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111.
Manuel Porcar Anne-Marie Grenier Brian Federici Yvan Rahbé 《Applied and environmental microbiology》2009,75(14):4897-4900
Four Bacillus thuringiensis δ-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive buffer-based controls.Many species of aphids are important sucking-insect pests that feed on plant vascular fluids. Their feeding mechanism makes these insects excellent vectors for many plant pathogens, especially viruses, yet less amenable to standard, nonsystemic chemical control by insecticides. Minor effects on the survival and fecundity of aphids reared on Bacillus thuringiensis (Bt) crops have been noted in some studies but not in others (1, 3, 6). However, the sensitivity of aphids to Bt toxins, or the lack thereof, has not been previously tested through artificial-diet bioassays with exhaustive buffer-based controls.Bt δ-endotoxins Cyt1A, Cry4A/Cry4B, and Cry11, obtained from three recombinant strains of B. thuringiensis subsp. israelensis, as well as Cry1Ab and Cry3A, obtained from recombinant Escherichia coli, were purified by ultracentrifugation in a discontinuous sucrose gradient as described previously (9). Cry proteins were solubilized in solubilization buffer (50 mM Na2CO3, 100 mM NaCl, pH 10) with dithiothreitol (10 mM) added before use. Cyt1A was first solubilized on 10 mM Na2CO3 (pH 11) buffer and then neutralized at pH 7.5 to 8 with 10 μl HCl (1 N). Both solubilized and trypsin-digested samples (1:30 over toxin weight) were used at different concentrations (32, 125, and 500 μg/ml; trypsin-activated toxin concentrations were calculated on the basis of the preactivation concentrations of the protoxins) to supplement the AP3 aphid synthetic diet (7) used to feed Acyrthosiphon pisum (LL01 green clone). Ampicillin (100 μg/ml), an ineffective antibiotic for A. pisum or its obligate symbiont Buchnera, was added to the medium to avoid bacterial growth. For each concentration, 30 nymphs (10 nymphs/box and three repetitions) were bioassayed at 20°C and under a 16:8 (light-dark) photoperiod. Survival time was calculated from aphid deposition on the test diet (day 0). Mortality was surveyed daily, and body weights of survivors were noted at day 7. ST50 (median survival time after challenge) was calculated by using an actuarial survival analysis (Statview) with censoring values of survivors at the end of the experiments. The approximate concentrations resulting in a 50% decrease in mean body weight (IC50) and killing of 50% of the insects tested (LC50) were calculated at the end of the experiments from the growth reduction and mortality data, respectively, derived with the three doses by using Statview and the censoring values of survivors.All of the Cry δ-endotoxins tested were lethal to A. pisum and retarded the growth of survivors (Fig. (Fig.11 and and2).2). Mortalities ranged from only 25% (Cry1Ab) to 100% (Cry4 and Cry11) after 3 to 6 days of exposure to 500 μg/ml of solubilized protein (Fig. (Fig.1).1). When significant mortalities were achieved (Cry3A, Cry4, and Cry11), trypsin activation enhanced toxicity. Activation of Cry4 at the intermediate concentration tested (125 μg/ml) resulted in a twofold increase in mortality (Fig. (Fig.1D).1D). ST50s were calculated for both solubilized protoxins and activated Cry3A, Cry4, and Cry11. The ST50s (at 500 μg/ml) ranged from 1.8 ± 0.14 days for solubilized Cry4 and Cry11 to 3.7 ± 1.2 days for trypsin-activated Cry3A (Table (Table1).1). Control aphids fed buffer all survived for >8 days. The LC50 of Cry1Ab was not calculated, since mortality associated with Cry1Ab reached a plateau at 500 μg/ml. The LC50 of Cry4 was estimated to be 70 to 100 μg/ml (data not shown).Open in a separate windowFIG. 1.Mortality assays over the nymphal life stage of the pea aphid, A. pisum, upon ingestion of artificial diets containing purified Bt toxins after either solubilization (open symbols) or solubilization and trypsin activation (solid symbols). The toxins used were Cry1Ab (circles), Cry3A (squares), a mixture of Cry4A and Cry4B (diamonds), and Cry11A (triangles). The soluble-toxin doses used were low at 32 μg/ml (blue), intermediate at 125 μg/ml (violet), and high at 500 μg/ml (red). Assays were carried out with 30 initial neonate insects in three batches of 10 individuals.Open in a separate windowFIG. 2.Growth inhibition assays with purified Bt toxins Cry3A, Cry4, and Cry 11 (A) and Cry1Ab and Cyt1A (B) on the pea aphid, A. pisum. Toxins were added to the diet either after solubilization (open symbols) or after solubilization and trypsin activation (solid symbols). Error bars show the standard errors (SE) of individual weights at day 7 of experiments, standardized by the control group mean weight (toxin dose, 0; initial number, 30). Color coding of toxins: Cry3A, red squares; Cry4A and Cry4B mixture, violet diamonds; Cry11A, blue triangles; Cry1Ab, green circles; Cyt1A, yellow squares. In the experiment with Cry1Ab (B), the toxin was purified by high-performance liquid chromatography and activated toxin was provided as a salt-free lyophilisate by W. Moar (Auburn University, Auburn, AL).
