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41.
Kimi Araki Naoki Takeda Atsushi Yoshiki Yuichi Obata Naomi Nakagata Toshihiko Shiroishi Kazuo Moriwaki Ken-ichi Yamamura 《Mammalian genome》2009,20(1):14-20
MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide
substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed
in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity,
and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful
tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic
stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established.
They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three
lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 × BDF1 mice and found that blastocyst injection
resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated
with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 × BDF1 blastocyst injection.
This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome. 相似文献
42.
Hirata N Yanagawa Y Ogura H Satoh M Noguchi M Matsumoto M Togashi H Onoé K Iwabuchi K 《Cellular immunology》2011,(2):165-171
In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs. 相似文献
43.
Cloning of human muscle phosphofructokinase cDNA 总被引:7,自引:0,他引:7
Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency). 相似文献
44.
James R. P. Worth Shota Sakaguchi Nobuyuki Tanaka Michimasa Yamasaki Yuji Isagi 《Biological journal of the Linnean Society. Linnean Society of London》2013,108(2):263-277
Sciadopitys verticillata is amongst the most relictual of all plants, being the last living member of an ancient conifer lineage, the Sciadopityaceae, and is distributed in small and disjunct populations in high rainfall regions of Japan. Although mega‐fossils indicate the persistence of the species within Japan through the Pleistocene glacial–interglacial cycles, how the species withstood the colder and drier climates of the glacials is not well known. The present study utilized phylogeography and palaeodistribution modelling to test whether the species survived within pollen‐based coastal temperate forest glacial refugia or within previously unidentified refugia close to its current range. Sixteen chloroplast haplotypes were found that displayed significant geographical structuring. Unexpectedly, northern populations in central Honshu most distant from coastal refugia had the highest chloroplast diversity and were differentiated from the south, a legacy of glacial populations possibly in inland river valleys close to its current northern range. By contrast, populations near putative coastal refugia in southern Japan, harboured the lower chloroplast diversity and were dominated by a single haplotype. Fragment size polymorphism at a highly variable and homoplasious mononucleotide repeat region in the trnT‐trnL intergenic spacer reinforced the contrasting patterns of diversity observed between northern and southern populations. The divergent histories of northern and southern populations revealed in the present study will inform the management of this globally significant conifer. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 263–277. 相似文献
45.
Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. 总被引:25,自引:7,他引:25 下载免费PDF全文
T Hayashi H McMahon S Yamasaki T Binz Y Hata T C Südhof H Niemann 《The EMBO journal》1994,13(21):5051-5061
Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis. 相似文献
46.
Nishimoto E Yamashita S Yamasaki N Imoto T 《Bioscience, biotechnology, and biochemistry》1999,63(2):329-336
The fluorescence spectral distributions of four tryptophan residues of hen egg-white lysozyme were analyzed using time-resolved and quenching-resolved fluorescence spectroscopy. Trp62 and Trp108 gave the fluorescence maxima at 352 nm and 342 nm, respectively. The fluorescence of Trp28 and Trp111 occurred only at 300-360 nm and they were observed as an unresolved emission band with a maximum and shoulder at 320 nm and 330 nm. The fluorescence quenching and decay parameters of each tryptophan residue reconfirmed that Trp62 was fully exposed to the solvent but Trp108 was sealed in the cage of the peptide chains and furthermore showed that Trp28 and Trp111 are under the influence of the larger fluctuational motion at the hydrophobic matrix box. The fluorescence responses of each tryptophan residue to the lysozyme-ligand interaction suggested that the internal fluctuation was reduced by the binding of ligand to give a distorted conformation to the hydrophobic matrix box region. 相似文献
47.
Weiduan Xu Jianmin Chen Glenn Yamasaki John E. Murphy Baisong Mei 《Molecular biotechnology》2010,45(3):248-256
Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation
factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal
sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation
can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but
this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop
assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream
product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional
to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII
producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based
ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors
and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment.
To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing
bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation
between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be
rapidly performed, thereby making them effective for in-process monitoring of protein sialylation. 相似文献
48.
49.
Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes. 相似文献
50.
Limnology - The in-stream processing of nutrients plays an important role in the fluvial nutrient transport from lands to the ocean, but few empirical studies have addressed the temporal dynamics... 相似文献