全文获取类型
收费全文 | 3054篇 |
免费 | 152篇 |
国内免费 | 1篇 |
专业分类
3207篇 |
出版年
2024年 | 4篇 |
2023年 | 18篇 |
2022年 | 50篇 |
2021年 | 67篇 |
2020年 | 45篇 |
2019年 | 58篇 |
2018年 | 71篇 |
2017年 | 70篇 |
2016年 | 125篇 |
2015年 | 170篇 |
2014年 | 221篇 |
2013年 | 217篇 |
2012年 | 262篇 |
2011年 | 241篇 |
2010年 | 167篇 |
2009年 | 138篇 |
2008年 | 187篇 |
2007年 | 170篇 |
2006年 | 127篇 |
2005年 | 120篇 |
2004年 | 125篇 |
2003年 | 90篇 |
2002年 | 83篇 |
2001年 | 74篇 |
2000年 | 68篇 |
1999年 | 43篇 |
1998年 | 16篇 |
1997年 | 14篇 |
1996年 | 14篇 |
1995年 | 7篇 |
1994年 | 4篇 |
1993年 | 5篇 |
1992年 | 15篇 |
1991年 | 12篇 |
1990年 | 11篇 |
1989年 | 14篇 |
1988年 | 9篇 |
1987年 | 6篇 |
1986年 | 6篇 |
1985年 | 9篇 |
1984年 | 5篇 |
1983年 | 4篇 |
1982年 | 6篇 |
1979年 | 9篇 |
1978年 | 4篇 |
1975年 | 4篇 |
1974年 | 5篇 |
1973年 | 4篇 |
1972年 | 3篇 |
1968年 | 2篇 |
排序方式: 共有3207条查询结果,搜索用时 15 毫秒
141.
Mesenchymal stem/progenitor cells developed in cultures from UC blood 总被引:21,自引:0,他引:21
Background Whether umbilical cord blood (UCB) serves as a source of mesenchymal stem/progenitor cells (MSPC) is controversial. MSPC are the best candidates for cellular therapy of orthopedic skeletal tissues. In order to explore the possibility of UCB as a useful source of MSPC, we identified, expanded in culture, and characterized MSPC from UCB harvests on a large scale. Methods Mononuclear cells isolated from UCB harvests (n=411) were cultured in media supplemented with 10% FBS. MSPC-like cells cultured from each UCB harvest were expanded ex vivo by successive subcultivation. UCB harvests with a more than 1000-fold expanding capacity (n=9) were examined for surface Ag phenotypes and in vitro differentiation potentials into osteogenic, chondrogenic and adipogenic lineages. Results Ninety-five out of a total of 411 UCB units (23.1%) generated MSPC-like cells during cultivation. Nine UCB units (2.2%) yielded MSPC with more than 1000-fold expansion capacity. These cells positively expressed MSPC-related Ag, but did not express myeloid, histocompatibility or endothelial Ag. These cells also possessed multiple capacities for osteogenic, chondrogenic and adipogenic differentiation. Discussion Although the incidence of UCB harvests producing MSPC in culture was low, some of them showed a more than 1000-fold expanding capacity, which is enough in cell numbers to be an allogeneic source for cellular therapy. Our results may encourage the use of UCB as an attractive target for allogeneic cellular therapeutic options in tissue engineering. 相似文献
142.
To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation. 相似文献
143.
Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella 总被引:4,自引:0,他引:4
A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation. 相似文献
144.
The enzyme HemK (or PrmC) is one of the first identified methyltransferases that modify glutamine. It methylates the highly conserved GGQ motif in class I release factors (RF1 and RF2) in Escherichia coli. HemK from Thermotoga maritima was over-expressed and crystallized in the presence of S-adenosylmethionine at 296 K using ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystal is orthorhombic, belonging to the space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters of a = 104.24, b = 118.73, and c = 146.62 A. Two (or three) monomers of recombinant HemK are likely to be present in the crystallographic asymmetric unit, giving a V(M) of 3.62 A3 Da(-1) (or 2.41 A3 Da(-1)), with a solvent content of 62.7% (or 44.0%). 相似文献
145.
Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. 相似文献
146.
A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample.
This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular
MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and
gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7.
The maximum activity was observed at pH 8.7 and 50 °C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain.
The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of
the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
147.
Ha JS Ha CE Chao JT Petersen CE Theriault A Bhagavan NV 《Biochimica et biophysica acta》2003,1640(2-3):119-128
In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA-cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux. 相似文献
148.
Single-molecule fluorescence resonance energy transfer 总被引:18,自引:0,他引:18
Ha T 《Methods (San Diego, Calif.)》2001,25(1):78-86
Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation. 相似文献
149.
150.
Lowe E Rice P Ha T Li C Kelley J Ensley H Lopez-Perez J Kalbfleisch J Lowman D Margl P Browder W Williams D 《Microbes and infection / Institut Pasteur》2001,3(10):789-797
Glucans are fungal cell wall polysaccharides which stimulate innate immune responses. We determined the minimum unit ligand that would bind to glucan receptors on human U937 cells using laminarin-derived pentaose, hexaose, and heptaose glucan polymers. When U937 membranes were pretreated with the oligosaccharides and passed over a glucan surface, only the heptasaccharide inhibited the interaction of glucan with membrane receptors at a K(d) of 31 microM (95% CI 20-48 microM) and 100% inhibition. However, the glucan heptasaccharide did not stimulate U937 monocyte NFkappaB signaling, nor did it increase survival in a murine model of polymicrobial sepsis. Laminarin, a larger and more complex glucan polymer (M(w) = 7700 g/mol), only partially inhibited binding (61 +/- 4%) at a K(d) of 2.6 microM (99% CI 1.7-4.2 microM) with characteristics of a single binding site. These results indicate that a heptasaccharide is the smallest unit ligand recognized by macrophage glucan receptors. The data also indicate the presence of at least two glucan-binding sites on U937 cells and that the binding sites on human monocyte/macrophages can discriminate between glucan polymers. The heptasaccharide and laminarin were receptor antagonists, but they were not receptor agonists with respect to activation of NFkappaB-dependent signaling pathways or protection against experimental sepsis. 相似文献