全文获取类型
收费全文 | 17117篇 |
免费 | 1649篇 |
国内免费 | 2篇 |
专业分类
18768篇 |
出版年
2023年 | 56篇 |
2022年 | 161篇 |
2021年 | 320篇 |
2020年 | 159篇 |
2019年 | 238篇 |
2018年 | 301篇 |
2017年 | 267篇 |
2016年 | 541篇 |
2015年 | 845篇 |
2014年 | 908篇 |
2013年 | 1096篇 |
2012年 | 1360篇 |
2011年 | 1244篇 |
2010年 | 789篇 |
2009年 | 788篇 |
2008年 | 999篇 |
2007年 | 1013篇 |
2006年 | 921篇 |
2005年 | 903篇 |
2004年 | 861篇 |
2003年 | 849篇 |
2002年 | 805篇 |
2001年 | 187篇 |
2000年 | 136篇 |
1999年 | 203篇 |
1998年 | 217篇 |
1997年 | 177篇 |
1996年 | 140篇 |
1995年 | 153篇 |
1994年 | 139篇 |
1993年 | 141篇 |
1992年 | 147篇 |
1991年 | 114篇 |
1990年 | 116篇 |
1989年 | 92篇 |
1988年 | 94篇 |
1987年 | 91篇 |
1986年 | 84篇 |
1985年 | 102篇 |
1984年 | 111篇 |
1983年 | 63篇 |
1982年 | 93篇 |
1981年 | 70篇 |
1980年 | 73篇 |
1979年 | 55篇 |
1977年 | 52篇 |
1976年 | 41篇 |
1975年 | 43篇 |
1974年 | 49篇 |
1972年 | 40篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
101.
102.
Thuesen MH Nørgaard A Hansen AM Caspersen MB Christensen HE 《Protein expression and purification》2003,27(1):175-181
The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV-vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c(4). Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c(4) were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure. 相似文献
103.
Michel U Gerber J E O'Connor A Bunkowski S Brück W Nau R Phillips DJ 《Journal of neurochemistry》2003,86(1):238-245
Activin, a member of the transforming growth factor superfamily, is upregulated in a number of inflammatory episodes such as septicemia and rheumatoid arthritis. In the CNS, activin has been predominantly assessed in terms of a neuroprotective role. In this report we characterized the activin response in the CNS in a rabbit model of meningitis. In normal animals, cerebrospinal fluid (CSF) activin levels were higher than those in serum, indicating an intracranial secretion of this cytokine. Following intracisternal inoculation with Streptococcus pneumoniae, activin in CSF was unchanged for the first 12 h and then rose progressively; levels were increased approximately 15-fold within 24 h. Activin levels were correlated positively with CSF protein content and with the number of apoptotic neurons in the dentate gyrus. No apparent correlation was observed between CSF activin concentrations and bacterial titer, lactate concentrations or leukocyte density. Using immunohistochemistry, activin staining was localized to epithelial cells of the choroid plexus, cortical neurons and the CA3 region of the hippocampus, with similar staining intensities in both normal and meningitic brains. However, in meningitic brains there was also strong staining in activated microglia and infiltrating macrophages. Taken together, these results demonstrate that activin forms part of the CNS response to immune challenge and may be an important mediator to modulate inflammatory processes in the brain. 相似文献
104.
Grégory Lacraz Florence Figeac Jamileh Movassat Nadim Kassis Josiane Coulaud Anne Galinier Corinne Leloup Danielle Bailbé Fran?oise Homo-Delarche Bernard Portha 《PloS one》2009,4(8)
Background
Oxidative stress (OS), through excessive and/or chronic reactive oxygen species (ROS), is a mediator of diabetes-related damages in various tissues including pancreatic β-cells. Here, we have evaluated islet OS status and β-cell response to ROS using the GK/Par rat as a model of type 2 diabetes.Methodology/Principal Findings
Localization of OS markers was performed on whole pancreases. Using islets isolated from 7-day-old or 2.5-month-old male GK/Par and Wistar control rats, 1) gene expression was analyzed by qRT-PCR; 2) insulin secretion rate was measured; 3) ROS accumulation and mitochondrial polarization were assessed by fluorescence methods; 4) antioxidant contents were quantified by HPLC. After diabetes onset, OS markers targeted mostly peri-islet vascular and inflammatory areas, and not islet cells. GK/Par islets revealed in fact protected against OS, because they maintained basal ROS accumulation similar or even lower than Wistar islets. Remarkably, GK/Par insulin secretion also exhibited strong resistance to the toxic effect of exogenous H2O2 or endogenous ROS exposure. Such adaptation was associated to both high glutathione content and overexpression (mRNA and/or protein levels) of a large set of genes encoding antioxidant proteins as well as UCP2. Finally, we showed that such a phenotype was not innate but spontaneously acquired after diabetes onset, as the result of an adaptive response to the diabetic environment.Conclusions
The GK/Par model illustrates the effectiveness of adaptive response to OS by β-cells to achieve self-tolerance. It remains to be determined to what extend such islet antioxidant defenses upregulation might contribute to GK/Par β-cell secretory dysfunction. 相似文献105.
