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321.
322.
E A Evans 《Biophysical journal》1980,31(3):425-431
An experimental procedure that can be used to measure the interfacial free energy density for the adhesion of membranes of large vesicles to other surfaces is outlined and analyzed. The approach can be used for both large phospholipid bilayer vesicles and red blood cells when the membrane force resultants are dominated by isotropic tension. The large vesicle or red cell is aspirated by a micropipet with sufficient suction pressure to form a spherical segment outside the pipet. The vesicle is then brought into close proximity of the surface to be tested and, the suction pressure reduced to permit adhesion, and the new equilibrium configuration is established. The mechanical analysis of the equilibrium shape provides the interfacial free energy density for the surface affinity. With this approach, the measurable range of membrane surface affinity is 10(-4)-3 erg/cm2 for large phospholipid bilayer vesicles and 10(-2)-10 erg/cm2 for red blood cells. 相似文献
323.
J D Shore S A Evans J J Holbrook D M Parker 《The Journal of biological chemistry》1979,254(18):9059-9062
The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms. 相似文献
324.
Temperature-Sensitive Mutants of Complementation Group E of Vesicular Stomatitis Virus New Jersey Serotype Possess Altered NS Polypeptides
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In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2, and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with (32)P(i) to approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. We propose a model in which the NS polypeptide consists of at least three loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and thus the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide. 相似文献
325.
Spleen focus-forming Friend virus: identification of genomic RNA and its relationship to helper virus RNA.
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The genome of the defective, murine spleen focus-forming Friend virus (SFFV) was identified as a 50S RNA complex consisting of 32S RNA monomers. Electrophoretic mobility and the molecular weights of unique RNase T1-resistant oligonucleotides (T1-oligonucleotides) indicated that the 32S RNA had a complexity of about 7.4 kilobases. Hybridization with DNA complementary to Friend murine leukemia virus (Fr-MLV) has distinguished two sets of nucleotide sequences in 32S SFFV RNA, 74% which were Fr-MLV related and 26% which were SFFV specific. By the same method, SFFV RNA was 48% related to Moloney MLV. We have resolved 23 large T1-oligonucleotides of SFFV RNA and 43 of Fr-MLV RNA. On the basis of the relationship between SFFV and Fr-MLV RNAs, the 23 SFFV oligonucleotides fell into four classes: (i) seven which had homologous equivalents in Fr-MLV RNA; (ii) six more which could be isolated from SFFV RNA-Fr-MLV cDNA hybrids treated with RNases A and T1; (iii) eight more which were isolated from hybrids treated with RNases A and T1; and (iv) two which did not have Fr-MLV-related counterparts. Surprisingly, the two class iv oligonucleotides had homologous counterparts in the RNA of six amphotropic MLV's including mink cell focus-forming and HIX-MLVs analyzed previously. The map locations of the 23 SFFV T1-oligonucleotides relative to the 3' polyadenylic acid coordinate of SFFV RNA were deduced from the size of the smallest polyadenylic acid-tagged RNA fragment from which a given oligonucleotide was isolated. The resulting oligonucleotide map could be divided roughly into three segments: two terminal segments which are mosaics of oligonucleotides of classes i, ii, and iii and an internal segment between 2 and 2.5 kilobases from the 3' end containing the two oligonucleotides shared with amphotropic MLVs. Since SFFV RNA consists predominantly of sequence elements related to ecotropic and amphotropic helper-independent MLVs, it would appear that the transforming gene of SFFV is not a major specific sequence unrelated to genes of helper viruses, as is the case with Rous sarcoma and probably withe other defective sarcoma and acute leukemia viruses. 相似文献
326.
Summary Millet plants (Pennisetum glaucum) were grown at three levels of nitrogen fertilization with and without an inoculum of live nitrogen-fixing Azospirillum cells. The highest average rate of nitrogen fixation as estimated from acetylene reduction by excised preincubated roots was only 23g N2 fixed per ha per day and occurred after treatment with low levels of nitrogen amendment. The average rates of acetylene reduction for intact plants at all treatments were also low. The lack of significant nitrogen fixation due to an Azospirillum-millet association in this study was substantiated by plant dry weight analysis, and determination of the nitrogen content of plants, pot leachate, and soil. There was significant correlation between the total nitrogen content of the plants per pot at the termination of the experiment and the amount of nitrogen fertilizer added initially, but there was no effect of inoculum on final total nitrogen content. 相似文献
327.
I. Marta Evans Ronald R. D. Croy Philippa Hutchinson Donald Boulter Peter I. Payne Margaret E. Gordon 《Planta》1979,144(5):455-462
Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.Abbreviations DTT
dithiothreitol
- SDS-PAGE
SDS-polyacrylamide gel electrophoresis
- TCA
tricarboxylic acid 相似文献
328.
329.
Protoplasts have been obtained from vegetative thallus of the green seaweed Enteromorpha following enzymic digestion with driselase and pectinase. The viability of purified protoplast fractions was assessed by staining and measurements of O2 uptake and evolution.Abbreviations MES
2-(N-morpholino) ethanesulphonic acid
- TES
N-tris(hydoxymethyl) methyl-2 aminoethanesulphonic acid 相似文献
330.