全文获取类型
收费全文 | 3704篇 |
免费 | 105篇 |
国内免费 | 139篇 |
出版年
2023年 | 42篇 |
2022年 | 75篇 |
2021年 | 68篇 |
2020年 | 77篇 |
2019年 | 120篇 |
2018年 | 123篇 |
2017年 | 63篇 |
2016年 | 50篇 |
2015年 | 56篇 |
2014年 | 299篇 |
2013年 | 274篇 |
2012年 | 219篇 |
2011年 | 299篇 |
2010年 | 225篇 |
2009年 | 219篇 |
2008年 | 243篇 |
2007年 | 206篇 |
2006年 | 154篇 |
2005年 | 154篇 |
2004年 | 58篇 |
2003年 | 48篇 |
2002年 | 42篇 |
2001年 | 28篇 |
2000年 | 29篇 |
1999年 | 38篇 |
1998年 | 37篇 |
1997年 | 18篇 |
1996年 | 15篇 |
1995年 | 21篇 |
1994年 | 26篇 |
1993年 | 28篇 |
1992年 | 14篇 |
1991年 | 19篇 |
1990年 | 16篇 |
1989年 | 10篇 |
1988年 | 12篇 |
1986年 | 10篇 |
1985年 | 29篇 |
1984年 | 62篇 |
1983年 | 56篇 |
1982年 | 68篇 |
1981年 | 57篇 |
1980年 | 44篇 |
1979年 | 52篇 |
1978年 | 28篇 |
1977年 | 25篇 |
1976年 | 18篇 |
1975年 | 15篇 |
1974年 | 26篇 |
1973年 | 14篇 |
排序方式: 共有3948条查询结果,搜索用时 31 毫秒
111.
Kohjiro Nagao Minami Maeda Noralyn B. Mañucat Kazumitsu Ueda 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(2):398-406
ATP-binding cassette protein A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL). However, the detailed mechanism of HDL formation remains unclear; in order to reveal it, chemicals that specifically block each step of HDL formation would be useful. Cyclosporine A inhibits ABCA1-mediated cholesterol efflux, but it is not clear whether this is mediated via inhibition of calcineurin. We analyzed the effects of cyclosporine A and related compounds on ABCA1 function in BHK/ABCA1 cells. Cyclosporine A, FK506, and pimecrolimus inhibited ABCA1-mediated cholesterol efflux in a concentration-dependent manner, with IC50 of 7.6, 13.6, and 7.0 μM, respectively. An mTOR inhibitor, rapamycin also inhibited ABCA1, with IC50 of 18.8 μM. The primary targets for these drugs were inhibited at much lower concentrations in BHK/ABCA1 cells, suggesting that they were not involved. Binding of [3H] cyclosporine A to purified ABCA1 could be clearly detected. Furthermore, a non-immunosuppressive cyclosporine, PSC833, inhibited ABCA1-mediated cholesterol efflux with IC50 of 1.9 μM, and efficiently competed with [3H] cyclosporine A binding to ABCA1. These results indicate that cyclosporine A and PSC833 inhibit ABCA1 via direct binding, and that the ABCA1 inhibitor PSC833 is an excellent candidate for further investigations of the detailed mechanisms underlying formation of HDL. 相似文献
112.
Shahzad Siddiqi Atur Sheth Feenalie Patel Matthew Barnes Charles M. Mansbach II 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(8):1311-1321
How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound 3H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the 3H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the 3H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with 3H-oleate or 3H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed 3H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles. 相似文献
113.
Imre Berger Frederic Garzoni Maxime Chaillet Matthias Haffke Kapil Gupta Alice Aubert 《Journal of visualized experiments : JoVE》2013,(77)
Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects. 相似文献
114.
Qurra-tul-Ann Afza Gardner Hooria Younas Muhammad Akhtar 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):182-190
Human M-proinsulin was cleaved by trypsin at the R31R32–E33 and K64R65–G66 bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K59R60–G61 bond but at the B/C junction cleavage occurred at the R31R32–E33 as well as the R31–R32E33 bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the kcat./Km values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 105 s− 1 M− 1) and the cleavage of the K29–T30 bond of M-insulin-RR (1.3 ± 0.07 × 105 s− 1 M− 1). However, the K29–T30 bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (kcat./Km values around 1000 s− 1 M− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K29–T30 bond becomes shielded, exposed then shielded again respectively. 相似文献
115.
Jennifer J. Scott Luis A. Buatois M. Gabriela Mángano Robin W. Renaut R. Bernhart Owen 《Lethaia: An International Journal of Palaeontology and Stratigraphy》2020,53(1):62-71
Modern burrowing organisms feed on microbial organic matter in matgrounds near hot springs on the margins of Lake Bogoria, a saline alkaline lake in the Kenya Rift Valley. The burrowers produce a low-diversity trace assemblage similar to those produced by undermat miners during the Ediacaran–Cambrian transition. Despite obvious differences in body plans and phylogenetic affinities, these modern animals feed on microbes in similar ways to those inferred for primitive bilaterians. With increasing distance from hot-spring vents, outflow channels and adjacent matgrounds, the diversity and depth of the traces increase and mixgrounds become dominant. This modern extreme environment gives clues for interpreting the heterogeneous early Cambrian seafloor, with: (1) the restriction of ‘pre-agronomic revolution’ matground substrates; and (2) expansion of adjacent ‘post-agronomic revolution’ mixground areas. 相似文献
116.
Xianghao Wu D. Brisbane Tovilla-Coutiño Mark A. Eiteman 《Biotechnology and bioengineering》2020,117(9):2781-2790
The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals. 相似文献
117.
Jorge Soriano María García-Díaz Margarita Mora Maria Lluïsa Sagristá Santi Nonell Angeles Villanueva Juan Carlos Stockert Magdalena Cañete 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.Methods
Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.Results
m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.Conclusions
Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.General significance
These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment. 相似文献118.
Background
The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL–(GroES)2 (football-shaped) and asymmetric GroEL–GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split.Methods
Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated.Results
Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning.Conclusions
This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL–GroES–ADP homolog) from T. thermophilus.General significance
The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL–GroES system. 相似文献119.
Lukáš Kubala Hana Kolářová Jan Víteček Silvie Kremserová Anna Klinke Denise Lau Anna L.P. Chapman Stephan Baldus Jason P. Eiserich 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood.Methods
We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested.Results
MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed.Conclusions
Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins.General significance
Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins. 相似文献120.
Atanas G. Atanasov Jian N. Wang Shi P. Gu Jing Bu Matthias P. Kramer Lisa Baumgartner Nanang Fakhrudin Angela Ladurner Clemens Malainer Anna Vuorinen Stefan M. Noha Stefan Schwaiger Judith M. Rollinger Daniela Schuster Hermann Stuppner Verena M. Dirsch Elke H. Heiss 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013