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51.
Sarrah M'Barek Ziad Fajloun Sandrine Cestle Christiane Devaux Pascal Mansuelle Amor Mosbah Besma Jouirou Massimo Mantegazza Jurphaas van Rietschoten Mohamed El Ayeb Herv Rochat Jean‐Marc Sabatier Francois Sampieri 《Journal of peptide science》2004,10(11):666-677
Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time. 相似文献
52.
The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca2+-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte
plants. The large-conductance (170 pS), voltage- and Ca2+-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the
Maxi-K channel from animal cells.
This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates
as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes
of action of- and different bindings sites for these calmodulin antagonists.
Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though
both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten
curve.
W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the
channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent.
In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the
closed state at −50 mV but is distinguishable from it at voltages more negative than −100 mV. This substate may represent
times that trifluoperazine is bound to the channel.
Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect
open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect
of Ca2−. This is good kinetic evidence against the view that the channel is activated by Ca2+-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca2−-calmodulin less available.
Received: 26 August 1996/Revised: 7 October 1996 相似文献
53.
Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry. 总被引:5,自引:1,他引:4 下载免费PDF全文
A. M. Krezel C. Kasibhatla P. Hidalgo R. MacKinnon G. Wagner 《Protein science : a publication of the Protein Society》1995,4(8):1478-1489
The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions. 相似文献
54.
55.
David W. Michaels 《生物化学与生物物理学报:生物膜》1979,555(1):67-78
The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17 500. 相似文献
56.
C. DOUGLAS MINCHEW 《The Journal of eukaryotic microbiology》1977,24(2):213-220
SYNOPSIS. Four new species of Henneguya (myxosporidan) are described from Ictalurus punctatus Rafinesque (channel catfish). They are as follows: Henneguya postexilis sp. n. and Henneguya longicauda sp. n. from the gills; Henneguya adiposa sp. n. from the adipose fin; and Henneguya diversis sp. n. from the liver, kidney, connective tissue of muscles and fins, and tumor-like external growths. Henneguya pellis sp. n. is described from the dermis of Ictalurus furcatus (LeSueur) (blue catfish). The development stages of all described species are discussed. Henneguya exilis Kudo was found on the gills of one I. punctatus; notes on its spore characteristics are presented. 相似文献
57.
Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth 总被引:13,自引:0,他引:13
Parmender P. Mehta Agnes Hotz-Wagenblatt Birgit Rose David Shalloway Warner R. Loewenstein 《The Journal of membrane biology》1991,124(3):207-225
Summary Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals. 相似文献
58.
Developmental changes in the calcium currents in embryonic chick ventricular myocytes 总被引:1,自引:0,他引:1
Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI
Ca activated from a holding potential (V
h) of –80 mV. L-type current (I
L) was activated by depolarizing steps fromV
h –30 or –40 mV. The difference current (I
T) was obtained by subtractingI
L, fromI
Ca.I
T could also be distinguished pharmacologically fromI
L in these cells.I
T was selectively blocked by 40–160 m Ni2+, whereasI
L was suppressed by 1 m D600 or 2 m nifedipine. The Ni2+-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI
T andI
L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I
T andI
L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I
Ca was partly blocked by Ni2+ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI
T andI
L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V
1/2) of the Ni2+-resistant currentI
L averaged –18 mV. In contrast,V
1/2 of the Ni2+-sensitiveI
T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI
L at both ages, but that ofI
T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI
Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI
T along the voltage axis. 相似文献
59.
Bruce C. Spalding Patricia Taber John G. Swift Paul Horowicz 《The Journal of membrane biology》1991,123(3):223-233
Summary Efflux of36Cl– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl– efflux. The pH-dependent Cl– efflux can be described by the relationu=1/(1+10n(pK
a
-pH)), whereu is the Cl– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK
a
is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK
a
at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK
a
suggests that the critical pH-sensitive moiety producing the pH-dependent Cl– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k
–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q10 of 2.5. The Cl– efflux inhibitor SCN– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl– efflux by DEPC. The findings that the apparent pK
a
is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl– efflux, and that this action is retarded by SCN– supports the notion that protonation of histidine groups associated with Cl– channels is the controlling reaction for the pH-dependent Cl– efflux. 相似文献
60.
Summary Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8±8.7 pS (n=16, 21°C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 m) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide-binding, anion selective mitochondrial channel, that is not the UCP. 相似文献