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81.
Dirk Steinritz Jana Weber Frank Balszuweit Horst Thiermann Annette Schmidt 《Chemico-biological interactions》2013
Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300 μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300 μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard. 相似文献
82.
Veronika Slancarova Jana Zdanska Bohuslav Janousek Martina Talianova Christian Zschach Jitka Zluvova Jiri Siroky Viera Kovacova Hana Blavet Jiri Danihelka Bengt Oxelman Alex Widmer Boris Vyskot 《Evolution; international journal of organic evolution》2013,67(12):3669-3677
The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex‐determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex‐determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family. 相似文献
83.
Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pKa values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λi} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pKa values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:
Open in a separate windowaTaken from Wallace and Shen (12). The pKa''s of BBL were recalculated.bSampling time per pH replica.Breaking the simulations in two halves, we noticed that the second 5-ns sampling gave better agreement with experiment. The RMS deviation is reduced from 1.2 to 0.9 pH units, while the average absolute deviation is reduced from 1.0 to 0.6 pH units. The linear regression against experimental data is also improved, with the slope decreasing from 1.4 to 1.1 although R2 remains the same. Comparing these second-half results with the GB-based simulations, we find that the RMS and average absolute deviations are about the same as the GB-CpHMD results; however, the all-atom simulations show a small systematic overestimation (regression slope >1), whereas GB simulations show a systematic underestimation (regression slope <1).The improvement in the second halves of the simulations are seen mainly for residues involved in attractive electrostatic interactions, including Asp44 and Asp46 of HP36, Asp129 of BBL, and Asp48, Asp66, and Asp87 of HEWL. These residues are initially locked in salt-bridges or hydrogen bonds. However, in the second 5 ns, the attractive interactions weakened, leading to a decrease in the calculated pKa shifts relative to the model values and better agreement with experiment. For instance, Asp44 was initially in a salt-bridge distance from Arg55. However, the salt-bridge positions were sampled less often in the second 5 ns (see Fig. S5), which explains the 1-unit reduction in the calculated pKa shift. Significant fluctuation in ion-pair interactions was also observed in the work by Alexov (11). The carboxyl oxygen of Asp46 was a hydrogen-bond acceptor with both the backbone amide and hydroxyl of Ser43. These hydrogen bonds were less frequently sampled in the second 5 ns (see Fig. S6), leading to a decrease of the pKa shift for Asp46 by 1.3 units. These results indicate that extensive conformational sampling is necessary to give an accurate estimate of the ratio between the charged and neutral populations.Limited conformational sampling is also a contributing factor to the overestimation of the pKa shifts for buried residues (Fig. S7 and Fig. S8). The increase in SASA is correlated with the more frequent sampling of the states with λ close to 1, i.e., the deprotonated form (see Fig. S9). However, because Glu35 was buried in the starting conformation and the transition between buried and exposed states is slow compared to the simulation length, the exposed state may not be sufficiently sampled, leading to overestimation of the pKa shift.In contrast to Glu35, the SASA of Asp52 in HEWL is almost identical for both protonation states. The lack of conformational fluctuation is due to the strong hydrogen bonding with the side-chain amino group of Asn46 and Asn59 (data not shown). Overestimation of the