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41.
In vitro compartmentalization (IVC) was employed for the first time to select for novel bacteriophage λ integrase variants displaying significantly enhanced recombination activity on a non-cognate target DNA sequence. These variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type DNA substrate. The in vitro specificity phenotype extended to the intracellular recombination of episomal vectors in HEK293 cells. Surprisingly, mutations conferring the strongest phenotype do not occur in the λ integrase core-binding domain, which is known to interact directly with cognate target sequences. Instead, they locate to the N-terminal domain which allosterically modulates integrase activity, highlighting a previously unknown role for this domain in directing integrase specificity. The method we describe provides a robust, completely in vitro platform for the development of novel integrase reagent tools for in vitro DNA manipulation and other biotechnological applications. 相似文献
42.
In budding yeast the DNA helicase Mph1 prevents genome rearrangements during ectopic homologous recombination (HR) by suppressing the formation of crossovers (COs). Here we show that during ectopic HR repair, the anti-CO function of Mph1 is intricately associated with the mismatch repair (MMR) factor, MutSα. In particular, during HR repair using a completely homologous substrate, we reveal an MMR-independent function of MutSα in generating COs that is specifically antagonized by Mph1, but not Sgs1. In contrast, both Mph1 and MutSα are required to efficiently suppress COs in the presence of a homeologous substrate. Mph1 acts redundantly with Sgs1 in this respect since mph1Δ sgs1Δ double mutant cells pheno-copy MutSα mutants and completely fail to discriminate homologous and homeologous sequences during HR repair. However, this defect of mph1Δ sgs1Δ cells is not due to an inability to carry out MMR but rather is accompanied by elevated levels of gene conversion (GC) and bi-directional GC tracts specifically in non-crossover products. Models describing how Mph1, MutSα and Sgs1 act in concert to suppress genome rearrangements during ectopic HR repair are discussed. 相似文献
43.
Bing Zhang Zhou Cheng Su Tong Earn Tay Vincent B. C. Tan 《Journal of molecular modeling》2010,16(6):1159-1168
In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD)
simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations
showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular
mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is
the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding
of the protein–protein recognition characteristics between the kinase and its activator. 相似文献
44.
Background
Although there is a growing body of evidence showing that patients with type 2 diabetes mellitus (T2DM) have poor glycemic control in general, it is not clear whether T2DM patients with pre-existing cardiovascular diseases (CVD) are more or less likely to have good glycemic control than patients without pre-existing CVD. Our aim was to examine the degree of glycemic control among T2DM patients in Europe with and without pre-existing CVD.Methods
This is a matched cohort study based on a multi-center, observational study with retrospective medical chart reviews of T2DM patients in Spain, France, United Kingdom, Norway, Finland, Germany, and Poland. Included patients were aged >= 30 years at time of diagnosis of T2DM, had added a SU or a PPARγ agonist to failing metformin monotherapy (index date) and had pre-existing CVD (cases). A control cohort with T2DM without pre-existing CVD was identified using 1:1 propensity score matching. With difference-in-difference approach, logistic and linear regression analyses were applied to identify differences in glycemic control by CVD during the follow up period, after controlling for baseline demographics, clinical information, and concurrent anti-hyperglycemic medication use.Results
The percentage of case patients with adequate glycemic control relative to control patients during the 1st, 2nd, 3rd, and 4th years after the index date was 19.9 vs. 26.5, 16.8 vs. 26.5, 18.8 vs. 28.3, and 16.8 vs. 23.5 respectively. Cases were significantly less likely to have adequate glycemic control (odds ratio: 0.62; 95% confidence interval: 0.46-0.82) than controls after adjusting for baseline differences, secular trend, and other potential confounding covariates.Conclusions
T2DM patients with pre-existing CVD tended to have poorer glycemic control than those without pre-existing CVD, all other factors being equal. It suggests that clinicians may need to pay more attention to glycemic control among T2DM patients with CVD. 相似文献45.
Multiple fluorochrome experiments with as many fluorochromes as possible are desired for exploring the detailed structure
of bioaggregates. Spectral peak interference and other practical limitations, however, restrict the maximum number of stains
used simultaneously to three. This current study proposes a sixfold labelled scheme to stain the total cells, dead cells,
proteins, lipids, and α- and β-polysaccharides in bioaggregates. Two aerobic granule systems, the phenol-fed and the acetate-fed
granules, were utilized as the testing samples for demonstrating the use of the proposed scheme. 相似文献
46.
Bioaugmentation and coexistence of two functionally similar bacterial strains in aerobic granules 总被引:2,自引:0,他引:2
The survival of the inoculated microbial culture is critical for successful bioaugmentation but impossible to predict precisely.
