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31.
32.
Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma
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Yuste E Sanford HB Carmody J Bixby J Little S Zwick MB Greenough T Burton DR Richman DD Desrosiers RC Johnson WE 《Journal of virology》2006,80(6):3030-3041
To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies. 相似文献
33.
W E Jack L Greenough L F Dorner S Y Xu T Strzelecka A K Aggarwal I Schildkraut 《Nucleic acids research》1991,19(8):1825-1829
The type II restriction endonuclease BamHI has been expressed in E. coli, producing 100-fold more enzyme than the wild type Bacillus amyloliquefaciens H strain. This high yield has facilitated purification to homogeneity of large amounts of the enzyme, along with its crystallization in a form which diffracts to at least 1.9 A in X-ray analysis. 相似文献
34.
The activity and diurnal variation of 3-hydroxy-3-methyglutaryl-CoA reductase (EC 1.1.1.34; HMG-CoA reductase), the rate-limiting enzyme in the cholesterol-biosynthetic pathway, of normal and dystrophic hamsters was determined. Liver enzyme activity showed a diurnal pattern in the normal male, but not in the dystrophic male. Enzyme values in normal males at the midpoint of the 12 h dark period were 10 times those in dystrophic males. No evidence for diurnal variation in the HMG-CoA reductase of the brain was observed, and similar activities were found for normal and dystrophic animals. The apparent Km for HMG-CoA reductase from the liver of normal or dystrophic hamsters was approx. 9 microM, and the Vmax. was 5.9 and 21.7 pmol/min per mg of protein for dystrophic and normal hamsters respectively. 相似文献
35.
36.
Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2 总被引:11,自引:0,他引:11
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Li W Greenough TC Moore MJ Vasilieva N Somasundaran M Sullivan JL Farzan M Choe H 《Journal of virology》2004,78(20):11429-11433
Replication of viruses in species other than their natural hosts is frequently limited by entry and postentry barriers. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV) utilizes the receptor angiotensin-converting enzyme 2 (ACE2) to infect cells. Here we compare human, mouse, and rat ACE2 molecules for their ability to serve as receptors for SARS-CoV. We found that, compared to human ACE2, murine ACE2 less efficiently bound the S1 domain of SARS-CoV and supported less-efficient S protein-mediated infection. Rat ACE2 was even less efficient, at near background levels for both activities. Murine 3T3 cells expressing human ACE2 supported SARS-CoV replication, whereas replication was less than 10% as efficient in the same cells expressing murine ACE2. These data imply that a mouse transgenically expressing human ACE2 may be a useful animal model of SARS. 相似文献
37.
38.
C. J. McNeil Katharine R. Greenough P. A. Weeks C. H. Self J. M. Cooper 《Free radical research》1992,17(6):399-406
Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised. 相似文献
39.
Carlum Shiu Coleen K. Cunningham Thomas Greenough Petronella Muresan Victor Sanchez-Merino Vincent Carey J. Brooks Jackson Carrie Ziemniak Lawrence Fox Marvin Belzer Stuart C. Ray Katherine Luzuriaga Deborah Persaud and the Pediatric AIDS Clinical Trials Group P Team 《Journal of virology》2009,83(19):9731-9742
40.
Gabriel Castrillo Eduardo Sánchez-Bermejo Laura de Lorenzo Pedro Crevillén Ana Fraile-Escanciano Mohan TC Alfonso Mouriz Pablo Catarecha Juan Sobrino-Plata Sanna Olsson Yolanda Leo del Puerto Isabel Mateos Enrique Rojo Luis E. Hernández Jose A. Jarillo Manuel Pi?eiro Javier Paz-Ares Antonio Leyva 《The Plant cell》2013,25(8):2944-2957