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181.
182.
To gain more insights about the biological roles of PDK1, we have used the yeast two-hybrid system and in vivo binding assay to identify interacting molecules that associate with PDK1. As a result, serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor-beta (TGF-beta) receptor-interacting protein, was identified as an interacting partner of PDK1. STRAP was found to form in vivo complexes with PDK1 in intact cells. Mapping analysis revealed that this binding was only mediated by the catalytic domain of PDK1 and not by the pleckstrin homology domain. Insulin enhanced a physical association between PDK1 and STRAP in intact cells, but this insulin-induced association was prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. In addition, the association between PDK1 and STRAP was decreased by TGF-beta treatment. Analysis of the activities of the interacting proteins showed that PDK1 kinase activity was significantly increased by coexpression of STRAP, probably through the inhibition of the binding of 14-3-3, a negative regulator, to PDK1. Consistently, knockdown of the endogenous STRAP by the transfection of the small interfering RNA resulted in the decrease of PDK1 kinase activity. PDK1 also exhibited an inhibition of TGF-beta signaling with STRAP by contributing to the stable association between TGF-beta receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-beta. Knockdown of endogenous PDK1 with small interfering RNA has an opposite effect. Taken together, these results suggested that STRAP acts as an intermediate signaling molecule linking between the phosphatidylinositol 3-kinase/PDK1 and the TGF-beta signaling pathways. 相似文献
183.
The unique morphology of the adult human carotid bifurcation and its sinus has been investigated extensively, but its long-term, age-dependent development has not. It is important fundamentally and clinically to understand the hemodynamics and developmental forces that play a role in remodeling of the carotid bifurcation and maturation of the sinus in association with brain maturation. This understanding can lead to better prognostication and therapy of carotid disease. We analyzed the change of sinus morphology and the angle of the carotid bifurcation in four postnatal developmental stages (Group I: 0-2 years, Group II: 3-9 years, Group III: 10-19 years, and Group IV: 20-36 years, respectively) using multiprojection digital subtraction angiograms and image post-processing techniques. The most significant findings are the substantial growth of the internal carotid artery (ICA) with age and the development of a carotid sinus at the root of the ICA during late adolescence. The bifurcation angle remains virtually unchanged from infancy to adulthood. However, the angle split between the ICA and external carotid artery (ECA) relative to the common carotid artery (CCA) undergoes significant changes. Initially, the ICA appears to emanate as a side branch. Later in life, to reduce hydraulic resistance in response to increased flow demand by the brain, the bifurcation is remodeled to a construct in which both daughter vessels are a skewed continuation of the parent artery. This study provides a new analysis method to examine the development of the human carotid bifurcation over the developmental years, despite the small and sparse database. A larger database will enable in the future a more extensive analysis such as gender or racial differences. 相似文献
184.
Kang HK Seo MY Seo ES Kim D Chung SY Kimura A Day DF Robyt JF 《Biochimica et biophysica acta》2005,1727(1):5-15
Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene (m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 degrees C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 micromol min-1 mg-1. 相似文献
185.
A series of polyguanidylated dendritic structures that can be used as molecular translocators have been designed and synthesized based on nonpeptide units. The dendritic oligoguanidines conjugated with fluorescein or with a green fluorescent protein (GFP) mutant as cargos were isolated and characterized. Quantification and time-course analyses of the cellular uptake of the conjugates using HeLa S3 and human cervical carcinoma cells reveal that the polyguanidylated dendrimers have comparable translocation efficiency to the Tat(49-57) peptide. Furthermore, the deconvolution microscopy image analysis shows that they are located inside the cells. These results clearly show that nonlinear, branched dendritic oligoguanidines are capable of translocation through the cell membrane. This work also demonstrates the potential of these nonpeptidic dendritic oligoguanidines as carriers for intracellular delivery of small molecule drugs, bioactive peptides, and proteins. 相似文献
186.
Mesenchymal stem cells from cryopreserved human umbilical cord blood 总被引:32,自引:0,他引:32
Lee MW Choi J Yang MS Moon YJ Park JS Kim HC Kim YJ 《Biochemical and biophysical research communications》2004,320(1):273-278
Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages, but the presence of mesenchymal stem cells (MSCs) in UCB has been disputed and it remains to be validated. In this study, we examined the ability of cryopreserved UCB harvests to produce cells with characteristics of MSCs. We were able to obtain homogeneous plastic adherent cells from the mononuclear cell fractions of cryopreserved UCB using our culture conditions. These adherent cell populations exhibited fibroblast-like morphology and typical mesenchymal-like immunophenotypes (CD73+, CD105+, and CD166+, etc.). These cells presented the self-renewal capacity and the mesenchymal cell-lineage potential to form bone, fat, and cartilage. Moreover, they expressed mRNAs of multi-lineage genes including SDF-1, NeuroD, and VEGF-R1, suggesting that the obtained cells had the multi-differentiation capacity as bone marrow-derived MSCs. These results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and therapeutic applications. 相似文献
187.
Heng BC Ye CP Liu H Toh WS Rufaihah AJ Yang Z Bay BH Ge Z Ouyang HW Lee EH Cao T 《Journal of biomedical science》2006,13(3):433-445
Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols. 相似文献
188.
Several studies examining DNA deamination have published levels of 2'-deoxyinosine that illustrated a large variation between studies. Most of them are the result of artifactual DNA deamination that occurs during the process of sample preparation, particularly acid hydrolysis. This protocol for measurement of 2'-deoxyinosine describes the use of nuclease P1 and alkaline phosphatase to achieve release of nucleosides from DNA, followed by HPLC prepurification with subsequent gas chromatography-mass spectrometry analysis of the nucleosides. It has been used in the measurement of the levels of 2'-deoxyinosine in DNA of commercial sources and DNA from cells and animal tissues, and gives values ranging from 3 to 7 2'-deoxyinosine per 10(6) 2-deoxyadenosine. This protocol should take approximately 7 days to complete. 相似文献
189.
In Koo Hwang Sun Shin Yi Jae Hoon Shin Ki-Yeon Yoo Jung Hoon Choi Choong Hyun Lee Je Kyung Seong Yeo Sung Yoon Jeong Ho Park Moo-Ho Won 《Neurochemical research》2010,35(3):465-472
In the present study, we observed the effects of cyclosporine A (CsA), an efficient immunosuppressant, on cell proliferation
and neuroblast differentiation in the subgranular zone of the dentate gyrus (SZDG) in normal C57BL/6 mice using Ki67 and doublecortin
(DCX) immunohistochemical staining, respectively. At 8 weeks of age, vehicle (physiological saline) or CsA was daily administered
(40 mg/kg, i.p.) for 1 week. Animals were sacrificed at 2 weeks after last administration. CsA treatment did not show any
influences in neurons, astrocytes and microglia based on immunohistochemistry for its markers, respectively. However, in the
CsA-treated group, Fluoro-Jade B, a marker for neurodegeneration, positive cells were found in the SZDG, not in the vehicle-treated
group. In the vehicle-treated group, Ki67 immunoreactive (+) nuclei were clustered in the SZDG, whereas in the CsA-treated group Ki67+ nuclei were scattered in the SZDG, showing no difference in cell numbers. Numbers of DCX+ neuroblasts with well-developed processes (tertiary dendrites) were much lower in the CsA-treated group than those in the
vehicle-treated group; however, numbers of DCX+ neuroblasts with secondary dendrites were similar in both the groups. These results suggest that CsA significantly reduces
dendritic outgrowth and complexity from neuroblasts in the SZDG without any affecting in neurons, astrocytes and microglia
in normal mice. 相似文献
190.