全文获取类型
收费全文 | 8769篇 |
免费 | 685篇 |
国内免费 | 813篇 |
出版年
2024年 | 30篇 |
2023年 | 150篇 |
2022年 | 329篇 |
2021年 | 589篇 |
2020年 | 385篇 |
2019年 | 430篇 |
2018年 | 438篇 |
2017年 | 325篇 |
2016年 | 407篇 |
2015年 | 578篇 |
2014年 | 659篇 |
2013年 | 686篇 |
2012年 | 841篇 |
2011年 | 734篇 |
2010年 | 457篇 |
2009年 | 385篇 |
2008年 | 422篇 |
2007年 | 370篇 |
2006年 | 314篇 |
2005年 | 263篇 |
2004年 | 214篇 |
2003年 | 182篇 |
2002年 | 149篇 |
2001年 | 106篇 |
2000年 | 112篇 |
1999年 | 105篇 |
1998年 | 84篇 |
1997年 | 85篇 |
1996年 | 55篇 |
1995年 | 56篇 |
1994年 | 71篇 |
1993年 | 34篇 |
1992年 | 38篇 |
1991年 | 33篇 |
1990年 | 25篇 |
1989年 | 31篇 |
1988年 | 16篇 |
1987年 | 15篇 |
1986年 | 10篇 |
1985年 | 20篇 |
1984年 | 6篇 |
1983年 | 8篇 |
1982年 | 7篇 |
1981年 | 1篇 |
1980年 | 4篇 |
1979年 | 4篇 |
1978年 | 2篇 |
1975年 | 1篇 |
1950年 | 1篇 |
排序方式: 共有10000条查询结果,搜索用时 187 毫秒
921.
Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Here, we show that myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin. 相似文献
922.
Han J Farnsworth RL Tiwari JL Tian J Lee H Ikonomi P Byrnes AP Goodman JL Puri RK 《Genomics》2006,87(4):552-559
Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product. 相似文献
923.
In vitro fertilization as a tool for investigating sexual reproduction of angiosperms 总被引:5,自引:5,他引:0
Ya Ying Wang Anxiu Kuang Scott D. Russell Hui Qiao Tian 《Sexual plant reproduction》2006,19(3):103-115
In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants. 相似文献
924.
Zhang T Sun Y Tian E Deng H Zhang Y Luo X Cai Q Wang H Chai J Zhang H 《Development (Cambridge, England)》2006,133(6):1023-1033
We describe the identification and characterization of a novel PcG gene in C. elegans, sor-1, which is involved in global repression of Hox genes. sor-1 encodes a novel protein with an RNA-binding activity. We provide evidence that SOR-1 and the previously identified RNA-binding protein SOP-2 may constitute an RNA-binding complex in Hox gene repression. SOR-1 and SOP-2 directly interact with each other and are colocalized in nuclear bodies. The localization of SOR-1 depends on SOP-2. Surprisingly, homologs of SOR-1 and SOP-2 are not found in other organisms, including the congeneric species C. briggsae, suggesting an unexpected lack of evolutionary constraint on an essential global gene regulatory system. 相似文献
925.
Yao W Farmer R Cooper R Chmielewski PA Tian XY Setterberg RB Jee WS Lundy MW 《Journal of musculoskeletal & neuronal interactions》2006,6(3):277-283
Current published results on whether statins have beneficial effects on bone metabolism have been conflicting so far. In order to further investigate if statins were promising candidates for the treatment for osteoporosis, we conducted a study in which rats were ovariectomized (OVX) at 6 months of age, allowed to lose bone for 60 days and followed by oral administration of simvastatin at the dose levels of 0.3-10 mg/kg/d for 60 days. PGE2 (6 mg/kg) was used as a positive control. Study endpoints included bone histomorphometry on the proximal tibial metaphysis (PTM) and the tibial diaphysis (TX), dual-energy X-ray absorptiometry on the right femur and micro computed tomography (ICT) on the 5th lumbar vertebra (LV). After 120 days of OVX, cancellous bone lost by 80% in the PTM and 18% in the LV accompanied by increased bone formation and resorption. Simvastatin at all dose levels did not affect bone volume, bone formation rate and bone erosion surface when compared to 120 day ovariectomized animals at all bone sites studied. By contrast, PGE2 restored cancellous and cortical bone area to sham control levels. In conclusion, this study demonstrated that unlike PGE2, oral administration of simvastatin did not have effects on cancellous or cortical bone formation and resorption; and consequently was not able to prevent further bone loss or restore bone mass in the osteopenic, OVX rats. 相似文献
926.
