全文获取类型
收费全文 | 5184篇 |
免费 | 476篇 |
出版年
2023年 | 6篇 |
2022年 | 55篇 |
2021年 | 77篇 |
2020年 | 42篇 |
2019年 | 74篇 |
2018年 | 77篇 |
2017年 | 76篇 |
2016年 | 140篇 |
2015年 | 236篇 |
2014年 | 285篇 |
2013年 | 339篇 |
2012年 | 400篇 |
2011年 | 357篇 |
2010年 | 250篇 |
2009年 | 249篇 |
2008年 | 326篇 |
2007年 | 314篇 |
2006年 | 332篇 |
2005年 | 331篇 |
2004年 | 308篇 |
2003年 | 241篇 |
2002年 | 248篇 |
2001年 | 68篇 |
2000年 | 47篇 |
1999年 | 57篇 |
1998年 | 83篇 |
1997年 | 44篇 |
1996年 | 40篇 |
1995年 | 37篇 |
1994年 | 46篇 |
1993年 | 44篇 |
1992年 | 36篇 |
1991年 | 31篇 |
1990年 | 51篇 |
1989年 | 30篇 |
1988年 | 28篇 |
1987年 | 19篇 |
1986年 | 20篇 |
1985年 | 27篇 |
1984年 | 24篇 |
1983年 | 15篇 |
1982年 | 15篇 |
1981年 | 16篇 |
1980年 | 20篇 |
1979年 | 10篇 |
1978年 | 18篇 |
1976年 | 14篇 |
1974年 | 6篇 |
1973年 | 7篇 |
1971年 | 7篇 |
排序方式: 共有5660条查询结果,搜索用时 31 毫秒
921.
Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating 总被引:1,自引:0,他引:1
Maria Nyblom Yi Wang Karin Hallgren Richard Neutze Susanna Törnroth-Horsefield 《Journal of molecular biology》2009,387(3):653-1335
Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca2+ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the Cα atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating. 相似文献
922.
TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5′-(β,γ-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (∼ 10− 4 s− 1) and accelerated > 1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg2+-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by < 80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic. 相似文献
923.
924.
Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.Flaviviruses form a genus in the family Flaviviridae (52) and comprise a number of important human pathogens such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses (30). They are small, enveloped viruses with only three structural proteins, designated C (capsid), M (membrane), and E (envelope). The E protein is oriented parallel to the viral membrane and forms a head-to-tail homodimeric complex (Fig. 1A and B). The structure of the E ectodomain (soluble E [sE])—consisting of about 400 amino acids and lacking the 100 C-terminal amino acids (including the so-called stem and two transmembrane helices)—has been determined by X-ray crystallography for several flaviviruses (Fig. (Fig.1A)1A) (25, 34, 36, 38, 44, 55). Both of the essential entry functions—receptor-binding and membrane fusion after uptake by receptor-mediated endocytosis—are mediated by E, which is therefore the primary target for virus-neutralizing antibodies (11, 42, 43, 45).Open in a separate windowFIG. 1.Structures and schematic representations of the TBE virus E protein, virions, and RSPs. In all panels, DI, DII, and DIII of the E protein are shown in red, yellow, and blue, respectively, and the fusion peptide (FP) is in orange. (A) Ribbon diagram of the sE dimer (top view). (B) Schematic of the full-length E dimer in a top view (upper panel) and side view (lower panel). The position of the two transmembrane helices of the membrane anchor and the two helices of the stem are based on Zhang et al. (54) and are shown in green and purple, respectively. (C) Pseudo-atomic structure of the virion based on cryo-EM reconstructions of dengue and West Nile viruses (27, 37, 54). One of the 30 rafts, each consisting of three parallel dimers, is highlighted. DIIIs of three monomers belonging to one icosahedral asymmetric unit are labeled by white stars. (D) Pseudo-atomic structure of RSP based on cryo-EM reconstructions (12).As revealed by cryo-electron microscopy (cryo-EM), mature infectious virions have smooth surfaces, comparable to a golf ball (27, 37). Their envelopes are icosahedrally symmetric and consist of a closed shell of 180 E monomers that are arranged in a herringbone-like