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151.
Hyeng-Il Cheun Kyungjin Kim Sejoung Yoon Won-Ja Lee Woo-Yoon Park Seobo Sim Jae-Ran Yu 《The Korean journal of parasitology》2013,51(3):353-355
There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections. 相似文献
152.
Oleg V. Kovalenko Andrea Olland Nicole Piché-Nicholas Adarsh Godbole Daniel King Kristine Svenson Valerie Calabro Mischa R. Müller Caroline J. Barelle William Somers Davinder S. Gill Lidia Mosyak Lioudmila Tchistiakova 《The Journal of biological chemistry》2013,288(24):17408-17419
The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs. 相似文献
153.
Klara Ivankova Rostislav Turecek Thorsten Fritzius Riad Seddik Laurent Prezeau La?titia Comps-Agrar Jean-Philippe Pin Bernd Fakler Valerie Besseyrias Martin Gassmann Bernhard Bettler 《The Journal of biological chemistry》2013,288(34):24848-24856
GABAB receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABAB receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABAB1a, GABAB1b, and GABAB2 form fully functional heteromeric GABAB(1a,2) and GABAB(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABAB(1a,2) and GABAB(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABAB receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABAB receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABAB receptor-mediated K+ current response. In summary, our experiments support that the up-regulation of functional GABAB receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12. 相似文献
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155.
Alessandra Podda Giovanni Checcucci Wafa Mouhaya Delphine Centeno Valerie Rofidal Renata Del Carratore François Luro Raphael Morillon Patrick Ollitrault Bianca Elena Maserti 《Journal of plant physiology》2013
To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na+ and Cl− accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found. 相似文献
156.
157.
158.
Kaat Kehoe Robert Verkerk Yani Sim Yannick Waumans Pieter Van der Veken Anne-Marie Lambeir Ingrid De Meester 《Analytical biochemistry》2013
Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 ± 0.02 and 0.72 ± 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome. 相似文献
159.
Lai-Ping Wong Rick?Twee-Hee Ong Wan-Ting Poh Xuanyao Liu Peng Chen Ruoying Li Kevin?Koi-Yau Lam Nisha?Esakimuthu Pillai Kar-Seng Sim Haiyan Xu Ngak-Leng Sim Shu-Mei Teo Jia-Nee Foo Linda?Wei-Lin Tan Yenly Lim Seok-Hwee Koo Linda?Seo-Hwee Gan Ching-Yu Cheng Sharon Wee Eric?Peng-Huat Yap Pauline?Crystal Ng Wei-Yen Lim Richie Soong Markus?Rene Wenk Tin Aung Tien-Yin Wong Chiea-Chuen Khor Peter Little Kee-Seng Chia Yik-Ying Teo 《American journal of human genetics》2013,92(1):52-66
Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. 相似文献
160.
Jung-eun Park Chiman Song Keehyun Choi Taebo Sim Bongjin Moon Eun Joo Roh 《Bioorganic & medicinal chemistry letters》2013,23(20):5515-5518
A series of 3,4-diarylpyrrolidin-2-one was designed, prepared and evaluated as triple reuptake inhibitors for antidepressant. Most compounds exhibited comparable in vitro efficacy as norepinephrine and dopamine transporter reuptake inhibitors. Especially, 2i showed better potency than GBR-12909 (IC50 = 14 nM) which was used as reference compound for dopamine transporter. In addition, 2a and 2b showed inhibition (5.17 μM–85.6 nM) for three transporters. 相似文献