Open in a separate windowaNL, nonlethal; >8, survival for >8 days.Aphids that survived ingestion of the Cry and Cyt proteins in the bioassays had markedly reduced growth rates compared to those of the control group (Fig. (Fig.2).2). Growth inhibition by each Cry protein correlated with mortality. Cry4 inhibited growth the most (Fig. (Fig.2A),2A), whereas Cry1Ab inhibited growth the least (Fig. (Fig.2B).2B). The IC50 of Cry4 was calculated to be 135 μg/ml. The growth of aphids surviving Cyt1A ingestion was strongly inhibited, with an average weight at the end of the assay, for doses of 125 μg/ml or higher, corresponding to less than 40% of that of the control group (Fig. (Fig.2B).2B). This decrease in aphid weight associated with the ingestion of Cyt1A is in contrast to the low mortality (about 10%) produced by the same dose of this protein. Most of the surviving insects did not reach adulthood as a result of feeding on Cyt1A, whereas control insects completed their nymphal development by the end of the bioassay.Cofeeding experiments with a mixture of toxins (Cry and Cyt1A) currently under way suggest that there is no identifiable synergy between Cry and Cyt toxins in this model, at least in the concentration range of 32 to 500 μg/ml (A.-M. Grenier et al., unpublished data).In two previous studies (10, 11), sensitivity of another aphid, Macrosiphum euphorbiae, to suspensions of Cry2, Cry3A, and Cry4 crystals was reported but no sensitivity to solubilized endotoxins was found. This may be explained by the lack of complete solubilization of the Bt crystals (10) and by the fact that control groups were fed a water-based artificial diet instead of a diet containing the buffer used to solubilize the crystals. Our bioassays, performed with buffer-based controls, show that A. pisum is indeed sensitive to Bt δ-endotoxins, although to a low degree. In fact, the IC50s and LC50s we calculated are very high compared to those of highly susceptible targets of B. thuringiensis (http://www.glfc.forestry.ca/bacillus/) but similar to those of organisms with low sensitivity, such as nematodes. For example, in feeding bioassays in which growth inhibition was measured against Caenorhabditis elegans fed E. coli/Cry strains, IC50s ranged from 16 μg/ml for Cry14A to as high as 230 μg/ml for Cry6A (12). The low activity of Bt endotoxins against aphids suggests that these proteins have not evolved to kill aphids. In fact, the ecological niches of B. thuringiensis and these insects are very different and it is unlikely that aphids, feeding on a virtually germfree environment such as plant phloem, come in contact with bacteria living either in other susceptible insects or on the plant surface. It might be hypothesized that the sensitivity of pea aphids to these Bt endotoxins is a consequence of similarities among midgut microvillar proteins and lipids, especially the surface molecules that compose the sugar residues known to serve as the initial binding sites for Bt toxins (4), rather than a result of direct selection for aphid sensitivity.The low sensitivity of aphids to Bt toxins is not in contrast to recent reports on the lack of deleterious effects of genetically modified crops on aphid populations (5). A recent report confirms the presence of Cry1Ac in the phloem of transgenic oilseed rape and in aphids feeding on these plants (2). However, the concentration of Cry1Ac in phloem, being low, is compatible with the absence of deleterious effects of transgenic oilseed rape on aphids, as well as with previous studies reporting no detectable levels of Cry toxins in phloem translocated through sieves of commercial transgenic plants (8). Although low, the susceptibility of aphids to B. thuringiensis we report here could theoretically lead to the development of effective strategies for controlling these and other sucking insect pests with genetically modified crops expressing appropriate toxins. However, two conditions should concur. (i) Toxins must be present in the plant phloem to be accessible to these pests and vectors, and (ii) more effective toxins should be found, and thus screening programs with a range of natural and engineered toxins should be performed in order to determine their activity on sucking insects. Although a wide range of further studies are still needed to assess the potential of Bt crops for controlling aphids and other sucking insect pests, the substantial economic losses sucking insects cause to agriculture worldwide clearly merit exploration of the possibilities our results suggest. 相似文献
TABLE 1.
ST50s of pea aphids feeding on solubilized Cry toxins and solubilized Cry toxins activated with trypsinToxin | Mean ST50a ± SE (days) at dose of:
| ||
---|---|---|---|
32 mg/ml | 125 mg/ml | 500 mg/ml | |
Cry1Ab | |||
Solubilized | NL | >8 | >8 |
Trypsinized | NL | >8 | >8 |
Cry3A | |||
Solubilized | NL | >8 | >8 |
Trypsinized | NL | >8 | 3.7 ± 1.2 |
Cry4A | |||
Solubilized | NL | >8 | 1.8 ± 0.14 |
Trypsinized | >8 | 1.8 ± 0.15 | 1.9 ± 0.17 |
Cry11A | |||
Solubilized | NL | >8 | 1.8 ± 0.14 |
Trypsinized | NL | >8 | 2.5 ± 0.10 |
112.
Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential
metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this
in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as
organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as
siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe,
Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation
of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high
concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores
extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component
of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment
rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem. 相似文献
113.
Yvan Zivanovic Jean Armengaud Arnaud Lagorce Christophe Leplat Philippe Guérin Murielle Dutertre Véronique Anthouard Patrick Forterre Patrick Wincker Fabrice Confalonieri 《Genome biology》2009,10(6):R70-23
Background
Thermococcus gammatolerans was isolated from samples collected from hydrothermal chimneys. It is one of the most radioresistant organisms known amongst the Archaea. We report the determination and annotation of its complete genome sequence, its comparison with other Thermococcales genomes, and a proteomic analysis. 相似文献114.
Arnaud Augert Christine Payré Yvan de Launoit Jesus Gil Gérard Lambeau David Bernard 《EMBO reports》2009,10(3):271-277
Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway. 相似文献
115.
Sophie Reuse Miriam Calao Kabamba Kabeya Allan Guiguen Jean-Stéphane Gatot Vincent Quivy Caroline Vanhulle Aurélia Lamine Dolores Vaira Dominique Demonte Valérie Martinelli Emmanuelle Veithen Thomas Cherrier Véronique Avettand Solène Poutrel Jacques Piette Yvan de Launoit Michel Moutschen Arsène Burny Christine Rouzioux Stéphane De Wit Georges Herbein Olivier Rohr Yves Collette Olivier Lambotte Nathan Clumeck Carine Van Lint 《PloS one》2009,4(6)
116.
117.
Nicole M Gerardo Boran Altincicek Caroline Anselme Hagop Atamian Seth M Barribeau de Martin Vos Elizabeth J Duncan Jay D Evans Toni Gabaldón Murad Ghanim Adelaziz Heddi Isgouhi Kaloshian Amparo Latorre Andres Moya Atsushi Nakabachi Benjamin J Parker Vincente Pérez-Brocal Miguel Pignatelli Yvan Rahbé John S Ramsey Chelsea J Spragg Javier Tamames Daniel Tamarit Cecilia Tamborindeguy Caroline Vincent-Monegat Andreas Vilcinskas 《Genome biology》2010,11(2):1-17
118.
Mortier E Quéméner A Vusio P Lorenzen I Boublik Y Grötzinger J Plet A Jacques Y 《The Journal of biological chemistry》2006,281(3):1612-1619
Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets. 相似文献
119.
Background
A significant proportion of myocardial infarction (MI) patients undergo complex, coordinated perturbations at the molecular level that may eventually drive the occurrence of ventricular dysfunction and heart failure. Despite advances in the elucidation of key processes implicated in this condition, traditional methods relying on gene expression data and the identification of individual biomarkers in isolation pose major limitations not only for improving prediction power, but also for model interpretability. Mechanisms underlying clinical responses after MI remain elusive and there is no biomarker with the capacity to accurately predict ventricular dysfunction after MI. This calls for the exploration of system-level modeling of ventricular dysfunction in post-MI patients. Within this discovery framework key perturbations and predictive patterns are characterized by the integrated biological activity levels observed in pathways, rather than in individual genes.Methodology/Principal Findings
Here we report an integrative approach to identifying pathways related with ventricular dysfunction post MI with potential prognostic and therapeutic value. We found that a diversity of pathway-level perturbations can be profiled in samples of patients with ventricular dysfunction post MI, most of which represent major reductions of gene expression. Highly perturbed pathways included those implicated in antigen-dependent B-cell activation and the synthesis of leucine. By analyzing patient-specific samples encoded with information derived from highly-perturbed pathways, it is possible to visualize differential prognostic patterns and to perform computational classification of patients with areas under the receiver operating characteristic curve above 0.75. We also demonstrate how the integration of the outcomes generated by different pathway-based analysis models may improve ventricular dysfunction prediction performance.Significance
This research offers an alternative, comprehensive view of key relationships and perturbations that may trigger the emergence or prevention of ventricular dysfunction post-MI. 相似文献120.
Darcy L. McRose Oliver Baars François M. M. Morel Anne M. L. Kraepiel 《Environmental microbiology》2017,19(9):3595-3605
Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co‐factors Mo and V. However, regulation of siderophore production by Mo‐ and V‐limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N2‐fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe‐limitation the production of all siderophores increases, while under Mo‐limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo‐ and V‐limitation. An examination of the potential nitrogen ‘cost’ of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo‐ as opposed to the less efficient V‐nitrogenase. 相似文献