
  1986年   17511篇
  1985年   17198篇
  1984年   14519篇
  1983年   12567篇
  1982年   9730篇
  1981年   8790篇
  1980年   8184篇
  1979年   13674篇
  1978年   10844篇
  1977年   9835篇
  1976年   9163篇
  1975年   10173篇
  1974年   11110篇
  1973年   10885篇
  1972年   9784篇
  1971年   9040篇
  1970年   7599篇
  1969年   7452篇
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
1.
Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.  相似文献   
2.
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   
3.

Aims

This study investigated Cu uptake and accumulation as well as physiological and biochemical changes in grapevines grown in soils containing excess Cu.

Methods

The grapevines were collected during two productive cycles from three vineyards with increasing concentrations of Cu in the soil and at various growth stages, before and after the application of Cu-based fungicides. The Cu concentrations in the grapevine organs and the macronutrients and biochemical parameters in the leaf blades were analyzed.

Results

At close to the flowering stage of the grapevines, the concentration and content of Cu in the leaves were increased. However, the Cu concentrations in the roots, stem, shoots and bunches did not correlate with the metal concentrations in the soil. The application of Cu-based fungicides to the leaves increased the Cu concentrations in the shoots, leaves and rachis; however, the effect of the fungicides on the Cu concentration in the berries was not significant. The biochemical analyses of the leaf blades demonstrated symptoms of oxidative stress that correlated with the Cu concentrations in soil.

