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121.
Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile(170), Ser(172), and Ser(226) with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys(30), located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi. 相似文献
122.
E2F1 mediates DNA damage and apoptosis through HCF‐1 and the MLL family of histone methyltransferases 下载免费PDF全文
E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti‐oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1‐to‐S phase transition, E2F1 associates through a short DHQY sequence with the cell‐cycle regulator HCF‐1 together with the mixed‐lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF‐1‐binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF‐1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF‐1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF‐1‐binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF‐1 association with E2F1 is a regulator of E2F1‐induced apoptosis. 相似文献
123.
Martínez-Páramo S Pérez-Cerezales S Gómez-Romano F Blanco G Sánchez JA Herráez MP 《Theriogenology》2009,71(4):594-604
Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0 ± 3.32% to 77.9 ± 3.59% and 77.6 ± 6.53% to 76.6 ± 2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7 ± 0.23% to 6.0 ± 0.28%), but not those from Duerna males.After freezing/thawing, the percentage of eyed embryos drops from 66.8 ± 6.77% to 16.1 ± 3.46% and from 50 ± 8.97% to 11.5 ± 2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure.Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations. 相似文献
124.
The B30.2/SPRY domain is present in many proteins, including various members of the tripartite motif (TRIM) protein family such as TRIM5α, which mediates innate intracellular resistance to retroviruses in several primate species.
This resistance is dependent on the integrity of the B30.2 domain that evolves rapidly in primates and exhibits species-specific
anti-viral activity. TRIM22 is another positively selected TRIM gene. Particularly, the B30.2 domain shows rapid evolution in the primate lineage and recently published data indicate an
anti-viral function of TRIM22. We show here that human and rhesus TRIM22 localise to different subcellular compartments and
that this difference can be assigned to the positively selected B30.2 domain. Moreover, we could demonstrate that amino acid
changes in two variable loops (VL1 and VL3) are responsible for the different subcellular localisations. 相似文献
125.
Acetylene, Not Ethylene, Inactivates the Uptake Hydrogenase of Actinorhizal Nodules during Acetylene Reduction Assays 总被引:1,自引:0,他引:1 下载免费PDF全文
Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C2H2 and not by C2H4. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcl1 in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cell-free extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene. 相似文献
126.
Paul D. Pratt John C. Herr Raymond I. Carruthers Michael J. Pitcairn Baldo Viellgas M. Brent Kelley 《Biocontrol Science and Technology》2019,29(7):686-705
This report summarises efforts to establish Diorhabda carinulata (Desbrochers) and D. elongata (Brullé) in California for the control of invasive saltcedars (Tamarix spp.), which degrade riparian ecosystems in the western United States. Over 14,000 D. carinulata individuals were released in California among four locations between 1999 and 2002 but beetles only established at the Tinemaha Reservoir site, the most eastern release location. More than 236,000 D. elongata individuals were released between 13 sites from 2003–2009 and establishment was limited to two sites, along the Cache and Pope creeks in northwestern California. The D. carinulata population did not disperse beyond the release area despite the presence of nearby (ca. 20?km) patches of the host plant. In contrast, D. elongata spread along Cache Creek and branches of related tributaries within the same watershed at ca. 14?km per year. A survey of 122 Tamarix stands across 15 California counties revealed that neither introduced beetle colonised other host patches, including those in neighbouring watersheds. Despite exclusive use of T. parviflora for ca. 36 generations, field collected D. elongata adults demonstrated strong preferences for T. ramosissima over T. parviflora when selecting both resting and ovipositional sites in caged choice tests. The proportion of D. elongata ovipositing on T. parviflora varied over time but with no clear trend of shifting host preference despite strong selection pressure. Explanations for the limited establishment and spread of Diorhabda spp. as well as impact to the target weeds are discussed. 相似文献
127.
Sysmex UF‐1000i flow cytometer to screen urinary tract infections: the URISCAM multicentre study 下载免费PDF全文
128.
Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus latent membrane protein 2B: a new modality for vaccination 总被引:9,自引:0,他引:9 下载免费PDF全文
Ranieri E Herr W Gambotto A Olson W Rowe D Robbins PD Kierstead LS Watkins SC Gesualdo L Storkus WJ 《Journal of virology》1999,73(12):10416-10425
Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8(+) T lymphocytes from HLA-A2.1(+), EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-gamma spot formation (T-cell frequency, <0.003%). IFN-gamma ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8(+) effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies. 相似文献
129.
130.
Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography 总被引:3,自引:0,他引:3
This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine. 相似文献