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991.
Hyperpolarization-activated K channels (K
H
channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell
mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K
H
channels in isolated guard cell protoplasts from Vicia faba leaves.
Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K
H
channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK
a
of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits
caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing
of these charges was ∼30 ? and their apparent dissociation constant was 10−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the
framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence
of all four transition rate constants.
Received: 26 April 1996/Revised: 29 June 1996 相似文献
992.
T. Ishikawa 《The Journal of membrane biology》1996,153(2):147-159
A Ca2+-activated Cl− conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When
the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca2+ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of
the cells with pipette solutions containing 300 nm or less than 1 nm free Ca2+ strongly reduced the Cl− currents, indicating the currents were Ca2+-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent.
The anion permeability sequence of the Cl− channels was: NO−
3 (2.00) > I− (1.85) ≥ Br− (1.69) > Cl− (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette
solutions was increased from 0 to 10 mm, the Ca2+-dependency of the Cl− current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca2+-activated Cl− currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca2+-activated Cl− currents. The outward Cl− currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable
Cl− conductance controlled by cytosolic Ca2+ and ATP levels in rat submandibular acinar cells.
Received: 9 January 1996/Revised: 20 May 1996 相似文献
993.
994.
We studied the specificities of human red cell membrane bindings of three long chain fatty acids, palmitic- arachidonic-
and oleic acid, using resealed membranes, ghosts. Previously estimated binding capacities, affinities and inside/outside distributions
[6, 10, 11, 12], suggest separated binding sites. This possibility is explored by estimating the binding properties of one
fatty acid in the presence of one or two of the others. Binding capacities, nmol g−1 ghosts, of palmitic and arachidonic acid estimated simultaneously vs. separately are 27.4 ± 2.7 vs. 29.0 ± 2.1 (P < 0.6) and 6.5 ± 0.6 vs. 5.5 ± 0.5 (P < 0.2) respectively. The corresponding estimates for oleic- and palmitic acid are 36.5 ± 2.0 vs. 34.0 ± 2.2 (P < 0.4) and 28.4 ± 1.8 versus 29.1 ± 2.1 (P < 0.8). The binding sites are therefore independent. For each of the three fatty acids in the absence or in the presence of
one or two of the others, the inside/outside distributions of the binding sites and the membrane transfer rate constants are
elucidated by exchange efflux kinetics at 0°C from ghosts with and without enclosed albumin. Packed ghosts loaded with radioactive
acids are injected rapidly into a large volume of vigorously stirred buffer with albumin. With a resolution time of about
1-sec serial filtered ghost-free aliquots are collected and counted. The analyses show that palmitic- and oleic acid sites
of transport are entirely independent but do not exclude that palmitic- and/or oleic acid binding may diminish the arachidonic
acid affinity a little. The diversity combined with specificity suggests that the transport sites for long chain fatty acids
are protein-determined microdomains of phospholipids.
Received: 26 June 1995/Revised: 11 October 1995 相似文献
995.
Received: 18 April 1996/Revised: 26 June 1996 相似文献
996.
The mechanosensitive properties of large-conductance Ca2+-activated K+ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of
the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the
patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed
in on-cell patches. This mechanosensitivity was not mediated by Ca2+ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity
in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time
of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous
results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment.
Received: 1 February 1996/Revised: 25 March 1996 相似文献
997.
Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 n M phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4α-PDBu analogue at 100 n M had no effect on release. Furthermore, 100 n M bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 n M or 1-oleoyl-2-acetyl- sn -glycerol at 50 µ M did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 n M staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia. 相似文献
998.
999.
Hidetaka Kosako Yukiko Gotoh Eisuke Nishida 《Development, growth & differentiation》1996,38(6):577-582
Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis . Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos. 相似文献
1000.
The helminth fauna of Litoria genimaculata, a rainforest frog from northern Queensland, was quantified from 53 adult male frogs collected at monthly intervals between April 1990 and March 1991. The helminth fauna of this species was depauperate (6 species: Mesocoelium sp., Parapolystoma bulliense, Austraplectana sp., Onchocercidae gen. sp., Cosmocerca sp. and an unidentified nematode larva). The most commonly encountered species was P. bulliense, but the intestinal infracommanity was dominated by the digenean Mesocoelium sp. Fifty-five per cent of frogs were infected with only 1 helminth species and only 1 frog had more than 2 species, resulting in low diversity values. These results support previous studies which indicate that amphibians have depauperate helminth communities. 相似文献