Conclusions

The increased availability of Cu in soil had a slight effect on the levels and accumulation of Cu in mature grapevines during the productive season and did not alter the nutritional status of the plant. However, increased Cu concentrations were observed in the leaves. The evidence of oxidative stress in the leaves correlated with the increased levels of Cu in soil.  相似文献   
4.
The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.Introducing DNA-modifying enzymes rather than DNA-based expression cassettes is an attractive alternative for genetic engineering and genome-editing applications such as gene targeting or site-specific recombination. It offers a transient presence of the enzymes, and the process can be coordinated with high levels of enzymatic activity at the time and sites of the desired DNA recombination events. Many DNA-metabolizing enzymes (endonucleases, transposases, and topoisomerases), when delivered in an unrestrained manner, show adverse effects on cell viability. Delivery in the form of protein or RNA may help to mitigate these effects (Cui et al., 2011; Sander et al., 2011; Watanabe et al., 2012). In addition, by introducing proteins, one can avoid the need to remove the protein-encoding DNA fragments from the engineered plant genome. This may help shorten the time from laboratory to field for future improved germplasms.Site-specific recombinases such as Cre or FLP have been widely used in genetic engineering applications (Sorrell and Kolb, 2005). The 38-kD Cre enzyme specifically binds to and recombines the 34-bp loxP sequences, allowing the removal, integration, or inversion of the DNA fragment flanked by these sequences (for review, see Wang et al., 2011). There are a number of established methodologies designed to provide the Cre recombinase activity for site-specific recombination in eukaryotic cells that do not involve the delivery of DNA. These methods include lipofection (Baubonis and Sauer, 1993), microinjection of protein or mRNA (de Wit et al., 1998; Luckow et al., 2009), electroporation of protein or mRNA (Kolb and Siddell, 1996; Ponsaerts et al., 2004), or using modified microorganisms for Cre delivery to their host cells (Vergunst et al., 2000; Koshy et al., 2010). Another strategy that has been used is the incubation or injection of tissues/cell cultures with cell-permeant Cre, a modified Cre protein fused to protein transduction domains or cell-penetrating peptides (Jo et al., 2001; Will et al., 2002; Lin et al., 2004; Nolden et al., 2006).For biotechnological applications in plant sciences, protein delivery systems have been developed, including microinjection (Wymer et al., 2001), protein immobilization to gold particles (Wu et al., 2011), and protein transduction through cell-penetrating peptides (for review, see Chugh et al., 2010). The cell-penetrating peptides were shown to enable intracellular delivery of the Cre recombinase protein to rice (Oryza sativa) callus tissues (Cao et al., 2006). Nanobiotechnology is offering an attractive alternative, since nanoparticles can be precisely tailored to deliver a particular biomolecule to the cell, tissue, or organism of interest when needed (for review, see Du et al., 2012). Mesoporous silica nanoparticles (MSNs) are particularly suited for this purpose. These porous nanoparticles are formed by a matrix of well-ordered pores that confers high loading capacity of molecules like proteins (for review, see Popat et al., 2011). Additionally, surfaces of MSNs can be readily modified, permitting the customization of nanoparticles to particular experimental needs (for review, see Trewyn et al., 2007). In our previous studies, it was shown that MSNs can be used for the codelivery of DNA and chemicals (Torney et al., 2007) as well as DNA and proteins (Martin-Ortigosa et al., 2012a) to plant cells via biolistics. To improve MSN performance as a projectile, gold plating of MSN surfaces was performed, increasing nanoparticle density and, subsequently, the ability to pass through the plant cell wall upon bombardment (Martin-Ortigosa et al., 2012b).In this work, the Cre recombinase enzyme was loaded into the pores of gold-plated MSNs and delivered through the biolistic method to maize (Zea mays) cells containing loxP sites integrated into chromosomal DNA (Lox-corn; Fig. 1A). Lox-corn expressed the glyphosate acetyltransferase gene (gat) and the Anemonia majano cyan fluorescent protein gene (AmCyan1) flanked by loxP sites. The MSN-released Cre enzyme recombined the loxP sites, thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of a variant of Discosoma sp. red fluorescent protein gene (DsRed2) and the loss of the selectable marker gene (Fig. 1A). Visual selection was used to recover the recombination events. Subsequently, fertile maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues, leading to successful genome editing.Open in a separate windowFigure 1.A, Schematic representation of the MSN-based bombardment technology. Cre protein is loaded into the pores of gold-plated MSN (Cre-6x-MSN) and subsequently bombarded onto immature embryos of a transgenic maize line carrying a loxP construct (Lox-corn). The parental transgenic Lox-corn tissues are blue fluorescence and herbicide resistant because they harbor a cassette with the glyphosate acetyltransferase (gat) selection gene and the AmCyan1 (cyan) marker gene flanked by the loxP sites. The DsRed2 (dsred) gene for the expression of a red fluorescent protein is placed downstream of the cassette. Once Cre recombinase is released inside the cell, it performs the recombination, excising gat-AmCyan1 genes and leading to the expression of the DsRed2 gene, switching the cell fluorescence pattern from blue to red. P, Promoter; T, terminator. UBINTRF, CYANF, and DSRED2R are primers for DNA analysis. B, Transmission electron microscope image showing the typical hexagonal shape and the well-ordered pore structure of a 6x-MSN. C, Scanning electron microscope image showing gold nanoparticle deposition (white dots) in all surfaces of 6x-MSN. D, Western blot showing Cre protein loading and release dynamics from 6x-MSN. The protein loading is almost immediate, even though some protein can be detected in the buffer even after 1 h of loading. For the release, some Cre protein can be observed after 24 h of incubation. Most of the protein remains in the 6x-MSN pellet. C+, 400 ng of Cre protein; Empty, a lane with no protein loading. The bands observed in the Empty lane were the spillover from the neighboring Pellet lane, which represents Cre-loaded 6x-MSN after the release experiment resuspended in Laemmli loading buffer (see “Materials and Methods”).  相似文献   
5.
6.
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.  相似文献   
7.
8.
The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination.  相似文献   
9.
Toll-like receptors (TLRs) are best characterized for their roles in mediating dorsoventral patterning and the innate immune response. However, recent studies indicate that TLRs are also involved in regulating neuronal growth and development. Here, we demonstrate that the TLR Tollo positively regulates growth of the Drosophila melanogaster larval neuromuscular junction (NMJ). Tollo mutants exhibited NMJ undergrowth, whereas increased expression of Tollo led to NMJ overgrowth. Tollo expression in the motoneuron was both necessary and sufficient for regulating NMJ growth. Dominant genetic interactions together with altered levels of phosphorylated c-Jun N-terminal kinase (JNK) and puc-lacZ expression revealed that Tollo signals through the JNK pathway at the NMJ. Genetic interactions also revealed that the neurotrophin Spätzle3 (Spz3) is a likely Tollo ligand. Spz3 expression in muscle and proteolytic activation via the Easter protease was necessary and sufficient to promote NMJ growth. These results demonstrate the existence of a novel neurotrophin signaling pathway that is required for synaptic development in Drosophila.  相似文献   
10.
Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.  相似文献   
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号