As an alternative strategy, bioaugmentation of a group of microorganisms may improve reliability of bioaugmentation. This
study evaluated simultaneous bioaugmentation of two functionally similar bacterial strains in aerobic granules. The two strains,
Pandoraea sp. PG-01 and Rhodococcus erythropolis PG-03, showed high phenol degradation and growth rates in phenol medium, but they were characterized as having a poor aggregation
activity and weak bioflocculant-producing and biofilm-forming abilities. In the spatially homogeneous batch conditions, strain
PG-01 with higher growth rates outcompeted strain PG-03. However, the two strains could stably coexist in the spatially heterogeneous
conditions. Then the two strains were mixed and bioaugmented into activated sludge in two sequencing batch reactors, which
were operated with the different settling times of 5 and 30 min, respectively. Aerobic granules were developed only in the
reactor with a settling time of 5 min. Fluorescence in situ hybridization and denaturing gradient gel electrophoresis showed
that the two strains could coexist in aerobic granules but not in activated sludge. These findings suggested that the compact
structure of aerobic granules provided spatial isolation for coexistence of competitively superior and inferior strains with
similar functions. 相似文献
47.
48.
Inhibition of Fc epsilon RI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes 总被引:2,自引:0,他引:2
Melendez AJ Harnett MM Pushparaj PN Wong WS Tay HK McSharry CP Harnett W 《Nature medicine》2007,13(11):1375-1381
Atopic allergy is characterized by an increase in IgE antibodies that signal through the high-affinity Fcepsilon receptor (FcepsilonRI) to induce the release of inflammatory mediators from mast cells. For unknown reasons, the prevalence of allergic diseases has recently increased steeply in the developed world. However, this increase has not been mirrored in developing countries, even though IgE concentrations are often greatly elevated in individuals from these countries, owing to nonspecific IgE induction by universally present parasitic worms. Here we offer one explanation for this paradox based on the properties of ES-62, a molecule secreted by filarial nematodes. We found that highly purified, endotoxin-free ES-62 directly inhibits the FcepsilonRI-induced release of allergy mediators from human mast cells by selectively blocking key signal transduction events, including phospholipase D-coupled, sphingosine kinase-mediated calcium mobilization and nuclear factor-kappaB activation. ES-62 mediates these effects by forming a complex with Toll-like receptor 4, which results in the sequestration of protein kinase C-alpha (PKC-alpha). This causes caveolae/lipid raft-mediated, proteasome-independent degradation of PKC-alpha, a molecule important for the coupling of FcepsilonRI to phospholipase D and mast cell activation. We also show that ES-62 is able to protect mice from mast cell-dependent hypersensitivity in the skin and lungs, indicating that it has potential as a novel therapeutic for allergy. 相似文献
49.
Dinesh Srinivasan Kumar S Thameem Dheen Samuel Sam Wah Tay 《Cardiovascular diabetology》2007,6(1):1-14
Background
Congenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice.Methods and results
We analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos.Conclusion
It is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects. 相似文献50.
The effect of abatacept, a selective T-cell co-stimulation modulator, on vaccination has not been previously investigated.
In this open-label, single-dose, randomized, parallel-group, controlled study, the effect of a single 750 mg infusion of abatacept
on the antibody response to the intramuscular tetanus toxoid vaccine (primarily a memory response to a T-cell-dependent peptide
antigen) and the intramuscular 23-valent pneumococcal vaccine (a less T-cell-dependent response to a polysaccharide antigen)
was measured in 80 normal healthy volunteers. Subjects were uniformly randomized to receive one of four treatments: Group
A (control group), subjects received vaccines on day 1 only; Group B, subjects received vaccines 2 weeks before abatacept;
Group C, subjects received vaccines 2 weeks after abatacept; and Group D, subjects received vaccines 8 weeks after abatacept.
Anti-tetanus and anti-pneumococcal (Danish serotypes 2, 6B, 8, 9V, 14, 19F and 23F) antibody titers were measured 14 and 28
days after vaccination. While there were no statistically significant differences between the dosing groups, geometric mean
titers following tetanus or pneumococcal vaccination were generally lower in subjects who were vaccinated 2 weeks after receiving
abatacept, compared with control subjects. A positive response (defined as a twofold increase in antibody titer from baseline)
to tetanus vaccination at 28 days was seen, however, in ≥ 60% of subjects across all treatment groups versus 75% of control
subjects. Similarly, over 70% of abatacept-treated subjects versus all control subjects (100%) responded to at least three
pneumococcal serotypes, and approximately 25–30% of abatacept-treated subjects versus 45% of control subjects responded to
at least six serotypes. 相似文献