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21.
To understand force generation under a wide range of loads, the stepping of single kinesin molecules was measured at loads from −20 to 42 pN by optical tweezers with high temporal resolution. The optical trap has been improved to halve positional noise and increase bandwidth by using 200-nm beads. The step size of the forward and backward steps was 8.2 nm even over a wide range of loads. Histograms of the dwell times of backward steps and detachment fit well to two independent exponential equations with fast (~0.4 ms) and slow (>3 ms) time constants, indicating the existence of a fast step in addition to the conventional slow step. The dwell times of the fast steps were almost independent of the load and ATP concentration, while those of the slow backward steps and detachment depended on those. We constructed the kinetic model to explain the fast and slow steps under a wide range of loads. 相似文献
22.
Manfred J. Sippl 《Proteins》1993,17(4):355-362
A major problem in the determination of the three-dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. New developments in X-ray crystallography and nuclear magnetic resonance spectroscopy have acceleratedd the process of structure determination and the biological community is confronted with a steadily increasing number of experimentally determined protein folds. However, in the recent past several experimentally determined protein structures have been proven to contain major errors, indicating that in some cases the interpretation of experimental data is difficult and may yield incorrect models. Such problems can be avoided when computational methods are employed which complement experimental structure determinations. A prerequisite of such computational tools is that they are independent of the parameters obtained from a particular experiment. In addition such techniques are able to support and accelerate experimental structure determinations. Here we present techniques based on knowledge based mean fields which can be used to judge the quality of protein folds. The methods can be used to identify misfolded structures as well as faulty parts of structural models. The techniques are even applicable in cases where only the Cα trace of a protein conformation is available. The capabilities of the technique are demonstrated using correct and incorrect protein folds. © 1993 Wiley-Liss, Inc. 相似文献
23.
用原子力显微镜(AFM)研究了磷脂DMPC三层Langmuir-Blodgett(LB)膜的分子排列结构,结果表明:在磷脂LB膜的两相(液体压缩相Liquid-condensedphase和液体扩张相Liquid-expandedphase)共存时,液体压缩相中的磷脂分子排列紧密,取向一致,分子间作用力较大,因而能够得到分子图像。而液体扩张相中的磷脂分子排列松散,取向混乱。分子间的作用力较弱,难于得到分子图像。在液体压缩相中磷脂分子以单斜晶格结构排列,分子间隔为0.72nm.分子高度为2.1nm。这一结果和DMPC的单晶结构进行了比较。 相似文献
24.
Properties of microfiltration membranes: Mechanisms of flux loss in the recovery of an enzyme 总被引:3,自引:0,他引:3
The transmission and rate of filtration of the enzyme yeast alcohol dehydrogenase (YADH) has been studied at capillary pore microfiltration membranes. Photon correlation spectroscopy (PCS) with nanometer resolution showed that the enzyme existed as discreate molecules only for a narrow range of pH and ionic strength. Under such conditions, the transmission of the enzyme was high. However, the rate of filtration still decreased continuously with time. Analyssis of the time dependence of the rate of filtration indicated that this decrease was due to in-pore enzyme deposition at low concentration ("standard blocking model") and suface depositon at high concentration ("cake filtration model"). Use of atomic force microscopy (AFM) gave unequivocal and quantitative confirmation of these inferences. The work shows the great advantage of using advanced physical characterization techniques, both for the identification of the optimum conditions for filtration (PCS) and for the elucidation of mechanisms giving rise to inefficiencies in the filtration process (AFM). (c) 1995 John Wiley & Sons, Inc. 相似文献
25.
Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a
process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into
the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which
a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly,
solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport
with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by
fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient,
are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation
product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into
the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume
cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different
types of secondary metabolic energy generation will be discussed. 相似文献
26.
27.
Lawrence W. Adler Tomio Ichikawa Syed M. Hasan Tomofusa Tsuchiya Barry P. Rosen 《Journal of cellular biochemistry》1977,7(1):15-27
Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ). 相似文献
28.
L.M. Chailakhyan A.N. Glagolev T.N. Glagoleva G.V. Murvanidze T.V. Potapova V.P. Skulachev 《BBA》1982,679(1):60-67
An attempt at demonstrating lateral power transmission over millimeter distances along a coupling membrane has been undertaken. Trichomes of the multicellular filamentous cyanobacteria Phormidium uncinatum were illuminated with a very narrow light beam forming a light spot that covered only 4–5% of a 1–2 mm long cyanobacterial trichome. Such illumination was found to support motility (gliding along agar surface) of the trichome under conditions when the light was the only energy source. It was also shown that illumination with the light spot caused rotation of rings of slime (accompanying the operation of the ‘motors’ responsible for the motility of cyanobacteria) not only in the illuminated, but also in the distal, nonilluminated part of the trichome. Electric potential transmission along trichomes was revealed by means of the extracellular electrode technique. The light spot was found to induce generation of an electric potential difference between two electrodes in the dark region of the trichomes, which were placed at different distances from the illuminated end. Cutting the trichomes between the light spot and the closest ‘dark’ electrode abolished this effect. Valinomycin + K+ and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected the potential difference formation between two ‘dark’ electrodes much stronger than that between a light and a dark electrode. All the light spot-induced effects develop in the seconds time scale. Both the amplitudes and the kinetics of the potential difference measured with four electrodes placed along the trichome prove to be in good agreement with the theoretical curves computed on the basis of the electric cable equation. It is concluded that transcellular power transmission in the form of Δψ takes place along trichomes of cyanobacteria. This confirms the hypothesis about the biological function of Δψ as a transportable form of energy. 相似文献
29.
A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca2+ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca2+-troponin interactions coincident with MgATP-induced force development. Over the free [Ca2+] range 6 · 10?8–1.2 · 10?5 M the bound Ca2+ varied from 0.25 to 1.65 μmol/g protein. The free [Ca2+] at half-maximal Ca2+ saturation was 2 · 10?7 M while that a half-maximal force was 5 · 10?7 M. Half-maximal Ca2+ saturation was associated with 20% maximal force. The force-[Ca2+] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca2+-binding sites is essential for initiation of cross-bridge cycling. 相似文献
30.
Roger Wigren Hans Elwing Ragnar Erlandsson Stefan Welin Ingemar Lundstrm 《FEBS letters》1991,280(2):225-228
It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule. 相似文献