全文获取类型
收费全文 | 5523篇 |
免费 | 508篇 |
国内免费 | 1篇 |
出版年
2023年 | 24篇 |
2022年 | 67篇 |
2021年 | 135篇 |
2020年 | 83篇 |
2019年 | 113篇 |
2018年 | 119篇 |
2017年 | 104篇 |
2016年 | 172篇 |
2015年 | 315篇 |
2014年 | 325篇 |
2013年 | 405篇 |
2012年 | 489篇 |
2011年 | 478篇 |
2010年 | 280篇 |
2009年 | 249篇 |
2008年 | 353篇 |
2007年 | 326篇 |
2006年 | 298篇 |
2005年 | 328篇 |
2004年 | 300篇 |
2003年 | 255篇 |
2002年 | 216篇 |
2001年 | 46篇 |
2000年 | 32篇 |
1999年 | 43篇 |
1998年 | 48篇 |
1997年 | 29篇 |
1996年 | 32篇 |
1995年 | 21篇 |
1994年 | 37篇 |
1993年 | 21篇 |
1992年 | 26篇 |
1991年 | 24篇 |
1990年 | 14篇 |
1989年 | 14篇 |
1988年 | 10篇 |
1987年 | 13篇 |
1986年 | 18篇 |
1985年 | 10篇 |
1984年 | 21篇 |
1983年 | 7篇 |
1982年 | 10篇 |
1980年 | 11篇 |
1979年 | 10篇 |
1978年 | 9篇 |
1977年 | 25篇 |
1976年 | 12篇 |
1974年 | 6篇 |
1972年 | 8篇 |
1971年 | 7篇 |
排序方式: 共有6032条查询结果,搜索用时 15 毫秒
991.
Tip W. Loo M. Claire Bartlett David M. Clarke 《The Journal of biological chemistry》2009,284(36):24074-24087
P-glycoprotein (P-gp, ATP-binding cassette B1) is a drug pump that extracts toxic drug substrates from the plasma membrane and catalyzes their ATP-dependent efflux. To map the residues in the drug translocation pathway, we performed arginine-scanning mutagenesis on all transmembrane (TM) segments (total = 237 residues) of a P-gp processing mutant (G251V) defective in folding (15% maturation efficiency) (glycosylation state used to monitor folding). The rationale was that arginines introduced into the drug-binding sites would mimic drug rescue and enhance maturation of wild-type or processing mutants of P-gp. It was found that 38 of the 89 mutants that matured had enhanced maturation. Enhancer mutations were found in 11 of the 12 TM segments with the largest number found in TMs 6 and 12 (seven in each), TMs that are critical for P-gp-drug substrate interactions. Modeling of the TM segments showed that the enhancer arginines were found on the hydrophilic face, whereas inhibitory arginines were located on a hydrophobic face that may be in contact with the lipid bilayer. It was found that many of the enhancer arginines caused large alterations in P-gp-drug interactions in ATPase assays. For example, mutants A302R (TM5), L339R (TM6), G872R (TM10), F942R (TM11), Q946R (TM11), V982R (TM12), and S993R (TM12) reduced the apparent affinity for verapamil by ∼10-fold, whereas the F336R (TM6) and M986R (TM12) mutations caused at least a 10-fold increase in apparent affinity for rhodamine B. The results suggest that P-gp contains a large aqueous-filled drug translocation pathway with multiple drug-binding sites that can accommodate the bulky arginine side chains to promote folding of the protein.The human multidrug resistance P-glycoprotein (P-gp, ATP-binding cassette B1)2 is an ATP-dependent drug pump that mediates efflux of a broad range of hydrophobic compounds out of the cell (1). It is expressed in the epithelium of liver, kidney, and gastrointestinal tract and at the blood-brain or blood-testes barrier where it functions to protect us from cytotoxic compounds. It is clinically important because it contributes to multidrug resistance in diseases such as cancer and AIDS (1).P-gp is an ATP-binding cassette transporter of 1280 amino acids that consists of two homologous halves (2). Each half begins with a transmembrane domain (TMD) containing six TM segments followed by a nucleotide-binding domain (NBD).A key goal to understanding the mechanism of P-gp drug transport is to identify the amino acids that line the drug translocation pathway. Because P-gp extracts drug substrates from the lipid bilayer, the drug-binding pocket/drug translocation pathway are predicted to reside in the transmembrane (TM) segments. We previously showed that the TMDs alone were sufficient for drug binding (3). Expression of the TMDs as separate polypeptides showed that both TMD1 and TMD2 were required for binding drug substrate (4). The results of studies utilizing cysteine-scanning mutagenesis and labeling with thiol-reactive drug substrates suggested that all of the TM segments contribute to the drug-binding pocket/drug translocation pathway (reviewed in Ref. 5). The next step is to identify the specific amino acids that line the drug translocation pathway. It is important to identify amino acids that line the drug translocation pathway and to compare whether the biochemical evidence supports a model of P-gp structure in the closed conformation (6) (NBDs close together that was based on the bacterial Sav1866 crystal structure (7)) or the recent crystal structure of mouse P-gp in the open conformation (NBDs far apart) (8). There have been concerns that the mouse P-gp structure may be a non-native structure or in a conformation that exists very transiently (9).Our approach to map the drug translocation pathway has been to use arginine-scanning mutagenesis of the TM segments of a P-gp processing mutant (G251V) that shows partial maturation (∼15% maturation efficiency) (10). Maturation efficiency can be used to detect folding of P-gp in whole cells by monitoring the conversion of P-gp from a core-glycosylated (150 kDa) protein to a mature protein (170 kDa) that contains complex carbohydrate. Because mutant G251V shows partial maturation, we can detect whether an introduced arginine promotes, inhibits, or has a neutral effect on folding. The rationale for using arginine-scanning mutagenesis