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991.
Bovine anaplasmosis is a rickettsial disease of world-wide economic importance caused by Anaplasma marginale. Several major surface proteins with conserved gene sequences have been examined as potential candidates for vaccines and/or diagnostic assays. Major surface protein 1 (MSP1) is composed of polypeptides MSP1a and MSP1b. MSP1a is expressed from the single copy gene msp1 alpha and MSP1b is expressed by members of the msp1 beta multigene family. In order to determine if the msp1 genes are conserved, primers specific for msp1 alpha, msp1 beta(1), and msp1 beta(2) genes were synthesized and used to amplify msp1 sequences of A. marginale from tick cell cultures, from cattle during acute and chronic infections and from salivary glands of Dermacentor variabilis. Protein sequences of MSP1a, MSP1b(1) and MSP1b(2) were conserved during the life cycle of the parasite. No amino acid changes were observed in MSP1a. However, small variations were observed in the MSP1b(1) and MSP1b(2) protein sequences, which could be attributed to recombination, selection for sub-populations of A. marginale in the vertebrate host and/or PCR errors. Several isolate-specific sequences were also observed. Based on the information obtained in this study, the MSP1 protein appears to be fairly well conserved and a potential vaccine candidate. 相似文献
992.
993.
Xie L Pilbrough W Metallo C Zhong T Pikus L Leung J Auniņs JG Zhou W 《Biotechnology and bioengineering》2002,80(5):569-579
PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions. Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8. The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates. Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions. It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced. Adenovirus infection resulted in profound changes in cell growth and metabolism. Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection. Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH. The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study. Deviations from this optimum resulted in significant reductions of virus productivity. The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production. 相似文献
994.
Gap junctions and tumour progression 总被引:3,自引:0,他引:3
Naus CC 《Canadian journal of physiology and pharmacology》2002,80(2):136-141
Gap junctional intercellular communication has been implicated in growth control and differentiation. The mechanisms by which connexins, the gap junction proteins, act as tumor suppressors are unclear. In this review, several different mechanisms are considered. Since transformation results in a loss of the differentiated state, one mechanism by which gap junctions may control tumour progression is to promote or enhance differentiation. Processes of differentiation and growth control are mediated at the genetic level. Thus, an alternative or complimentary mechanism of tumour suppression could involve the regulation of gene expression by connexins and gap junctional coupling. Finally, gap junction channels form a conduit between cells for the exchange of ions, second messengers, and small metabolites. It is clear that the sharing of these molecules can be rather selective and may be involved in growth control processes. In this review, examples will be discussed that provide evidence for each of these mechanisms. Taken together, these findings point to a variety of mechanims by which connexins and the gap junction channels that they form may control tumour progression. 相似文献
995.
Lefeber DJ Gutiérrez Gallego R Grün CH Proietti D D'Ascenzi S Costantino P Kamerling JP Vliegenthart JF 《Carbohydrate research》2002,337(9):819-825
A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part. 相似文献
996.
To investigate structural requirements for the homophilic interaction between carbohydrates on planar model membranes, divalent derivatives with enforced proximity between the two carbohydrate epitopes (lactose, Lewis(X), and sialyl Lewis(X)) were synthesized by use of a dimeric membrane anchor as scaffold. 相似文献
997.
The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction. 相似文献
998.
The amino acid residues of human protein SRP54 which are required for binding to SRP RNA were identified by generating 40 nonoverlapping tri-alanine alterations within its methionine-rich M-domain (SRP54M). The mutant polypeptides were expressed in Escherichia coli, and their ability to bind to human and Methanococcus jannaschii SRP RNA were determined in vitro. Residues at positions 379-387, 394-396, 400-405, and 409-411 of human SRP54 were within the predicted RNA binding site, and their alteration abolished the binding activities of the mutant polypeptides as expected. Changes at positions 418-423 had intermediate effects. Polypeptides containing mutations of 328-TLR-330 were inactive although these residues were far away from the presumed RNA binding site in the crystal structure of the free protein. Using the structures of the E. coli Ffh/4.5S core and of the human SRP54m dimer as templates, a molecular model of the complex between human SRP RNA helix 8 and a single SRP54M molecule was constructed in which Leucine 329 was positioned in closer proximity to the RNA binding domain. This representation was supported by studies of the SRP54m monomer/dimer ratio using gel filtration. The results were consistent with a change in the shape of the signal peptide binding groove upon binding of SRP54 to SRP RNA. We propose that the SRP RNA and a small region centered at a bulky nonpolar amino acid residue at position 329 of protein SRP54 play a critical role in the SRP-dependent binding and release of signal peptides. 相似文献
999.
Vitrification of posterior corneal lamellae 总被引:6,自引:0,他引:6
Cryopreservation of corneas has not yet been established as a routine method. Unsatisfactory experimental results with conventional techniques prompted us to explore the possibilities of vitrification. The aim of the present study was to optimize the heat exchange between the corneal tissue and cooling medium by reducing the corneal tissue volume and using a suitable sample container. A further objective was to promote vitrification by developing a new device for rapid cooling to -140 degrees C, just below the vitrification temperature of the cryopreservation medium. Experiments were done using posterior lamellar discs from pig corneas with a diameter of 7.5 mm and a thickness of 250-350 microm. The volume of tissue to be vitrified was 88% lower with posterior corneal lamellae than with the previously used corneoscleral discs. A very thin-walled (0.05 mm), teflon-coated bag served as the sample container. Immersed in only 0.1 ml of the vitrification solution VS41a, the lamellae were cooled to a final storage temperature of -196 degrees C. After warming and organ-culturing for 24h, the endothelium was stained with trypan blue and alizarin red, to determine cell viability. Vitrification of corneal lamellae without apparent ice formation or cracking of the specimen was achieved. Despite the successful vitrification, only a maximum of 10% of the endothelial cells was vital after warming. Thus, the toxicity of the cryoprotective agents and the devitrification that occurred during the heating process require further optimization of the method. 相似文献
1000.
The objective of this study was to determine the cryobiological characteristics of canine red blood cells (RBC). These included the hydraulic conductivity (L(p)), the permeability coefficients (P(s)) of common cryoprotectant agents (CPAs), the associated reflection coefficient (sigma), the activation energies (E(a)) of L(p) and P(s) and the osmotic tolerance limits. By using a stopped-flow apparatus, the changes of fluorescence intensity emitted by intracellularly entrapped 5-carboxyfluorescein diacetate (CFDA) were recorded when cells were experiencing osmotic volume changes. After the determination of the relationship between fluorescence intensity and cell volume, cell volume changes were calculated. These volume changes were used in three-parameter fitting calculations to determine the values of L(p), P(s), and sigma for common CPAs. These volume measurements and data analyses were repeated at three different temperatures (22, 14, 7 degrees C). Using the Arrhenius equation, the activation energies of L(p) and P(s) in the presence of CPAs were determined. The osmotic tolerance limits for canine RBC were determined by measuring the percentage of free hemoglobin in NaCl solutions with various osmolalities compared to that released by RBC incubated in double distilled water. The upper and lower osmotic tolerance limits were found to be 150mOsm (1.67V(iso)) and 1200mOsm (0.45V(iso)), respectively. These parameters were then used to calculate the amount of non-permeating solute needed to keep cell volume excursions within the osmotic tolerance limits during CPA addition and removal. 相似文献