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151.
Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34 总被引:1,自引:0,他引:1
Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function. 相似文献
152.
153.
Pancreatic cancer is a leading cause of cancer-related death, largely due to metastatic dissemination. We investigated pancreatic cancer progression by utilizing a mathematical framework of metastasis formation together with comprehensive data of 228 patients, 101 of whom had autopsies. We found that pancreatic cancer growth is initially exponential. After estimating the rates of pancreatic cancer growth and dissemination, we determined that patients likely harbor metastases at diagnosis and predicted the number and size distribution of metastases as well as patient survival. These findings were validated in an independent database. Finally, we analyzed the effects of different treatment modalities, finding that therapies that efficiently reduce the growth rate of cells earlier in the course of treatment appear to be superior to upfront tumor resection. These predictions can be validated in the clinic. Our interdisciplinary approach provides insights into the dynamics of pancreatic cancer metastasis and identifies optimum therapeutic interventions. 相似文献
154.
Schäfer F Schuster J Würz B Härtig C Harms H Müller RH Rohwerder T 《Applied and environmental microbiology》2012,78(17):6280-6284
The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed. 相似文献
155.
The osmotic potential and effects of plasmolysis were investigated in two different Klebsormidium strains from alpine habitats by incubation in 300–2,000 (3,000) mM sorbitol. Several members of this genus were previously found to tolerate desiccation in the vegetative state yet information was lacking on the osmotic potentials of these algae. The strains were morphologically determined as Klebsormidium crenulatum and Klebsormidium nitens. These species belong to distinct clades, as verified by phylogenetic analysis of the rbcL gene. K. crenulatum is part of to the K. crenulatum/mucosum (‘F’ clade) and K. nitens of the ‘E2’ clade. Plasmolysis occurred in K. crenulatum at 800 mM sorbitol (961 mOsmol kg?1, Ψ?=??2.09 MPa) and in K. nitens at 600 mM sorbitol (720 mOsmol kg?1, Ψ?=??1.67 MPa). These are extraordinarily high osmotic values (very negative osmotic potentials) compared with values reported for other green algae. In K. crenulatum, the maximum photosynthetic rate (Pmax) in the light-saturated range was 116 μmol O2 h?1 mg?1 chl a. Incubation in 1,000 mM sorbitol decreased Pmax to 74.1% of the initial value, whereas 2,000 mM sorbitol (Ψ?=??5.87 MPa) lead to an almost complete loss of oxygen production. In K. nitens, Pmax was 91 μmol O2 h?1 mg?1 chl a under control conditions and incubation in 800 mM sorbitol did not decrease Pmax, 2,000 mM sorbitol decreased Pmax only to about 62.6% of the initial value whereas 3,000 mM sorbitol stopped oxygen evolution. This indicated a broader amplitude for photosynthesis in the examined strain of K. nitens. Control samples and samples plasmolysed for 3 h in 800 mM sorbitol (K. nitens), 1,000 mM sorbitol (K. crenulatum), or 2,000 mM sorbitol were investigated by transmission electron microscopy after chemical or high-pressure freeze fixation. In cells undergoing plasmolysis the protoplasts were retracted from the cell wall, the cytoplasm appeared dense, vacuoles were small and fragmented, and the cytoplasm was filled with ribosomes. Thin cytoplasmic strands were connected to the cell wall; 2,000 mM sorbitol increased the effect. The content of soluble carbohydrates in these two strains was investigated by HPLC, as this is one known mechanism for cells to maintain high osmotic pressure of the cytosol. Both Klebsormidium species contained diverse soluble carbohydrates, including a dominant mixed peak of unidentified oligosaccharides, and more minor amounts of raffinose, sucrose, glucose, xylose, galactose, mannose, inositol, fructose, glycerol, mannitol, and sorbitol. The total content of soluble carbohydrates was approximately 1.2% of the dry weight, indicating that this is not a major factor contributing to the high osmotic potential in these strains of Klebsormidium. 相似文献
156.
