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Using polyclonal and monoclonal antibodies to visualize under a confocal microscope type-1 cannabinoid receptors (CB1) and acetylcholine (ACh) receptors, respectively, or α-bungarotoxin conjugated to Alexa-Fluor 555 for Ach receptors, we found that they colocalize on twitch muscle fibers in the frog (Rana pipiens). We show that both the CB1 and ACh receptors are present on the fast skeletal muscle motor end-plate. The CB1 receptor is present along the entire membrane of the muscle fiber, whereas the ACh receptor is expressed primarily at the motor end-plate. Analysis of the colocalization produced a cross-correlation coefficient of 0.519 ± 0.021 (n = 9) for both receptors at the muscle motor end-plate. This study suggests a close proximity between these two types of receptor proteins and that they could interact. CB1 could function at some stage of excitation–contraction coupling in these muscle fibers. However, further investigation is needed in order to clarify these issues.  相似文献   
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Cytoplasmic dynein play an important role in transporting various intracellular cargos by coupling their ATP hydrolysis cycle with their conformational changes. Recent experimental results showed that the cytoplasmic dynein had a highly variable stepping pattern including “hand-over-hand”, “inchworm” and “nonalternating-inchworm”. Here, we developed a model to describe the coordinated stepping patterns of cytoplasmic dynein, based on its working cycle, construction and the interaction between its leading head and tailing head. The kinetic model showed how change in the distance between the two heads influences the rate of cytoplasmic dynein under different stepping patterns. Numerical simulations of the distribution of step size and striding rate are in good quantitative agreement with experimental observations. Hence, our coordinated stepping model for cytoplasmic dynein successfully explained its diverse stepping patterns as a molecular motor. The cooperative mechanism carried out by the two heads of cytoplasmic dynein shed light on the strategies adopted by the cytoplasmic dynein in executing various functions.  相似文献   
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Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to promote mammogenesis. To this end, we constructed a mammary gland-specific expression vector, pcGH, and demonstrated effective GH expression in goat mammary epithelial cells in vitro by ELISA. Then, to produce transgenic offspring that were capable of stable GH expression in vivo, the linearized pcGH vector was electroporated into goat fetal fibroblasts. Cell colonies that were positive for GH were used as donors for nuclear transfer to enucleated oocytes. A total of 253 morulae or blastocytes developed from the reconstructed embryos were transferred to 56 recipients, resulting in 24 pregnancies at day 35. Finally, six transgenic goats were born. PCR detection confirmed the success of the cloning procedure. To observe the mammogenesis of dairy goats, the GH transgenic goats were mated with a completely healthy buck. In the later pregnancy period, the mammary gland of the GH transgenic goats were extensive than non-transgenic goats. These experiments indicated that the pcGH vector was incorporated into the transgenic goats and affected mammogenesis, which laid a solid foundation for elucidating the impact of GH on mammogenesis and lactation performance.  相似文献   
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Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, β-glucosidase/β-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of β-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-β-d-xylopyranoside (pNPX) and p-nitrophenyl-α-d-arabinofuranoside (pNPAf), suggesting it is a multifunctional β-glucosidase/β-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of β-glucosidase activity was 100× greater than β-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0–10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.  相似文献   
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Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.  相似文献   
8.
Li XZ  Oaks A 《Plant physiology》1993,102(4):1251-1257
Zea mays (cv W64A x W182E) was used to investigate the induction and turnover of nitrate reductase (NR). In our system, 5 or 10 mM KNO3 gave the best growth over a 6-d growing period. With these NO3- levels, NR reached steady-state levels after 24 h. For the turnover experiments, the seedlings were transferred to a NO3--free medium after a 24-h induction. Shoot NR was less sensitive to the removal of NO3- than root NR, which declined almost as soon as NO3- was removed when the seedlings were induced with 5 or 10 mM NO3-. With 1 mM NO3-, however, removal of NO3- from medium resulted in declines in both NR activity and NO3- in shoot and root. Although there was a delay in the degradation of NR protein relative to the loss of NR activity, this protein was not reactivated when NO3- was resupplied. These results indicate that NO3- regulates NR by influencing the de novo synthesis of the NR protein and not by a reversible activation-inactivation of that protein.  相似文献   
9.
Ab initio calculations are used to investigate the proton transfer process in bacteriorhodopsin. HN = CH2 serves as a small prototype of the Schiff base while HCOO- models its carboxylate-containing counterion and HO- the hydroxyl group of water of tyrosine, leading to the HCOO-..H+..NHCH2 and HO-..H+..NHCH2 complexes. In isolation, both complexes prefer a neutral pair configuration wherein the central proton is associated with the anion. However, the Schiff base may be protonated in the former complex, producing the HCOO-..+HNHCH2 ion pair, when there is a high degree of dielectric coupling with an external polarizable medium. Within a range of intermediate level coupling, the equilibrium position of the proton (on either the carboxylate or Schiff base) can be switched by suitable changes in the intermolecular angle. pK shift resulting from a 60 degrees reorientation are calculated to be some 5-12 pK U within the coupling range where proton transfers are possible. The energy barrier to proton transfer reinforces the ability of changes in angle and dielectric coupling to induce a proton transfer.  相似文献   
10.
The hydrolysis of [3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sacroplasmic reticulum and soluble fraction; Km and Vmax were 25.2 microM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol, indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.  相似文献   
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