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51.
Magdalena Kanabus Adrianna Nowicka Ewa Sledziewska-Gójska Piotr Jonczyk Zygmunt Ciesla 《Molecular & general genetics : MGG》1995,247(2):216-221
It has previously been suggested that inhibition of the proofreading 3-5 exonuclease activity of DNA polymerase may play an important role in generation of UV-induced mutations inEscherichia coli. Our previous work showing that overproduction of , the proofreading subunit of DNA polymerase III, counteracts the SOS mutagenic response ofE. coli seemed to be consistent with this hypothesis. To explore further the nature of the antimutagenic effect of we constructed plasmid pMK17, which encodes only two of the three highly conserved segments of — Exol and ExoII; the third segment, ExoIII, which is essential for 3–5 exonuclease activity, is deleted. We show that at 40°C, over-production of the truncated e subunit significantly delays production of M13 phage, suggesting that the protein retains its capacity to bind to DNA. On the other hand, the presence of pMK17 in atrpE65 strain growing at 40°C causes a 10-fold decrease in the frequency of UV-induced Trp+ mutations. This antimutagenic effect of the truncated s is effectively relieved by excess UmuD,C proteins. We also show that the presence of plasmid pIP21, which contains thednaQ49 allele encoding an subunit that is defective in proofreading activity, almost completely prevents generation of UV-induced mutations in thetrpE65 strain. We propose that the DNA binding ability of free , rather than its 3–5 exonuclease activity, affects processing of premutagenic UV-induced lesions, possibly by interfering with the interaction between the UmuC-UmuD-RecA complex and Pol III holoenzyme. This interaction is probably a necessary condition for translesion synthesis. 相似文献
52.
Protein phosphorylation was investigated in [32P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both 1- and 2-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the 2-AR only marginally increased while the stimulation of 1-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M2-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated Gi-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of 1-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported. 相似文献
53.
Subunit interaction in extracellular superoxide dismutase: effects of mutations in the N-terminal domain. 总被引:1,自引:0,他引:1
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P. Stenlund D. Andersson L. A. Tibell 《Protein science : a publication of the Protein Society》1997,6(11):2350-2358
Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a well-defined three-dimensional structure, which probably contains alpha-helical elements and is responsible for the tetramerization of the protein. In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic alpha-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction. 相似文献
54.
Francesco Frati Chris Simon Jack Sullivan David L. Swofford 《Journal of molecular evolution》1997,44(2):145-158
The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic
levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic
relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all
insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the
basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions
appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in
some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels
observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or
exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity
of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information
was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical
support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well
supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from
an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data.
Received: 12 January 1996 / Accepted: 10 August 1996 相似文献
55.
Rika Morishita Shinsuke Saga Noriko Kawamura †Yoshio Hashizume ‡Toshiaki Inagaki Kanefusa Kato Tomiko Asano 《Journal of neurochemistry》1997,68(2):820-827
Abstract: The localization of two forms of the γ subunit of G proteins, γ3 and γ12, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ3 tended to decrease with age, whereas that of γ12 in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ3 was abundant in the neuropil, whereas γ12 was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ12-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ12-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ12 is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ12 was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ3 in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ3 and γ12 are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ3 and γ12 in the brain are related to the numbers and/or extent of maturation of these cells. 相似文献
56.
Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus 总被引:3,自引:0,他引:3
Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. 相似文献
57.
58.
Urea-induced dissociation and unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric and spectral studies. 总被引:1,自引:1,他引:0
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M. Zolkiewski N. J. Nosworthy A. Ginsburg 《Protein science : a publication of the Protein Society》1995,4(8):1544-1552
Urea-induced dissociation and unfolding of manganese.glutamine synthetase (Mn.GS) have been studied at 37 degrees C (pH 7) by spectroscopic and calorimetric methods. In 0 to approximately 2 M urea, Mn.GS retains its dodecameric structure and full catalytic activity. Mn.GS is dissociated into subunits in 6 M urea, as evidenced by a 12-fold decrease in 90 degrees light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies. The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra-ring and inter-ring subunit contacts, respectively, in the layered hexagonal structure of Mn.GS. The enthalpies for transferring Mn.GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn.GS is 14 +/- 4 cal/g. A net proton uptake of approximately 50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn.GS and the number of binding sites increasing approximately 9-fold during the protein unfolding. 相似文献
59.
Crystallization of alpha 1-acid glycoprotein 总被引:1,自引:0,他引:1
A McPherson M L Friedman H B Halsall 《Biochemical and biophysical research communications》1984,124(2):619-624
A possible link between cellular cyclic AMP content and Na+K+ATPase activity was investigated in homogenates of rat kidney. Enzyme kinetics of Mg2+ and Na+K+ATPase were run in the presence of cyclic AMP, dibutyryl cAMP and compounds expected to elevate cyclic AMP levels such as forskolin, a potent adenylate cyclase activator, IBMX, an inhibitor of phosphodiesterases, and the beta-agonist isoproterenol. Medullary Na+K+ATPase is strongly inhibited by cyclic AMP whereas cortical Na+K+ATPase was stimulated in the same conditions. The correlation between ATPase activity and cellular cyclic AMP content supports the concept of a possible regulation of the enzyme by cyclic AMP. 相似文献
60.
Stimulation of phospholipase A2 activity by high osmotic pressure on cholesterol-containing phospholipid vesicles 总被引:1,自引:0,他引:1
Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 10(7) M-1 at 4 degrees C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography. 相似文献