全文获取类型
收费全文 | 549篇 |
免费 | 33篇 |
专业分类
582篇 |
出版年
2022年 | 1篇 |
2021年 | 6篇 |
2020年 | 3篇 |
2019年 | 3篇 |
2018年 | 12篇 |
2017年 | 24篇 |
2016年 | 10篇 |
2015年 | 11篇 |
2014年 | 26篇 |
2013年 | 33篇 |
2012年 | 40篇 |
2011年 | 32篇 |
2010年 | 12篇 |
2009年 | 18篇 |
2008年 | 24篇 |
2007年 | 33篇 |
2006年 | 30篇 |
2005年 | 29篇 |
2004年 | 23篇 |
2003年 | 19篇 |
2002年 | 19篇 |
2001年 | 28篇 |
2000年 | 27篇 |
1999年 | 18篇 |
1998年 | 5篇 |
1997年 | 7篇 |
1996年 | 3篇 |
1995年 | 2篇 |
1994年 | 4篇 |
1993年 | 1篇 |
1992年 | 6篇 |
1991年 | 11篇 |
1990年 | 7篇 |
1989年 | 6篇 |
1988年 | 6篇 |
1987年 | 8篇 |
1986年 | 5篇 |
1985年 | 5篇 |
1984年 | 2篇 |
1983年 | 1篇 |
1981年 | 4篇 |
1979年 | 2篇 |
1977年 | 2篇 |
1975年 | 2篇 |
1974年 | 1篇 |
1973年 | 5篇 |
1972年 | 2篇 |
1971年 | 2篇 |
1969年 | 1篇 |
1968年 | 1篇 |
排序方式: 共有582条查询结果,搜索用时 13 毫秒
1.
The fluorescence of the voltage sensitive dye, diS-C3-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C3-(5) fluorescence from cells and from the supernatant. It has been found that diS-C3-(5) fluorescence in the supernatant can be selectively monitored at exc = 630 nm and em= 650 nm, while the cell associated fluorescence can be observed at exc= 690 nm and em = 710 nm. A modified theory for the diSC3-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, p=p-°p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K+ gradients. It has been demonstrated that the membrane potential change, p,can be measured on an absolute scale.
Offprint requests to: J. Plasek 相似文献
2.
Effects of extracellular nucleotides on electrical properties of subconfluent Madin Darby canine kidney cells 总被引:1,自引:0,他引:1
ATP and ADP but not AMP lead to sustained hyperpolarization of Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for an influence of other nucleotides on the potential difference across the cell membrane (PD) in subconfluent MDCK cells. PD has been continuously monitored with conventional microelectrodes during rapid exchange of extracellular fluid. Application of 1 mumol/1 UTP leads to a rapid (less than 2 s) hyperpolarization of the cell membrane by -17.0 +/- 0.4 mV (from -50.1 +/- 0.6 mV), a reduction of cell membrane resistance and an increase of the sensitivity of PD to alterations of extracellular potassium. The concentration needed for half maximal effect of UTP is approximately equal to 0.2 mumol/1. ITP is similarly effective, whereas UDP, GTP and GDP are less effective. Up to 1 mmol/1 UMP, GMP, TTP or CTP do not significantly alter PD. In calcium-free extracellular fluid the hyperpolarizing effect of UTP is blunted (-11.6 +/- 2.3 mV) and only transient. In conclusion, UTP similar to purine triphosphates hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium, which is apparently recruited from both intra- and extracellular sources. 相似文献
3.
Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli 总被引:1,自引:0,他引:1
The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI. 相似文献
4.
Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. 总被引:9,自引:0,他引:9
We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli. 相似文献
5.
Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3′-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3′-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential inSaccharomyces cerevisiœ in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with
probe concentration in the range of 60–3000 nmol/L. The optimum staining period was 15–20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop
in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with
different fluorescence properties. 相似文献
6.
Stefan N Martin-Killias P Wyss-Stoeckle S Honegger A Zangemeister-Wittke U Plückthun A 《Journal of molecular biology》2011,413(4):826-843
Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (Kd = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting. 相似文献
7.
Phylogeny of calcareous dinoflagellates as inferred from ITS and ribosomal sequence data 总被引:6,自引:0,他引:6
Gottschling M Keupp H Plötner J Knop R Willems H Kirsch M 《Molecular phylogenetics and evolution》2005,36(3):3-455
The phylogenetic relationships of calcareous dinoflagellates (i.e., Calciodinellaceae and Thoracosphaera) are investigated. Molecular data from the ribosomal 5.8S rRNA and highly conserved motifs of the ITS1 show Calciodinellaceae s.l. to be monophyletic when few non-calcareous taxa are included. They segregate into three monophyletic assemblages in a molecular analysis that considers the 5.8S rRNA and both the Internal Transcribed Spacer regions ITS1 and ITS2: a clade comprising species of Ensiculifera and Pentapharsodinium (E/P-clade), Scrippsiella s.l. (including fossil-based taxa such as Calciodinellum and Calcigonellum), and a heterogeneous group (T/P-clade) of calcareous (e.g., Thoracosphaera) and non-calcareous taxa (e.g., the highly toxic Pfiesteria). The potential to produce calcareous structures is considered as apomorphic within alveolates, and non-calcareous taxa nesting with calcareous dinoflagellates may have reduced calcification secondarily. Molecular results do not contradict general evolutionary scenarios provided by previous morphological (mainly paleontological) investigations. 相似文献
8.
Pretreatment of wheat straw using combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose 总被引:12,自引:0,他引:12
Bjerre AB Olesen AB Fernqvist T Plöger A Schmidt AS 《Biotechnology and bioengineering》1996,49(5):568-577
The wet oxidation process of wheat straw has been studied as a pretreatment method to attain our main goal: To break down cellulose to glucose enzymatic, and secondly, to dissolve hemicellulose (e.g., for fermentation) without producing microbial inhibitors. Wet oxidation combined with base addition readily oxidizes lignin from wheat straw facilitating the polysaccharides for enzymatic hydrolysis. By using a specially constructed autoclave system, the wet oxidation process was optimized with respect to both reaction time and temperature. The best conditions (20 g/L straw, 170 degrees C, 5 to 10 min) gave about 85% w/w yield of converting cellulose to glucose. The process water, containing dissolved hemicellulose and carboxylic acids, has proven to be a direct nutrient source for the fungus Aspergillus niger producing exo-beta-xylosidase. Furfural and hydroxymethyl-furfural, known inhibitors of microbial growth when other pretreatment systems have been applied, were not observed following the wet oxidation treatment. (c) 1996 John Wiley & Sons, Inc. 相似文献
9.
Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis
下载免费PDF全文
![点击此处可从《Molecular systems biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Paul J Boersema Jan‐Oliver Jost Katharina Kramer Ahmet Bakirbas Julia Sindlinger Magdalena Plöchinger Dario Leister Glen Uhrig Greg BG Moorhead Jürgen Cox Michael E Salvucci Dirk Schwarzer Matthias Mann Iris Finkemeier 《Molecular systems biology》2017,13(10)
Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions. 相似文献
10.
Yana Chen Kevork Hagopian Douglas Bibus José?M. Villalba Guillermo López-Lluch Plácido Navas Kyoungmi Kim Roger?B. McDonald Jon?J. Ramsey 《Bioscience reports》2013,33(1)
To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H2O2 production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals. 相似文献