pKa shifts for buried residues can also be attributed to the limitation of the additive force field which underestimates dielectric response in protein environment (more discussion see Supporting Material) of the pKa shifts for buried residues.Finally, to ascertain if the presence of hydroxide/hydronium introduces artifacts, we studied the interaction between hydroxide/hydronium and the titratable sites/ions. Comparing the hydroxide/hydronium with respective chloride/sodium ions, we find that the spatial distributions are nearly identical (see plots of distance distributions and radial distribution functions in Figs. S10–S13). However, the relative occupancy of the hydroxide around the neutral Asp/Glu, positive histidine, or sodium ion is 2–3 times as that of a chloride. The water-bridged interaction between sodium and chloride ions becomes much weaker when chloride is replaced by hydroxide or sodium is replaced by hydronium. By contrast, the occupancy of the hydronium around the solute is similar to that of the sodium. Furthermore, similar pKa results for these proteins were obtained when coions were used instead of titratable waters (data not shown). Thus, we believe that potential artifacts related to the ionized forms of water are negligible. Work is underway to further understand the limitations of the methodology and to explore applications to protein dynamics coupled to proton transfer.In summary, we have developed and tested titratable water models for use in all-atom CpHMD simulations. Although the benchmark pKa calculations indicate a comparable accuracy as the GB-CpHMD method, the all-atom method offers physical rigor and most importantly, it is applicable to systems that cannot be studied with GB-based simulations such as lipids and nucleic acids. We anticipate that the accuracy of this methodology can be further improved by incorporating the new-generation force fields that account for polarization. The coupling between proton titration of water and solute offers a computationally efficient way to model proton transfer in molecular mechanics simulations. 相似文献
- 1.Loss of a proton to give a negatively charged hydroxide,
- 2.Gain of a proton to give a positively charged hydronium,
Table 1
Calculated and experimental pKa values of three proteinsResidue | Experimenta | GBa | All-atom CpHMD | ||
---|---|---|---|---|---|
Time (ns)b | 0–1 | 0–5 | 5–10 | 0–10 | |
HP36 | |||||
Asp44 | 3.10 (0.01) | 3.2 (0.1) | 2.0 | 3.0 | 2.6 (0.5) |
Glu45 | 3.95 (0.01) | 3.5 (0.1) | 4.3 | 4.5 | 4.4 (0.1) |
Asp46 | 3.45 (0.12) | 3.5 (0.1) | 2.4 | 3.7 | 3.1 (0.6) |
Glu72 | 4.37 (0.03) | 3.5 (0.1) | 4.4 | 4.4 | 4.4 (0.0) |
BBL | |||||
Asp129 | 3.88 (0.02) | 3.2 (0.0) | 2.2 | 3.2 | 2.7 (0.5) |
Glu141 | 4.46 (0.04) | 4.3 (0.0) | 4.0 | 4.4 | 4.2 (0.2) |
His142 | 6.47 (0.04) | 7.1 (0.0) | 5.9 | 5.8 | 5.8 (0.0) |
Asp145 | 3.65 (0.04) | 2.8 (0.2) | 3.0 | 3.1 | 3.1 (0.0) |
Glu161 | 3.72 (0.05) | 3.6 (0.3) | 4.2 | 3.9 | 4.0 (0.2) |
Asp162 | 3.18 (0.04) | 3.4 (0.3) | 2.9 | 3.5 | 3.2 (0.3) |
Glu164 | 4.50 (0.03) | 4.5 (0.1) | 5.7 | 4.6 | 5.2 (0.6) |
His166 | 5.39 (0.02) | 5.4 (0.1) | 4.4 | 4.4 | 4.4 (0.0) |
HEWL | |||||
Glu7 | 2.6 (0.2) | 2.6 (0.1) | 3.6 | 3.4 | 3.5 (0.1) |
His15 | 5.5 (0.2) | 5.3 (0.5) | 5.1 | 5.1 | 5.1 (0.0) |
Asp18 | 2.8 (0.3) | 2.9 (0.0) | 2.5 | 3.3 | 2.9 (0.4) |
Glu35 | 6.1 (0.4) | 4.4 (0.2) | 8.5 | 8.7 | 8.6 (0.1) |
Asp48 | 1.4 (0.2) | 2.8 (0.2) | −0.1 | 1.1 | 0.6 (0.6) |
Asp52 | 3.6 (0.3) | 4.6 (0.0) | 5.4 | 5.6 | 5.5 (0.1) |
Asp66 | 1.2 (0.2) | 1.2 (0.4) | −0.6 | 0.8 | 0.3 (0.7) |
Asp87 | 2.2 (0.1) | 2.0 (0.1) | 0.8 | 2.1 | 1.5 (0.7) |
Asp101 | 4.5 (0.1) | 3.3 (0.3) | 6.1 | 5.7 | 5.9 (0.2) |
Asp119 | 3.5 (0.3) | 2.5 (0.1) | 3.0 | 3.3 | 3.2 (0.1) |
Maximum absolute deviation | 1.8 | 2.4 | 2.6 | 2.5 | |
Average absolute deviation (RMS deviation) | 0.5 (0.7) | 1.0 (1.2) | 0.6 (0.9) | 0.7 (0.9) | |
Linear fit R2 (slope) | 0.7 (0.8) | 0.8 (1.4) | 0.7 (1.1) | 0.8 (1.2) |
84.