was that arginine has a large free energy barrier (17 kcal/mol) for insertion into the lipid bilayer because it is highly charged (11). Therefore, introduction of an arginine into a lipid face of the G251V mutant would likely inhibit maturation, whereas an arginine introduced into the aqueous face of the drug translocation pathway would not inhibit maturation of the mutant P-gp.In an initial study on TM1, we demonstrated the feasibility of the approach (10). All arginines introduced into the predicted lipid-facing positions inhibited maturation, whereas those introduced into positions predicted to face the drug translocation pathway did not. A particularly intriguing observation was that some arginines promoted maturation. The residues at these positions were coincidentally at positions identical to those that reacted with thiol-reactive drug substrates in cysteine-scanning mutagenesis studies and were found to be within the drug-binding pocket (10, 12). This suggested that arginine-scanning mutagenesis could be a useful approach for identifying residues in the drug translocation pathway and for determining the orientation of the TM segments in the membrane.Arginines that promote maturation appear to identify positions that are important for P-gp-drug interactions because they appear to mimic drug rescue of P-gp. It was also found that the ability of arginines (such as I306R in TM5) to promote maturation involved global enhancement of P-gp folding rather than simply compensating for a localized mutation (such as G251V) because other processing mutants could also be rescued (12). Because these arginine mutations enhance folding of P-gp in general, they will be described as enhancer rather than suppressor arginines. In this study we performed arginine-scanning mutagenesis on TMs 2–12 of P-gp processing mutant G251V to determine their orientations in the membrane and to identify residues that line the drug translocation pathway. 相似文献
992.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
993.
Cherdsak Liewlaksaneeyanawin Jun Zhuang Michelle Tang Nima Farzaneh Gillian Lueng Claire Cullis Susan Findlay Carol E. Ritland Jörg Bohlmann Kermit Ritland 《Tree Genetics & Genomes》2009,5(1):247-255
Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express
sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified
a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers
and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product
in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species.
Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed
multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide
similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism
in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce
and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified
product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably
lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps
are offset by their lower polymorphism.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
994.
Background
The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells. 相似文献995.
Micha? Komorowski B?rbel Finkenst?dt Claire V Harper David A Rand 《BMC bioinformatics》2009,10(1):343
Background
Fluorescent and luminescent gene reporters allow us to dynamically quantify changes in molecular species concentration over time on the single cell level. The mathematical modeling of their interaction through multivariate dynamical models requires the deveopment of effective statistical methods to calibrate such models against available data. Given the prevalence of stochasticity and noise in biochemical systems inference for stochastic models is of special interest. In this paper we present a simple and computationally efficient algorithm for the estimation of biochemical kinetic parameters from gene reporter data. 相似文献996.
Pautus S Aboraia AS Bassett CE Brancale A Coogan MP Simons C 《Journal of enzyme inhibition and medicinal chemistry》2009,24(2):487-498
The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC(50) 8 and 12 microM) and comparable with liarozole (IC(50) 7 microM). 相似文献
997.
Crepidula convexa, a calyptreid gastropod with direct embryonic development, changes sex from male to female in the course of its lifetime (protandry). Under sex-allocation theory, male reproductive success should be independent from age and size (a proxy used for age). However, this may be counterbalanced by female cryptic choice or gregarious behaviour. Eleven polymorphic microsatellite loci were thus developed to examine paternity of embryos and larvae. This set of loci appears suitable to carry out paternity analyses due to the high exclusion probability of unrelated males given the maternal genotype. 相似文献
998.
Amynah A. A. Pradhan Jér?me A. J. Becker Grégory Scherrer Petra Tryoen-Toth Dominique Filliol Audrey Matifas Dominique Massotte Claire Gavériaux-Ruff Brigitte L. Kieffer 《PloS one》2009,4(5)
Background
GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.Methods and Findings
In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.Conclusions
Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs. 相似文献999.
1000.
Romain Marlin Marie-Thérèse Nugeyre Claire de Truchis Nadia Berkane Amélie Gervaise Fran?oise Barré-Sinoussi Elisabeth Menu 《PloS one》2009,4(6)