pattern of 30 rafts of three dimers each (Fig. (Fig.1C)1C) (27). On the other hand, capsid-lacking subviral particles, which can be produced in recombinant form by the coexpression of prM and E, have a different symmetry, with 30 E dimers in a T=1 icosahedral structure (Fig. (Fig.1D)1D) (12, 49).The peculiar organization of E in virions is reminiscent of the tight packing of capsid proteins in nonenveloped viruses, for which it was shown that the native antigenic structure is strongly dependent on the intact capsid structure and not completely represented by isolated forms of capsid proteins (1, 41, 53). Such modulations of antigenic structure may be due to conformational changes in the course of packaging the capsid proteins into virions and/or to the fact that antibody binding sites at the virion surface are composed of residues that come together only through the juxtaposition of capsid proteins or neighboring protein subunits. Even in the case of spiky viral envelope proteins, the dependence of certain epitopes on the quaternary organization of the envelope glycoproteins has been described (8, 47).For flaviviruses, structural studies provide evidence for the considerable flexibility of E, especially at the junctions between the individual domains I, II, and III (DI, DII, and DIII) (7, 35, 55), suggesting that soluble forms may display differences in antigenic structure compared to those fixed in the closed envelope shell of whole virions. Furthermore, because of the tight packing of E at the virion surface, certain epitopes may be cryptic in the context of whole virus particles but accessible in soluble forms of E (40, 51).Studies on the antigenic structure of flaviviruses have used different antigen preparations including virions, recombinant subviral particles (RSPs), and soluble forms and subunits of E (10, 15-17, 32, 39, 40, 46, 49, 51), but so far no systematic comparative analysis of E in different physical forms and quaternary arrangements has been conducted. It was therefore the objective of our study, using TBE virus as a model, to investigate possible structural and/or antigenic differences between (i) soluble dimeric forms of E, including C-terminally truncated sE and detergent-solubilized full-length E (Fig. 1A and B); (ii) E in the context of whole virions (Fig. (Fig.1C);1C); and (iii) E in the context of RSPs (Fig. (Fig.1D).1D). For this purpose we used, and further characterized, a set of monoclonal antibodies (MAbs) directed to each of the three domains of E. All of these MAbs have neutralizing activity (17, 24) and therefore, by definition, react with infectious virions.Through these analyses, we demonstrate that the reactivity of several MAbs is significantly dependent on the quaternary arrangement of E and differs between virions, RSPs, and/or sE dimers. We thus provide evidence for previously unrecognized structural factors that have an impact on the antigenicity of the flavivirus E protein. 相似文献
925.
Ryan P. Topping John C. Wilkinson Karin Drotschmann Scarpinato 《The Journal of biological chemistry》2009,284(21):14029-14039
Mismatch repair (MMR) proteins participate in cytotoxicity induced by
certain DNA damage-inducing agents, including cisplatin
(cis-diamminedichloroplatinum(II), CDDP), a cancer chemotherapeutic
drug utilized clinically to treat a variety of malignancies. MMR proteins have
been demonstrated to bind to CDDP-DNA adducts and initiate MMR
protein-dependent cell death in cells treated with CDDP; however, the
molecular events underlying this death remain unclear. As MMR proteins have
been suggested to be important in clinical responses to CDDP, a clear
understanding of MMR protein-dependent, CDDP-induced cell death is critical.
