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51.
52.
RNA aptamers specifically interact with the prion protein PrP.   总被引:9,自引:0,他引:9       下载免费PDF全文
We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.  相似文献   
53.
Nuclear import and export of influenza virus nucleoprotein.   总被引:11,自引:4,他引:7       下载免费PDF全文
Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.  相似文献   
54.
The glycopeptides 1 and 2 ), carrying the core structure of serine-linked cell-surface proteoglycans were synthesized in a stereocontrolled manner. The carbohydrate key imidate xylosyl donors 3 and glycotetraosyl donors 4 and 5, as well as a tetrapeptide glycosyl acceptor 6, were coupled in the crucial glycosylation step. In these reactions, the application of either trimethylsilyl trifluoromethanesulfonate (TMSOTf) or borontrifluoride etherate (BF3-Et2O) as catalysts proved to be highly efficient. The serine linked glycopeptides 34, 36 and 37 thus obtained yielded target compounds 1 and 2 on complete deprotection.  相似文献   
55.
Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectinmediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occured (pH ? 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.  相似文献   
56.
The conformation of d(A-T-G-G) and d(A-T-G-G)cisPt has been investigated by 1H-NMR at 500 MHz and 90 MHz under various experimental conditions of temperature and concentration. Analysis of the coupling constants between the deoxyribose protons shows that all the sugar rings of d(A-T-G-G) adopt the S(C2'-endo) conformation most of the time. By contrast, in the platinated tetramer, d(A-T-G-G)cisPt, the N(C3'-endo) conformation is highly predominant for the internal dG residue while the S(C2'-endo) conformation is largely favoured for the other residues as in the case of the unplatinated compound. The relaxation time and nuclear Overhauser effect measurements indicate that the orientation of the two guanines of d(A-T-G-G)cisPt is anti in agreement with the previous results obtained for the dimers: r(G-G)cisPt, d(G-G)cisPt. On lowering the temperature from 80 degrees C to 20 degrees C, several proton resonances of d(A-T-G-G)cisPt exhibit large chemical shift and linewidth variations. The most spectacular temperature effect was observed for the internal dG(H1') and dT(H4') protons. All the delta = f(t) curves display a sigmoid form with the same mid-point temperature of 44 +/- 2 degrees C. This mid-point temperature together with the observed chemical shift and linewidth variations were found to be independent of the d(A-T-G-G)cisPt concentration. These results suggest that d(A-T-G-G)cisPt can adopt two different conformations depending on the temperature. The enthalpy for the transition between the high and low temperature conformations is about 84 kJ/mol.  相似文献   
57.
The magnetic shielding produced by the double helix in a B-DNA and a Z-DNA conformation is calculated for each non exchangeable proton of the oligodeoxynucleotides d(CGm5CG)2 and d(CGm5CGCG)2. The differences between the values obtained for the two helical forms are in good agreement with the variations of chemical shift measured when the salt concentration of the solution is changed from 0.1 M to 2 or 4 M. The analysis of the theoretical chemical shift variations shows that the large upfield shift observed for some of the protons of the cytidine residues is due to the sum of the ring current and local magnetic anisotropy effects of the guanines of the two nearest neighbours residues.  相似文献   
58.
Marked differences in the AChE activity of myelinated nerve fibers of ventral and dorsal roots could be established in human post mortem material. After a fixation time of 3 h and a critical incubation period of 24 h, in the mean 96% of the myelinated ventral root but only 4% of dorsal root fibers showed reaction product, detectable by the light microscope. The percentage of stained fibres varies, to some extent, in the different segments. Groups of very thin myelinated fibres within the ventral roots between the segments C-8 and L-3, showing a conspicuous high enzyme activity, are interpreted as pre-ganglionic sympathetic fibres; similar elements in the sacral ventral roots may represent parasympathetic fibres. The method of Karnovsky, applied under conditions established in this study, can be used for analysis of fibre types in a given human peripheral nerve.  相似文献   
59.
Chromatin subunits from murine erythroleukemia cells were prepared by a method which releases actively transcribing genes. Two casein kinase activities (CK1 and CK2) were isolated from these nucleosomes by gel nitration in 0.5 m NaCl. CK1 (Mr ~ 200,000) and CK2 (Mr ~ 35,000) were further purified by phosphocellulose chromatography and characterized with regard to several parameters which may regulate their activity in vivo. CK1 has an NaCl optimum of 0.14 m, utilizes GTP as phosphate donor ~25% as efficiently as ATP, and phosphorylates a discrete group of high molecular weight nonhistone proteins in the unfractionated chromatin starting material. CK2 has an NaCl optimum of 0.24 m, cannot utilize GTP, and modifies a different group of nonhistones. Both kinases are inhibited by concentrations of hemin (<50 μm) which efficiently induce globin gene expression in erythroleukemia cells. A histone kinase resolved during the gel filtration step is unaffected by hemin. An investigation of the mode of hemin inhibition reveals that CK1 and CK2 interact in different fashions with the inhibitor.  相似文献   
60.
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