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101.
Lloyd-Evans E Morgan AJ He X Smith DA Elliot-Smith E Sillence DJ Churchill GC Schuchman EH Galione A Platt FM 《Nature medicine》2008,14(11):1247-1255
Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse. 相似文献
102.
103.
Linsen Li Antony Okumu Sheri Dellos-Nolan Zoe Li Soumendrakrishna Karmahapatra Anthony English Jack C. Yalowich Daniel J. Wozniak Mark J. Mitton-Fry 《Bioorganic & medicinal chemistry letters》2018,28(14):2477-2480
Novel bacterial type II topoisomerase inhibitors (NBTIs) constitute a promising new class of antibacterial agents. We report a series of NBTIs with potent anti-staphylococcal activity and diminished hERG inhibition. Dioxane-linked compound 9 demonstrated MICs ≤1?μg/mL against both methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA), accompanied by reduced hERG inhibition as compared to cyclohexane- or piperidine-linked analogs. 相似文献
104.
Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells 下载免费PDF全文
Margarida Ruas Lianne C Davis Cheng-Chang Chen Anthony J Morgan Kai-Ting Chuang Timothy F Walseth Christian Grimm Clive Garnham Trevor Powell Nick Platt Frances M Platt Martin Biel Christian Wahl-Schott John Parrington Antony Galione 《The EMBO journal》2015,34(13):1743-1758
The second messenger NAADP triggers Ca2+ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca2+ responses as assessed by single-cell Ca2+ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca2+-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca2+ release. High-affinity [32P]NAADP binding still occurs in Tpcn1/2−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+-permeable channels indispensable for NAADP signalling. 相似文献
105.
106.
The temperature dependence of the partial reactions leading to turn-over of the UQH2:cyt c
2 oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c
1 and c
2, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Qz-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)2
P870, primary donor of the photochemical reaction center
-
b/c
1 complex
ubiquinol: cytochrome c
2 oxidoreductase
- cyt b
H
cytochrome b-561 or higher potential cytochrome b
- cyt b
L
cytochrome b-566, or low potential cytochrome b
- cyt c
1, cyt c
2, cyt c
t
cytochromes c
1 and c
2, and total cytochrome c (cyt c
1 and cyt c
2)
- Fe.S
Rieske-type iron sulfur center, Q
- QH2
ubiquinone, ubiquinol
- Qz, QzH2, Qz
–
ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site
- Qz-site
ubiquinol oxidizing site (also called Qo-(outside)
- Qo
(Oxidizing)
- QP
(Positive proton potential) site)
- Qc-site
uubiquinone reductase site (also called the Qi-(inside)
- QR
(Reducing), or
- QN
(Negative proton potential) site)
- UHDBT
5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol 相似文献
107.
A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions.
Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus
induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed
on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet
genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions.
Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed
in four R1 progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R1 progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes
into the genome of finger millet in the future. 相似文献
108.
Antony J. Durston 《Genesis (New York, N.Y. : 2000)》2019,57(7-8)
This article is concerned with the roles of retinoids and other known anterior–posterior morphogens in setting up the embryonic vertebrate anterior–posterior axis. The discussion is restricted to the very earliest events in setting up the anterior–posterior axis (from blastula to tailbud stages in Xenopus embryos). In these earliest developmental stages, morphogen concentration gradients are not relevant for setting up this axis. It emerges that at these stages, the core patterning mechanism is timing: BMP‐anti BMP mediated time space translation that regulates Hox temporal and spatial collinearities and Hox‐Hox auto‐ and cross‐ regulation. The known anterior–posterior morphogens and signaling pathways––retinoids, FGF's, Cdx, Wnts, Gdf11 and others––interact with this core mechanism at and after space–time defined “decision points,” leading to the separation of distinct axial domains. There are also other roles for signaling pathways. Besides the Hox regulated hindbrain/trunk part of the axis, there is a rostral part (including the anterior part of the head and the extreme anterior domain [EAD]) that appears to be regulated by additional mechanisms. Key aspects of anterior–posterior axial patterning, including: the nature of different phases in early patterning and in the whole process; the specificities of Hox action and of intercellular signaling; and the mechanisms of Hox temporal and spatial collinearities, are discussed in relation to the facts and hypotheses proposed above. 相似文献
109.
(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b50 and cytochrome b?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q?I) and the secondary acceptor ubiquinone (QII) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b50 reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b50 reduction increased about 10-fold as the Eh(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with Em(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b50 reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q?I and QII. It was an effective inhibitor of cytochrome b50 photoreduction at Eh(7.0) 390 mV, but not at Eh(7.0) 100 mV. At the latter Eh, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q?I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At Eh(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b50 reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b50 oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high Eh values. However, at lower redox potentials, cytochrome b50 oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b?90 in the rapid reactions of the cyclic electron transfer chain 相似文献
110.
Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1-but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch. 相似文献