Effective generation of transgenic reporter and gene trap lines of the medaka (Oryzias latipes) using the Ac/Ds transposon system 总被引:1,自引:0,他引:1
Froschauer A Sprott D Gerwien F Henker Y Rudolph F Pfennig F Gutzeit HO 《Transgenic research》2012,21(1):149-162
In model teleost fishes like the medaka and the zebrafish many genes which have been identified in genome sequencing projects
await their functional characterization. Techniques for the effective generation of transgenic animals are a prerequisite
for this challenging task, and, due to their transparency, fish offer the possibility to combine the use of fluorescent proteins
and developmental analysis in vivo. Here we describe the application of the Ac/Ds transposon system to generate transgenic
medaka reporter and gene trap lines. We determined a germline transmission rate of 30% in our experiments using constructs
ranging in size from 1.8 to 6 kilobase pairs. The genomic integration site of the Ds-elements can be easily identified which
is an important feature for gene trap mutagenesis experiments and similar approaches. We constructed gene trap vectors with
functional elements of medaka sequences that produce in frame fusions of the endogenous sequence to EGFP. These vectors mimic
endogenous expression of the trapped allele in transgenic animals and are capable to interfere with the expression of the
wild type allele in the homozygous individuals. 相似文献
157.
In their widely noticed study, Gergely, Bekkering, and Király (2002) showed that 14-month-old infants imitated an unusual action only if the model freely chose to perform this action and not if the choice of the action could be ascribed to external constraints. They attributed this kind of selective imitation to the infants' capacity of understanding the principle of rational action. In the current paper, we present evidence that a simpler approach of perceptual distraction may be more appropriate to explain their results. When we manipulated the saliency of context stimuli in the two original conditions, the results were exactly opposite to what rational imitation predicts. Based on these findings, we reject the claim that the notion of rational action plays a key role in selective imitation in 14-month-olds. 相似文献
158.
Jacob F Ukegjini K Nixdorf S Ford CE Olivier J Caduff R Scurry JP Guertler R Hornung D Mueller R Fink DA Hacker NF Heinzelmann-Schwarz VA 《PloS one》2012,7(2):e31885
Background
Activation of the Wnt signaling pathway is implicated in aberrant cellular proliferation in various cancers. In 40% of endometrioid ovarian cancers, constitutive activation of the pathway is due to oncogenic mutations in β-catenin or other inactivating mutations in key negative regulators. Secreted frizzled-related protein 4 (SFRP4) has been proposed to have inhibitory activity through binding and sequestering Wnt ligands.Methodology/Principal Findings
We performed RT-qPCR and Western-blotting in primary cultures and ovarian cell lines for SFRP4 and its key downstream regulators activated β-catenin, β-catenin and GSK3β. SFRP4 was then examined by immunohistochemistry in a cohort of 721 patients and due to its proposed secretory function, in plasma, presenting the first ELISA for SFRP4. SFRP4 was most highly expressed in tubal epithelium and decreased with malignant transformation, both on RNA and on protein level, where it was even more profound in the membrane fraction (p<0.0001). SFRP4 was expressed on the protein level in all histotypes of ovarian cancer but was decreased from borderline tumors to cancers and with loss of cellular differentiation. Loss of membrane expression was an independent predictor of poor survival in ovarian cancer patients (p = 0.02 unadjusted; p = 0.089 adjusted), which increased the risk of a patient to die from this disease by the factor 1.8.Conclusions/Significance
Our results support a role for SFRP4 as a tumor suppressor gene in ovarian cancers via inhibition of the Wnt signaling pathway. This has not only predictive implications but could also facilitate a therapeutic role using epigenetic targets. 相似文献159.
E Schellenberger A Haeckel L Schoenzart F Appler J Schnorr M Taupitz B Hamm 《Molecular imaging》2012,11(5):383-388
Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology. 相似文献
160.
F Matzer UV Wisiak M Graninger W Söllner HP Stilling M Glawischnig-Goschnik A Lueger C Fazekas 《PloS one》2012,7(8):e41775