Mie Agermose Gram Hanne-Dorthe Emborg Astrid Blicher Schelde Nikolaj Ulrik Friis Katrine Finderup Nielsen Ida Rask Moustsen-Helms Rebecca Legarth Janni Uyen Hoa Lam Manon Chaine Aisha Zahoor Malik Morten Rasmussen Jannik Fonager Raphael Niklaus Sieber Marc Stegger Steen Ethelberg Palle Valentiner-Branth Christian Holm Hansen 《PLoS medicine》2022,19(9)
BackgroundThe continued occurrence of more contagious Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants and waning immunity over time require ongoing reevaluation of the vaccine effectiveness (VE). This study aimed to estimate the effectiveness in 2 age groups (12 to 59 and 60 years or above) of 2 or 3 vaccine doses (BNT162b2 mRNA or mRNA-1273) by time since vaccination against SARS-CoV-2 infection and Coronavirus Disease 2019 (COVID-19) hospitalization in an Alpha-, Delta-, or Omicron-dominated period.Methods and findingsA Danish nationwide cohort study design was used to estimate VE against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha, Delta, or Omicron variant. Information was obtained from nationwide registries and linked using a unique personal identification number. The study included all previously uninfected residents in Denmark aged 12 years or above (18 years or above for the analysis of 3 doses) in the Alpha (February 20 to June 15, 2021), Delta (July 4 to November 20, 2021), and Omicron (December 21, 2021 to January 31, 2022) dominated periods. VE estimates including 95% confidence intervals (CIs) were calculated (1-hazard ratio∙100) using Cox proportional hazard regression models with underlying calendar time and adjustments for age, sex, comorbidity, and geographical region. Vaccination status was included as a time-varying exposure. In the oldest age group, VE against infection after 2 doses was 90.7% (95% CI: 88.2; 92.7) for the Alpha variant, 82.3% (95% CI: 75.5; 87.2) for the Delta variant, and 39.9% (95% CI: 26.3; 50.9) for the Omicron variant 14 to 30 days since vaccination. The VE waned over time and was 73.2% (Alpha, 95% CI: 57.1; 83.3), 50.0% (Delta, 95% CI: 46.7; 53.0), and 4.4% (Omicron, 95% CI: −0.1; 8.7) >120 days since vaccination. Higher estimates were observed after the third dose with VE estimates against infection of 86.1% (Delta, 95% CI: 83.3; 88.4) and 57.7% (Omicron, 95% CI: 55.9; 59.5) 14 to 30 days since vaccination. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 98.1% or above for the Alpha and Delta variants. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 95.5% or above for the Omicron variant. The main limitation of this study is the nonrandomized study design including potential differences between the unvaccinated (reference group) and vaccinated individuals.ConclusionsTwo vaccine doses provided high protection against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha and Delta variants with protection, notably against infection, waning over time. Two vaccine doses provided only limited and short-lived protection against SARS-CoV-2 infection with Omicron. However, the protection against COVID-19 hospitalization following Omicron SARS-CoV-2 infection was higher. The third vaccine dose substantially increased the level and duration of protection against infection with the Omicron variant and provided a high level of sustained protection against COVID-19 hospitalization among the +60-year-olds.Mie Agermose Gram and colleagues estimate vaccine effectiveness against infection and COVID-19 hospitalization with the Alpha, Delta or Omicron variant in Denmark. 相似文献
106.
Significant differentiation in the apolipoprotein(a)/lipoprotein(a) trait between chimpanzees from Western and Central Africa 下载免费PDF全文
107.
108.
109.
Anne Chevrel Agnes Mesneau Dyana Sanchez Louisa Celma Sophie Quevillon-Cheruel Andrea Cavagnino Sylvie Nessler Ines Li de la Sierra-Gallay Herman van Tilbeurgh Philippe Minard Marie Valerio-Lepiniec Agathe Urvoas 《Journal of structural biology》2018,201(2):88-99
We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize. 相似文献
110.
Peterson CN Day S Wolfe BE Ellison AM Kolter R Pringle A 《Environmental microbiology》2008,10(9):2257-2266
The community of organisms inhabiting the water-filled leaves of the carnivorous pitcher-plant Sarracenia purpurea includes arthropods, protozoa and bacteria, and serves as a model system for studies of food web dynamics. Despite the wealth of data collected by ecologists and zoologists on this food web, very little is known about the bacterial assemblage in this microecosystem. We used terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify bacterial diversity within the pitchers as a function of pitcher size, pH of the pitcher fluid and the presence of the keystone predator in this food web, larvae of the pitcher-plant mosquito Wyeomyia smithii. Results were analysed at two spatial scales: within a single bog and across three isolated bogs. Pitchers were sterile before they opened and composition of the bacterial assemblage was more variable between different bogs than within bogs. Measures of bacterial richness and diversity were greater in the presence of W. smithii and increased with increasing pitcher size. Our results suggest that fundamental ecological concepts derived from macroscopic food webs can also be used to predict the bacterial assemblages in pitcher plants. 相似文献