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121.
Andreas Hoffmann Christiane Haas Stefan Hennig Kai Ostermann Thomas Bley Christian Lser Thomas Walther 《Engineering in Life Science》2019,19(6):400-411
Microbial consortia can be used to catalyze complex biotransformations. Tools to control the behavior of these consortia in a technical environment are currently lacking. In the present study, a synthetic biology approach was used to build a model consortium of two Saccharomyces cerevisiae strains where growth and expression of the fluorescent marker protein EGFP by the receiver strain is controlled by the concentration of α‐factor pheromone, which is produced by the emitter strain. We have developed a quantitative experimental and theoretical framework to describe population dynamics in the model consortium. We measured biomass growth and metabolite production in controlled bioreactor experiments, and used flow cytometry to monitor changes of the subpopulations and protein expression under different cultivation conditions. This dataset was used to parameterize a segregated mathematical model, which took into account fundamental growth processes, pheromone‐induced growth arrest and EGFP production, as well as pheromone desensitization after extended exposure. The model was able to predict the growth dynamics of single‐strain cultures and the consortium quantitatively and provides a basis for using this approach in actual biotransformations. 相似文献
122.
Kristine von Bargen Mirella Scraba Ina Krmer Maren Ketterer Christian Nehls Sina Krokowski Urska Repnik Michaela Wittlich Anna Maaser Pia Zapka Madeleine Bunge Martin Schlesinger Gitta Huth Annette Klees Philipp Hansen Andreas Jeschke Gerd Bendas Olaf Utermhlen Gareth Griffiths Thomas Gutsmann Jens Wohlmann Albert Haas 《Cellular microbiology》2019,21(1)
Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences. 相似文献
123.
124.
Mimuro H Suzuki T Nagai S Rieder G Suzuki M Nagai T Fujita Y Nagamatsu K Ishijima N Koyasu S Haas R Sasakawa C 《Cell host & microbe》2007,2(4):250-263
Colonization of the gastric pits in the stomach by Helicobacter pylori (Hp) is a major risk factor for gastritis, gastric ulcers, and cancer. Normally, rapid self-renewal of gut epithelia, which occurs by a balance of progenitor proliferation and pit cell apoptosis, serves as a host defense mechanism to limit bacterial colonization. To investigate how Hp overcomes this host defense, we use the Mongolian gerbil model of Hp infection. Apoptotic loss of pit cells induced by a proapoptotic agent is suppressed by Hp. The ability of Hp to suppress apoptosis contributed to pit hyperplasia and persistent bacterial colonization of the stomach. Infection with WT Hp but not with a mutant in the virulence effector cagA increased levels of the prosurvival factor phospho-ERK and antiapoptotic protein MCL1 in the gastric pits. Thus, CagA activates host cell survival and antiapoptotic pathways to overcome self-renewal of the gastric epithelium and help sustain Hp infection. 相似文献
125.
von Brunn A Teepe C Simpson JC Pepperkok R Friedel CC Zimmer R Roberts R Baric R Haas J 《PloS one》2007,2(5):e459
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies. 相似文献
126.
Genome sequence of Babesia bovis and comparative analysis of apicomplexan hemoprotozoa 总被引:1,自引:0,他引:1
Brayton KA Lau AO Herndon DR Hannick L Kappmeyer LS Berens SJ Bidwell SL Brown WC Crabtree J Fadrosh D Feldblum T Forberger HA Haas BJ Howell JM Khouri H Koo H Mann DJ Norimine J Paulsen IT Radune D Ren Q Smith RK Suarez CE White O Wortman JR Knowles DP McElwain TF Nene VM 《PLoS pathogens》2007,3(10):1401-1413
127.
Everley PA Gartner CA Haas W Saghatelian A Elias JE Cravatt BF Zetter BR Gygi SP 《Molecular & cellular proteomics : MCP》2007,6(10):1771-1777
Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques. 相似文献
128.
Juergen Haas Rafal Gumienny Alessandro Barbato Flavio Ackermann Gerardo Tauriello Martino Bertoni Gabriel Studer Anna Smolinski Torsten Schwede 《Proteins》2019,87(12):1378-1387
Critical blind assessment of structure prediction techniques is crucial for the scientific community to establish the state of the art, identify bottlenecks, and guide future developments. In Critical Assessment of Techniques in Structure Prediction (CASP), human experts assess the performance of participating methods in relation to the difficulty of the prediction task in a biennial experiment on approximately 100 targets. Yet, the development of automated computational modeling methods requires more frequent evaluation cycles and larger sets of data. The “Continuous Automated Model EvaluatiOn (CAMEO)” platform complements CASP by conducting fully automated blind prediction evaluations based on the weekly pre-release of sequences of those structures, which are going to be published in the next release of the Protein Data Bank (PDB). Each week, CAMEO publishes benchmarking results for predictions corresponding to a set of about 20 targets collected during a 4-day prediction window. CAMEO benchmarking data are generated consistently for all methods at the same point in time, enabling developers to cross-validate their method's performance, and referring to their results in publications. Many successful participants of CASP have used CAMEO—either by directly benchmarking their methods within the system or by comparing their own performance to CAMEO reference data. CAMEO offers a variety of scores reflecting different aspects of structure modeling, for example, binding site accuracy, homo-oligomer interface quality, or accuracy of local model confidence estimates. By introducing the "bestSingleTemplate" method based on structure superpositions as a reference for the accuracy of 3D modeling predictions, CAMEO facilitates objective comparison of techniques and fosters the development of advanced methods. 相似文献
129.
The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa. 相似文献
130.
CD5, a type I glycoprotein expressed by T cells and a subset of B cells, is thought to play a significant role in modulating Ag receptor signaling. Previously, our laboratory has shown that bovine B cells are induced to express this key regulatory molecule upon Ag receptor cross-linking. To date, a ligand has not been described for bovine CD5. Given the importance ligand binding presumably plays in the functioning of CD5 on this B cell subset and on T cells, we sought to characterize the ligand for this protein using a bovine CD5-human IgG1 (CD5Ig) fusion protein produced by both mammalian and yeast cells. As determined by CD5Ig binding, expression of this ligand is negative to low on freshly isolated lymphocytes, with low-density expression being limited to activated B cells. Activation with LPS, PMA, and calcium ionophore, or ligation of CD40 alone or in combination with anti-IgM, resulted in B cell-specific expression of this ligand. Interestingly, activation through B cell Ag receptor cross-linking alone, although able to induce CD5 expression, did not result in expression of CD5 ligand (CD5L). In addition, we demonstrate a functional role for CD5L as a costimulatory molecule that augments CD40L-stimulated B cell proliferation. Finally, immunoprecipitation with CD5Ig suggests that the ligand characterized in this study has a molecular mass of approximately 200 kDa. The data reported herein, as well as future studies aimed at further characterizing this newly identified bovine CD5L, will undoubtedly aid in understanding the role that the CD5-CD5L interaction plays in immune responses. 相似文献