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71.
Masashi Iwamoto Koichi Watashi Senko Tsukuda Hussein Hassan Aly Masayoshi Fukasawa Akira Fujimoto Ryosuke Suzuki Hideki Aizaki Takayoshi Ito Osamu Koiwai Hiroyuki Kusuhara Takaji Wakita 《Biochemical and biophysical research communications》2014
Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection. 相似文献
72.
Andrew W. Farrell Safina GadeockAleta Pupovac Bin WangIman Jalilian Marie RansonRonald Sluyter 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor.Methods
Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA.Results
RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~ 116 μM, and this uptake was reduced in the presence of extracellular Ca2+ and Mg2+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake.Conclusions
P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.General significance
RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes. 相似文献73.
Jihye Jung Hyun Joo Park Ki-Nam Uhm Dooil Kim Hyung-Kwoun Kim 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(9):1841-1849
Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively. 相似文献
74.
Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl− replacement by Br− in active PSII. In Br− substituted samples, Cl− is effectively replaced by Br− in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. The following results have been obtained. i) The oxygen-evolving activities of the Br−-PSII samples are significantly lower than that of the Cl−-PSII samples; ii) The same S2 multiline EPR signals are observed in both Br− and Cl−-PSII samples; iii) The amplitudes of the visible light induced S1TyrZ• and S2TyrZ• EPR signals are significantly decreased after Br− substitution; the S1TyrZ• EPR signal is up-shifted about 8 G, whereas the S2TyrZ• signal is down-shifted about 12 G after Br− substitution. These results imply that the redox properties of TyrZ and spin interactions between TyrZ• and Mn-cluster could be significantly modified due to Br− substitution. It is suggested that Cl−/Br− probably coordinates to the Ca2+ ion of the Mn-cluster in active photosystem II. 相似文献
75.
Syed Ahmad 《Biochemical and biophysical research communications》2010,397(3):441-378
PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. In vivo animal and human studies have indicated that estradiol (E2) supplementation enhances serum PON1 activity. In this study, we sought to determine if E2 directly up-regulates cell-associated PON1 activity in vitro and to characterize the mechanism of regulation. In vitro E2 treatment of both the human hepatoma cell line Huh7 and normal rat hepatocytes resulted in a 2- to 3-fold increase in cell-associated PON1 catalytic activity. E2 potently induced PON1 activity with average EC50 values of 15 nM for normal hepatocytes and 68 nM for Huh7. The enhancement of PON1 activity by E2 was blocked by the estrogen receptor (ER) antagonist ICI 182,780 indicating that E2 was acting through the ER. The up-regulation of PON1 activity by E2 did not involve enhancement of PON1 mRNA or protein levels and did not promote secretion of PON1. Thus, E2 can enhance cell-associated PON1 activity in vitro without altering PON1 gene expression or protein level. Our data suggest that E2 may regulate the specific activity and/or stability of cell surface PON1. 相似文献
76.
77.
Jada SR Subur GS Matthews C Hamzah AS Lajis NH Saad MS Stevens MF Stanslas J 《Phytochemistry》2007,68(6):904-912
The plant Andrographis paniculata found throughout Southeast Asia contains Andrographolide 1, a diterpenoid lactone, which has antitumour activities against in vitro and in vivo breast cancer models. In the present study, we report on the synthesis of andrographolide derivatives, 3,19-isopropylideneandrographolide (2), 14-acetyl-3,19-isopropylideneandrographolide (3) and 14-acetylandrographolide (4), and their in vitro antitumour activities against a 2-cell line panel consisting of MCF-7 (breast cancer cell line) and HCT-116 (colon cancer cell line). Compounds 2 and 4 were also screened at the US National Cancer Institute (NCI) for their activities against a panel of 60 human cancer cell lines derived from nine cancer types. Compound 2 was found to be selective towards leukaemia and colon cancer cells, and compound 4 was selective towards leukaemia, ovarian and renal cancer cells at all the dose-response parameters. Compounds 2 and 4 showed non-specific phase of the cell cycle arrest in MCF-7 cells treated at different intervals with different concentrations. NCI's COMPARE and SOM mechanistic analyses indicated that the anticancer activities of these new class of compounds were not similar to that of standard anticancer agents, suggesting novel mechanism(s) of action. 相似文献
78.
Kelly BN Kyere S Kinde I Tang C Howard BR Robinson H Sundquist WI Summers MF Hill CP 《Journal of molecular biology》2007,373(2):355-366
The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CAN) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CAN and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly. 相似文献
79.
Interactions of phage Mu enhancer and termini that specify the assembly of a topologically unique interwrapped transpososome 总被引:1,自引:0,他引:1
The higher-order DNA-protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end-enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1-E interaction is essential to promote transpososome assembly, R3-E interaction contributes to the native topology of the transpososome, and L1-E and R2-E interactions are not required for assembly. The data on L2-E and L3-E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R-E and perhaps one L-E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions. 相似文献
80.
The biosorption of tetradecyl benzyl dimethyl ammonium chloride (C14BDMA) onto activated sludge was examined in aqueous solution with respect to the contact time, temperature and particle size. Equilibrium reached in about 2 h contact time. An decrease in the temperature increases of biosorption capacity of C14BDMA onto activated sludge, which also increases with decreasing particle size. The experimental data fit the pseudo-second-order kinetics model well. The Langmuir and Freundlich models were applied to describe equilibrium isotherms, and the equilibrium partitioning data was described well by both models. Thermodynamic data showed that C14BDMA biosorption onto activated sludge was feasible, spontaneous and exothermic. The Fourier transform infrared (FT-IR) spectrophotometry results show that both physisorption and chemisorption were involved. The measured zeta potential values and the enhanced cation concentration indicate the presence of electrostatic interactions, hydrophobic interactions and ion exchange. 相似文献