In this report, we demonstrate MMR protein-dependent relocalization of
cytochrome c to the cytoplasm and cleavage of caspase-9, caspase-3,
and poly(ADP-ribose) polymerase upon treatment of cells with CDDP. Chemical
inhibition of caspases specifically attenuates CDDP/MMR protein-dependent
cytotoxicity, suggesting that a caspase-dependent signaling mechanism is
required for the execution of this cell death. p53 protein levels were
up-regulated independently of MMR protein status, suggesting that p53 is not a
mediator of MMR-dependent, CDDP-induced death. This work is the first
indication of a required signaling mechanism in CDDP-induced, MMR
protein-dependent cytotoxicity, which can be uncoupled from other CDDP
response pathways, and defines a critical contribution of MMR proteins to the
control of cell death.The MMR2 system of
proteins plays roles in diverse cellular processes, perhaps most notably in
preserving genomic integrity by recognizing and facilitating the repair of
post-DNA replication base pairing errors. Recognition of these errors and
recruitment of repair machinery is performed by the MutSα complex
(consisting of the MMR proteins MSH2 and MSH6) or MutSβ complex
(consisting of MSH2 and MSH3). Defects in MMR proteins render cells
hypermutable and promote microsatellite instability, a hallmark of MMR
defects. MMR protein defects are found in a wide variety of sporadic cancers,
as well as in hereditary non-polyposis colorectal cancer
(1).In addition to their role in DNA repair, MMR proteins also play a role in
cytotoxicity induced by specific types of DNA-damaging chemotherapeutic drugs,
such as CDDP, which is utilized clinically to treat a number of different
cancer types. MutSα recognizes multiple types of DNA damage, including
1,2-intrastrand CDDP adducts and O6-methylguanine lesions
(2). Treatment of cells with
compounds that induce these types of lesions, including CDDP and methylating
agents such as
N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),
results in MMR protein-dependent cell cycle arrest and cell death
(3–7).
This suggests that MMR proteins, in addition to their role in DNA repair, are
also capable of initiating cell death in response to certain types of DNA
damage.Cells treated with DNA-damaging agents frequently activate an apoptotic
cell death pathway mediated by the mitochondria. This intrinsic death
signaling pathway predominantly involves the coordinated activity of two
groups of proteins: pro-death members of the Bcl-2 family that control the
integrity of mitochondrial membranes, and members of the caspase family of
cysteinyl proteases that proteolytically cleave intracellular substrates,
giving rise to apoptotic morphology and destruction of the cell
(8,
9). Pro-death Bcl-2 family
members, such as Bax and Bak, target the outer mitochondrial membrane and
cause the cytosolic release of pro-death factors residing within the
mitochondria of unstressed cells
(8). Predominant among these
factors is cytochrome c, whose cytoplasmic localization results in
the formation of a caspase-activating platform known as the apoptosome
(10). This complex includes
the adaptor protein Apaf-1, and when formed the apoptosome promotes the
cleavage and activation of caspase-9
(11,
12). Once activated, this
apical caspase proceeds to cleave and activate caspase-3, the predominant
effector protease of apoptosis.A significant amount of evidence has been gathered illustrating MMR
protein-dependent pro-death signaling in response to methylating agents
(13–16,
3). In contrast, the MMR
protein-dependent cytotoxic response to CDDP is largely unknown, with only the
p53-related transactivator protein p73 and the c-Abl kinase clearly implicated
as potential mediators of CDDP/MMR protein-dependent cell death in human cells
(17,
18). Interestingly, ATM, Chk1,
Chk2, and p53, which are activated in an MMR protein-dependent manner after
treatment of cells with MNNG
(3,
13), are not involved in the
MMR-dependent response to CDDP
(7,
17). In addition, the
magnitude of MMR protein-dependent cell death induced by methylating agents
and CDDP differs (4). These
findings suggest that unique signaling pathways may be engaged by MMR proteins
depending upon the type of recognized lesion. As such, there is a requirement
for further study of the molecular events underlying MMR protein-dependent
cell death and cell cycle arrest for each type of recognized DNA lesion. This
is particularly relevant in the case of CDDP, as evidence from a limited
number of retrospective clinical studies suggests that MMR proteins play an
important role in patient response to CDDP. Several studies examining
immunohistochemical staining against MSH2 or MLH1 have demonstrated that
levels of these proteins are reduced in ovarian and esophageal tumor samples
following CDDP-based chemotherapy
(19,
20). Low levels of MMR protein
post-chemotherapy seem to be predictive of lower overall survival in a certain
subset of tumors (esophageal cancer), but not others (ovarian and non-small
cell lung cancer)
(19–21).
