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631.
The nucleobase-ascorbate transporter or nucleobase-cation symporter-2 (NAT/NCS2)
family is one of the five known families of transporters that use nucleobases as
their principal substrates and the only one that is evolutionarily conserved and
widespread in all major taxa of organisms. The family is a typical paradigm of a
group of related transporters for which conservation in sequence and overall
structure correlates with high functional variations between homologs.
Strikingly, the human homologs fail to recognize nucleobases or related
cytotoxic compounds. This fact allows important biomedical perspectives for
translation of structure-function knowledge on this family to the rational
design of targeted antimicrobial purine-related drugs. To date, very few
homologs have been characterized experimentally in detail and only two, the
xanthine permease XanQ and the uric acid/xanthine permease UapA, have been
studied extensively with site-directed mutagenesis. Recently, the
high-resolution structure of a related homolog, the uracil permease UraA, has
been solved for the first time with crystallography. In this review, I summarize
current knowledge and emphasize how the systematic Cys-scanning mutagenesis of
XanQ, in conjunction with existing biochemical and genetic evidence for UapA and
the x-ray structure of UraA, allow insight on the structure-function and
evolutionary relationships of this important group of transporters. The review
is organized in three parts referring to (I) the theory of use of Cys-scanning
approaches in the study of membrane transporter families, (II) the state of the
art with experimental knowledge and current research on the NAT/NCS2 family,
(III) the perspectives derived from the Cys-scanning analysis of XanQ. 相似文献
632.
Andre Bazzone Wagner Steuer Costa Markus Braner Octavian C?linescu Lina Hatahet Klaus Fendler 《Journal of visualized experiments : JoVE》2013,(75)
The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods. 相似文献
633.
《Journal of molecular biology》2023,435(11):167975
Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles. 相似文献
634.
Affinity purification of histidine-tagged proteins 总被引:11,自引:0,他引:11
Expression of recombinant proteins is a standard technique in molecular biology and a wide variety of prokaryotic as well as eukaryotic expression systems are currently in use. A limiting step is often the purification of the expressed recombinant protein, particularly if mammalian expression systems that yield low expression levels are employed. Here, we discuss the advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni2+-NTA chromatography.Abbreviations GST
glutathione S-transferase
- NTA
nitrilotriacetic acid
- His
histidine
- PAGE
polyacrylamide gel electrophoresis 相似文献
635.
636.
This article presents the results of an investigation that assessed the extent and effect of oxidation of Cr(III) in manganese-enriched clays on the electrokinetic remedial efficiency. Because chromium commonly exists along with nickel and cadmium at contaminated sites, the effects of changes in chromium redox chemistry on the migration of the coexisting nickel and cadmium was also studied. Bench-scale electrokinetic experiments were conducted using two different clays: kaolin, a typical low buffering soil, and glacial till, a high buffering soil. Tests were performed with 1000?mg/kg of Cr(III), 500?mg/kg of Ni(II), and 250?mg/kg of Cd(II), both with and without the presence of 1000?mg/kg of manganese. All of these experiments were conducted under a constant voltage gradient of 1.0?VDC/ cm. The experimental results showed that in the presence of manganese, percentages of oxidation of Cr(III) into Cr(VI) ranged from 67% in kaolin to 28% in glacial till even before the application of induced electric potential. The low extent of oxidation of Cr(III) in glacial till may be attributed to the initial precipitation of Cr(III) as Cr(OH)3 resulting from high soil pH, reducing aqueous Cr(III) concentrations present within the soil. In kaolin, Cr(III), Ni(II), and Cd(II) under electric potential migrated toward cathode and precipitated near the cathode due to high soil pH. When manganese was present in kaolin, Cr(VI) that was formed due to the oxidation of Cr(III) migrated toward anode and adsorbed to the soil surfaces near the anode region due to low soil pH. However, remaining Cr(III) as well as Ni(II), and Cd(II) migrated towards and precipitated near the cathode due to high soil pH. In kaolin, the migration of Ni(II) and Cd(II) was retarded in the presence of manganese due to a larger soil zone of elevated pH near the cathode. In glacial till, the migration of Cr(III), Ni(II) and Cd(II) was insignificant due to precipitation resulting from high soil pH caused by the high buffering capacity of the soil. Cr(VI) that resulted from the partial oxidation of Cr(III) in the presence of manganese, however, migrated toward the anode. Overall, this study demonstrated that the effects of manganese on Cr(III) oxidation in low buffering soils can be significant, which can in turn affect the extent and direction of chromium migration under induced electric potential. 相似文献
637.
Solar UV-B radiation increased to 20 % over ambient level at Madurai was given to cowpea (Vigna unguiculata L. cv. Pusa-152)
seedlings sufficiently supplied by potassium (0.88 mM K2SO4) and K-deficient (0.05 mM K2SO4). Leaf mineral composition was significantly changed due to both increased UV-B radiation and K-deficiency imposed independently
or jointly for 12 d. A severe reduction in 23 kDa chloroplast protein was seen only in seedlings encountered combined stress.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
638.
639.
ROBERT S. BOYD 《Insect Science》2007,14(3):201-205
Nickel hyperaccumulator plants contain unusually elevated levels of Ni (〉 1 000 μg Ni/g). Some insect herbivores, including Lygus hesperus (Western tarnished plant bug), have been observed feeding on the California Ni hyperaccumulator Streptanthus polygaloides. This bug may be able to utilize S. polygaloides as a host either through its feeding behavior or by physiological tolerance of Ni. This experiment determined the Ni tolerance of L hesperus by offering insects artificial diet amended with 0, 0.4, 1, 2, 4.5, 10, 20 and 40 mmol Ni/L and recording survival. Survival varied due to Ni concentration, with diets containing 10 mmol Ni/L and greater resulting in significantly lower survival compared to the control (0 mmol Ni/L) treatment. Insects tolerated diet containing as much as 4.5 mmol Ni/L, a relatively elevated Ni concentration. I conclude that L hesperus can feed on S. polygaloides because it is Ni-tolerant, probably due to physiological mechanisms that provide it with resistance to plant chemical defenses including elemental defenses such as hyperaccumulated Ni. 相似文献
640.
Brown fat mitochondria obtained from a hibernator, the golden hamster, were investigated in order to elucidate the significance of membrane permeability for metabolic functioning at different temperatures. The mitochondria were shown to have active permeases for phosphate and pyruvate, but very poorly developed permeases for di- and tricarboxylate substrate anions. This was shown with both osmotic swelling techniques and respiration-driven uptake studies. It was shown that the very limited malate permeation observed was compatible with it being a non-carrier-mediated diffusion process. The role of malate transport in supporting fatty-acid oxidation in vitro as a function of temperature was studied in detail. The results support our earlier suggestion that physiologically pyruvate carboxylase probably functions to generate oxaloacetate when high concentrations of condensing partner are needed during thermogenesis. They may also explain earlier observations that acetate was produced from palmitoyl-carnitine at low temperatures even when malate was present; this is here shown to be due to the limited malate permeability at these low temperatures. Thus, even at the body temperature of the hibernating hamster (4–5°C), brown fat is probably able to combust fatty acids totally. 相似文献