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21.
Garcia-Viloca M Poulsen TD Truhlar DG Gao J 《Protein science : a publication of the Protein Society》2004,13(9):2341-2354
A subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X-ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl-CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton-abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg2+ metal cofactor that plays an active role in catalysis. 相似文献
22.
By using a combined quantum-mechanical and molecular-mechanical potential in molecular dynamics simulations, we have investigated the effects of the enzyme electric field of dihydrofolate reductase on the electronic polarization of its 5-protonated dihydrofolate substrate at various stages of the catalyzed hydride transfer reaction. Energy decomposition of the total electrostatic interaction energy between the ligands and the enzyme shows that the polarization effect is 4% of the total electrostatic interaction energy, and, significantly, it accounts for 9kcal/mol of transition state stabilization relative to the reactant state. Therefore it is essential to take account of substrate polarization for quantitative interpretation of enzymatic function and for calculation of binding free energies of inhibitors to a protein. Atomic polarizations are calculated as the differences in the average atomic charges on the atoms in gas phase and in molecular simulations of the enzyme; this analysis shows that the glutamate tail and the pterin ring are the highly polarized regions of the substrate. Electron density difference plots of the reactant and product complexes at instantaneous configurations in the enzyme active center confirm the inferences made on the basis of partial atomic charges. 相似文献
23.
Microrheology of human lung epithelial cells measured by atomic force microscopy 总被引:9,自引:0,他引:9
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Alcaraz J Buscemi L Grabulosa M Trepat X Fabry B Farré R Navajas D 《Biophysical journal》2003,84(3):2071-2079
Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (G(*)(omega)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1-100 Hz) and at different loading forces (0.1-0.9 nN) with atomic force microscopy. G(*)(omega) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus G'(omega) increased with frequency following a power law with exponent approximately 0.2. The loss modulus G"(omega) was approximately 2/3 lower and increased similarly to G'(omega) up to approximately 10 Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of G'(omega) and G"(omega). G(*)(omega) conformed to the power-law model with a structural damping coefficient of approximately 0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture. 相似文献
24.
Ríos G Lossow A Hertel B Breuer F Schaefer S Broich M Kleinow T Jásik J Winter J Ferrando A Farrás R Panicot M Henriques R Mariaux JB Oberschall A Molnár G Berendzen K Shukla V Lafos M Koncz Z Rédei GP Schell J Koncz C 《The Plant journal : for cell and molecular biology》2002,32(2):243-253
To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks. 相似文献
25.
Theocharis SE Margeli AP Koutselinis A 《The International journal of biological markers》2003,18(3):162-169
The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed. 相似文献
26.
Emma Harding-Esch Mireia Jofre-Bonet Jaskiran K. Dhanjal Sarah Burr Tansy Edwards Martin Holland Ansumana Sillah Sheila West Tom Lietman Jeremy Keenan David Mabey Robin Bailey 《PLoS neglected tropical diseases》2015,9(4)
Background
Mass drug administration (MDA) treatment of active trachoma with antibiotic is recommended to be initiated in any district where the prevalence of trachoma inflammation, follicular (TF) is ≥10% in children aged 1–9 years, and then to continue for at least three annual rounds before resurvey. In The Gambia the PRET study found that discontinuing MDA based on testing a sample of children for ocular Chlamydia trachomatis(Ct) infection after one MDA round had similar effects to continuing MDA for three rounds. Moreover, one round of MDA reduced disease below the 5% TF threshold. We compared the costs of examining a sample of children for TF, and of testing them for Ct, with those of MDA rounds.Methods
The implementation unit in PRET The Gambia was a census enumeration area (EA) of 600–800 people. Personnel, fuel, equipment, consumables, data entry and supervision costs were collected for census and treatment of a sample of EAs and for the examination, sampling and testing for Ct infection of 100 individuals within them. Programme costs and resource savings from testing and treatment strategies were inferred for the 102 EAs in the study area, and compared.Results
Census costs were $103.24 per EA plus initial costs of $108.79. MDA with donated azithromycin cost $227.23 per EA. The mean cost of examining and testing 100 children was $796.90 per EA, with Ct testing kits costing $4.80 per result. A strategy of testing each EA for infection is more expensive than two annual rounds of MDA unless the kit cost is less than $1.38 per result. However stopping or deciding not to initiate treatment in the study area based on testing a sample of EAs for Ct infection (or examining children in a sample of EAs) creates savings relative to further unnecessary treatments.Conclusion
Resources may be saved by using tests for chlamydial infection or clinical examination to determine that initial or subsequent rounds of MDA for trachoma are unnecessary. 相似文献27.
Jose Manuel Trujillo Gómez Laura Díaz-Gete Carlos Martín-Cantera Mireia Fábregas Escurriola Maribel Lozano Moreno Raquel Burón Leandro Ana María Gomez Quintero Jose Luis Ballve María Lourdes Clemente Jiménez Elisa Puigdomènech Puig Ramón Casas More Beatriz Garcia Rueda Marc Casajuana Marga Méndez-Aguirre David Garcia Bonias Soraya Fernández Maestre Jessica Sánchez Fondevila 《PloS one》2015,10(9)
28.
Background
Tobacco use prevalence rates are high among Spanish adolescents. Programming to counteract tobacco use is needed.Methods and Findings
The current study provides a one-year follow-up outcome evaluation of Project EX, an eight-session classroom-based curriculum. The intervention was tested using a randomized controlled trial with 1,546 Spanish students, involving three program and three control schools. Compared to the control condition, the program condition revealed a greater reduction in nicotine dependence (p < .05) and CO ppm levels (p < .001), and lower consumption of cigarettes at last month (p = .03).Conclusions
Long-term outcomes of the Project EX classroom-based program are promising for adolescent prevention and possibly cessation in Spain. 相似文献29.
Mireia Martín-Satué Elise G. Lavoie Michel Fausther Joanna Lecka Elisabet Aliagas Filip Kukulski Jean Sévigny 《Histochemistry and cell biology》2010,133(6):659-668
Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction,
steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for
mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface
ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant
and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members
of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628,
2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main
enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified
by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific
CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic
cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal
cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis
in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests
close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence
with NTPDases, may impact male fertility. 相似文献
30.
An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay. 相似文献