Two recent studies examining MMR protein levels and microsatellite instability
in germ cell tumors from patients receiving platinum-based chemotherapy have
suggested a prognostic value for pre-chemotherapy MMR protein status in these
tumors (22,
23). This potential clinical
relevance underscores the need for a greater understanding of MMR
protein-dependent mechanisms of CDDP-induced cell death.In this study, we report that CDDP induces an MMR protein-dependent
decrease in cell viability and MMR protein-dependent signaling in the form of
cytochrome c release to the cytoplasm and cleavage of caspase-9,
caspase-3, and PARP. Chemical inhibition of caspases specifically attenuates
CDDP/MMR protein-dependent loss of cell viability, indicating a requirement
for caspase activation in this process and uncoupling MMR protein-dependent
cytotoxic signaling from other CDDP response pathways. Additionally, the
CDDP-induced, MMR protein-dependent cytotoxic response is independent of p53
signaling. Our results demonstrate for the first time an MMR protein-dependent
pro-death signaling pathway in cells treated with CDDP. 相似文献
926.
927.
Diversity and abundance of freshwater Actinobacteria along environmental gradients in the brackish northern Baltic Sea 总被引:1,自引:0,他引:1
Karin Holmfeldt Claudia Dziallas Josefin Titelman Kirsten Pohlmann Hans-Peter Grossart Lasse Riemann 《Environmental microbiology》2009,11(8):2042-2054
Actinobacteria are highly abundant in pelagic freshwater habitats and also occur in estuarine environments such as the Baltic Sea. Because of gradients in salinity and other environmental variables estuaries offer natural systems for examining factors that determine Actinobacteria distribution. We studied abundance and community structure of Bacteria and Actinobacteria along two transects in the northern Baltic Sea. Quantitative (CARD-FISH) and qualitative (DGGE and clone libraries) analyses of community composition were compared with environmental parameters. Actinobacteria accounted for 22–27% of all bacteria and the abundance changed with temperature. Analysis of 549 actinobacterial 16S rRNA sequences from four clone libraries revealed a dominance of the freshwater clusters ac I and ac IV, and two new subclusters ( ac I-B scB-5 and ac IV-E) were assigned. Whereas ac I was present at all stations, occurrence of ac II and ac IV differed between stations and was related to dissolved organic carbon (DOC) and chlorophyll a (Chl a ) respectively. The prevalence of the ac I-A and ac I-B subclusters changed in relation to total phosphorus (Tot-P) and Chl a respectively. Community structure of Bacteria and Actinobacteria differed between the river station and all other stations, responding to differences in DOC, Chl a and bacterial production. In contrast, the composition of active Actinobacteria (analysis based on reversely transcribed RNA) changed in relation to salinity and Tot-P. Our study suggests an important ecological role of Actinobacteria in the brackish northern Baltic Sea. It highlights the need to address dynamics at the cluster or subcluster phylogenetic levels to gain insights into the factors regulating distribution and composition of Actinobacteria in aquatic environments. 相似文献
928.
Carsten Schleh Christian Mühlfeld Karin Pulskamp Andreas Schmiedl Matthias Nassimi Hans D Lauenstein Armin Braun Norbert Krug Veit J Erpenbeck Jens M Hohlfeld 《Respiratory research》2009,10(1):90
Background
Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO2) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling in vitro. In addition, TiO2 effects on surfactant ultrastructure were visualized.Methods
A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 μg/ml) of TiO2 NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope.Results
TiO2 NSP, but not MSP, induced a surfactant dysfunction. For TiO2 NSP, adsorption surface tension (γads) increased in a dose-dependent manner from 28.2 ± 2.3 mN/m to 33.2 ± 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (γmin) slightly increased from 4.8 ± 0.5 mN/m up to 8.4 ± 1.3 mN/m (p < 0.01) at high TiO2 NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both γads (63.6 ± 0.4 mN/m) and γmin (21.1 ± 0.4 mN/m). Interestingly, TiO2 NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae.Conclusion
TiO2 nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung. 相似文献929.
David J Lee Lewis EH Bingle Karin Heurlier Mark J Pallen Charles W Penn Stephen JW Busby Jon L Hobman 《BMC microbiology》2009,9(1):252
Background
Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. 相似文献930.