Interactions between antiarrhythmic drugs and ion channels are important subjects in the field of cardiovascular electro-pharmacology. This study explores the relationship between propafenone and C-type inactivation of Kv1.4 channel. fKvl.4deltaN, a ferret Kv1.4 N-terminal deleted mutant, was employed in this study. fKvl.4deltaN cRNA was injected into Xenopus oocytes to express fKvl.4deltaN channel and two electrode voltage clamp technique was used to record the current. We found that fKvl.4deltaN channel current was rapidly depressed in a frequency-dependent manner and meanwhile, C-type inactivation in this channel was increased more than 7 folds in the presence of 100 microM propafenone. While propafenone has no effect on Kv1.4deltaN recovery. All the results indicate that propafenone blocks Kvl.4deltaN channel through intracellular bindings and that binding of propafenone with Kvl.4deltaN channel leads to a conformational change on the extracellular site which accelerates C-type inactivation, suggesting that propafenone, as an open channel blocker, may affect the mechanism of C-type inactivation. 相似文献
927.
Hui-Min Yu Yue Shi Hui Luo Zhuo-Ling Tian Yan-Qin Zhu Zhong-Yao Shen 《Journal of Molecular Catalysis .B, Enzymatic》2006,43(1-4):80-85
A superior novel recombinant strain, E. coli BL21(DE3)/pETNHM, containing the start codon mutation of the subunit, was constructed and selected as an overexpression and high efficient mutation platform for the genetic manipulation of the nitrile hydratase (NHase). Under optimal conditions, the specific activity of the recombinant strain reached as high as 452 U/mg dry cell. Enzymatic characteristics studies showed that the reaction activation energy of the recombinant NHaseM was 24.4 ± 0.5 kJ/mol, the suited pH range for catalysis was 5.5–7.5, and the Km value was 4.34 g/L (82 mM). To assess the feasibility of the NHase improvement by protein rational design using this E. coli, site-directed mutagenesis of S122A, S122C, S122D and βW47E of the NHaseM were carried out. The NHaseM (S122A) and NHaseM (S122D) mutants were entirely inactive due to the charge change of the side-chain group. The product tolerance of the NHaseM (S122C) mutant was enhanced while its activity decreased by 30%. The thermo-stability of the NHaseM (βW47E) mutant was significantly strengthened, while its activity reduced by nearly 50%. These results confirmed that the specific activity of the mutant NHase expressed by the recombinant E. coli BL21(DE3)/pETNHM can reasonably change with and without mutations. Therefore, this recombinant E. coli can be efficiently and confidently used for the further rational/random evolution of the NHase to simultaneously improve the activity, thermo-stability and product tolerance of the target NHase. 相似文献
928.
Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803 总被引:3,自引:0,他引:3
Ling H Zhang LY Su Q Song Y Luo ZY Zhou XT Zeng X He J Tan H Yuan JP 《Cellular & molecular biology letters》2006,11(3):408-423
Diallyl disulfide (DADS) is a major constituent of garlic. Previously, we found that DADS both inhibited proliferation in
human gastric cancer cells in vitro and in vivo, and induced G2/M arrest. In this study, we investigated whether this differentiation effect was induced by DADS in human
gastric cancer MGC803 cells, and whether it was related to an alteration in ERK activity. The results showed that the growth
of MGC803 cells was inhibited by DADS. Cells treated with DADS displayed a lower nucleocytoplasmic ratio and tended to form
gland and intercellular conjunction structures. The ConA-mediated cell agglutination ratio and cells’ ALP specific activity
decreased. In MGC803 cells, dye transfer was limited to a few cells neighbouring the dye-injected cell and to a depth of 1–2
layers beneath the scrape site. However, after treatment with DADS, the LY (Lucifer Yellow) was transferred to several cells
immediately neighbouring the microinjected cell and to a depth of 2–4 cell layers from the scrape site. This indicated that
DADS induced differentiation in MGC803 cells. Western blot analysis revealed that although DADS did not influence the quantity
of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls.
At 30 mg·L−1, DADS inhibited the activation of ERK1/2 in 15–30 min. These results suggested that the DADS-induced differentiation of MGC803
cells involved an alteration of the ERK1/2 signaling pathway. 相似文献
929.
Teng D Wang JH Fan Y Yang YL Tian ZG Luo J Yang GP Zhang F 《Applied microbiology and biotechnology》2006,72(4):705-712
β-1,3-1,4-Glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone β-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml−1 for 1% (w/v) barley β-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg−1 for these two substrates. The expression level of recombinant β-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40°C, respectively. 相似文献
930.
Ma Z Liu T Li X Zhou T Xiao L Que H Tian D Jing S Liu S 《Cellular and molecular neurobiology》2006,26(3):277-288
Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI. 相似文献