In November 2004 a catastrophic windstorm destroyed a large part of the spruce forest in the Tatra National Park (Slovakia). The majority of the windthrown area was cleared; only a small part was left uncleared, thereby allowing regeneration by natural succession. The aim of the present study was to assess the impact of the different forestry practices on soil Oribatida. Three different stands were selected for the study, where sampling took place in June and October 2006: control forest stands (REF), windthrown stands left for natural development (NEX) and clear-cut windthrown stands (EXT). The mean abundance of Oribatida recorded in REF and NEX stands was significantly higher than in EXT stands. Kruskal-Wallis test of mean abundance of adults as well as juveniles confirmed significant influence of treatment and date. The highest abundance of adults was found in control forest stands (REF). Post hoc multiple comparison proved significantly lower abundance of adults in clear-cut stands (EXT) compared with REF. The mean abundance of adults and juveniles was several times higher in stands left for natural development (NEX) than in EXT stands. The highest species richness was observed in REF, followed by NEX and EXT stands. Ordination method showed differences in species composition between studied treatments. Furthermore, a much lower abundance of Hermannia gibba, a dweller of leaf litter and upper soil layers, was recorded in cleared stands compared to the other stands. Indeed, windthrown stands had an obvious lower species richness than control stands. The ordination method used demonstrated a significant influence of both treatment and sampling date on the abundance and species richness of Oribatida. The present study showed that clear-cutting of wind-damaged spruce forest markedly decreases the abundance of soil Oribatida compared with windthrown forest stands left to natural succession. 相似文献
85.
Shailendra Kumar Dhar Dwivedi Krishnananda Samanta Manisha Yadav Amit Kumar Jana Abhishek Kumar Singh Bandana Chakravarti Sankalan Mondal Rituraj Konwar Arun Kumar Trivedi Naibedya Chattopadhyay Sabyasachi Sanyal Gautam Panda 《Bioorganic & medicinal chemistry letters》2013,23(24):6816-6821
Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride (LAH) reduction, debenzylation, bimolecular nucleophilic substitution (SN2) reaction. The present study investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF-7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hallmarks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depolarization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could serve as a base for the development of a new group of effective breast cancer therapeutics. 相似文献
86.
Vanessa S. Oliveira Cecília Pimenteira Diana C.B. da Silva-Alves Laylla L.L. Leal Ricardo A.W. Neves-Filho Daniela M.A.F. Navarro Geanne K.N. Santos Kamilla A. Dutra Janaína V. dos Anjos Thereza A. Soares 《Bioorganic & medicinal chemistry》2013,21(22):6996-7003
The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC50 measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid. 相似文献
87.
Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli. 相似文献
88.
Martin von Bergen Nico Jehmlich Martin Taubert Carsten Vogt Felipe Bastida Florian-Alexander Herbst Frank Schmidt Hans-Hermann Richnow Jana Seifert 《The ISME journal》2013,7(10):1877-1885
The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes (13C, 15N, 36S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed. 相似文献
89.
María-Eugenia Guazzaroni Florian-Alexander Herbst Iván Lores Javier Tamames Ana Isabel Peláez Nieves López-Cortés María Alcaide Mercedes V Del Pozo José María Vieites Martin von Bergen José Luis R Gallego Rafael Bargiela Arantxa López-López Dietmar H Pieper Ramón Rosselló-Móra Jesús Sánchez Jana Seifert Manuel Ferrer 《The ISME journal》2013,7(1):122-136
Microbial metabolism in aromatic-contaminated environments has important ecological implications, and obtaining a complete understanding of this process remains a relevant goal. To understand the roles of biodiversity and aromatic-mediated genetic and metabolic rearrangements, we conducted ‘OMIC'' investigations in an anthropogenically influenced and polyaromatic hydrocarbon (PAH)-contaminated soil with (Nbs) or without (N) bio-stimulation with calcium ammonia nitrate, NH4NO3 and KH2PO4 and the commercial surfactant Iveysol, plus two naphthalene-enriched communities derived from both soils (CN2 and CN1, respectively). Using a metagenomic approach, a total of 52, 53, 14 and 12 distinct species (according to operational phylogenetic units (OPU) in our work equivalent to taxonomic species) were identified in the N, Nbs, CN1 and CN2 communities, respectively. Approximately 10 out of 95 distinct species and 238 out of 3293 clusters of orthologous groups (COGs) protein families identified were clearly stimulated under the assayed conditions, whereas only two species and 1465 COGs conformed to the common set in all of the mesocosms. Results indicated distinct biodegradation capabilities for the utilisation of potential growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilisation and non-stimulated communities exhibiting a greater metabolic window than previously predicted. On the basis of comparing protein expression profiles and metagenome data sets, inter-alia interactions among members were hypothesised. The utilisation of curated databases is discussed and used for first time to reconstruct ‘presumptive'' degradation networks for complex microbial communities. 相似文献
90.
Tomá? Venit Rastislav Dzijak Al?běta Kalendová Michal Kahle Jana Roho?ková Volker Schmidt Thomas Rülicke Birgit Rathkolb Wolfgang Hans Alexander Bohla Oliver Eickelberg Tobias Stoeger Eckhard Wolf Ali ?nder Yildirim Valérie Gailus-Durner Helmut Fuchs Martin Hrabě de Angelis Pavel Hozák 《PloS one》2013,8(4)