全文获取类型
收费全文 | 24536篇 |
免费 | 569篇 |
国内免费 | 491篇 |
出版年
2023年 | 129篇 |
2022年 | 198篇 |
2021年 | 308篇 |
2020年 | 340篇 |
2019年 | 488篇 |
2018年 | 537篇 |
2017年 | 339篇 |
2016年 | 363篇 |
2015年 | 672篇 |
2014年 | 1938篇 |
2013年 | 2054篇 |
2012年 | 1698篇 |
2011年 | 2529篇 |
2010年 | 2091篇 |
2009年 | 1088篇 |
2008年 | 1083篇 |
2007年 | 1012篇 |
2006年 | 891篇 |
2005年 | 797篇 |
2004年 | 723篇 |
2003年 | 696篇 |
2002年 | 457篇 |
2001年 | 256篇 |
2000年 | 255篇 |
1999年 | 307篇 |
1998年 | 318篇 |
1997年 | 325篇 |
1996年 | 308篇 |
1995年 | 332篇 |
1994年 | 296篇 |
1993年 | 263篇 |
1992年 | 238篇 |
1991年 | 214篇 |
1990年 | 163篇 |
1989年 | 186篇 |
1988年 | 145篇 |
1987年 | 143篇 |
1986年 | 111篇 |
1985年 | 166篇 |
1984年 | 241篇 |
1983年 | 186篇 |
1982年 | 203篇 |
1981年 | 108篇 |
1980年 | 135篇 |
1979年 | 101篇 |
1978年 | 37篇 |
1977年 | 41篇 |
1976年 | 28篇 |
1974年 | 21篇 |
1973年 | 14篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
51.
Mengnan Wang Dongjie Li Mingyue Zhang Wenzhi Yang Yali Cui Shijie Li 《Animal genetics》2015,46(4):354-360
The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting. 相似文献
52.
53.
Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: Strategies of protein stabilization 总被引:2,自引:0,他引:2
Rainer Jaenicke 《FEMS microbiology reviews》1996,18(2-3):215-224
Abstract: The molecular origin of protein stability has been the subject of active research for more than a generation (R. Jaenicke (1991) Eur. J. Biochem. 202, 715–728). Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential. In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima , it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds ( ΔΔG stab ≈ 50 kJ/mol). As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology. In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents. 相似文献
54.
In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted. 相似文献
55.
Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc. 相似文献
56.
Victor M. Luque-Almagro Verity J. Lyall Stuart J. Ferguson M. Dolores Roldán David J. Richardson Andrew J. Gates 《The Journal of biological chemistry》2013,288(41):29692-29702
Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression. 相似文献
57.
Markus Pech Thomas Spreter Roland Beckmann Birgitta Beatrix 《The Journal of biological chemistry》2010,285(25):19679-19687
Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome. 相似文献
58.
An inhibitor of the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100-fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE-Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. alpha-Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment. 相似文献
59.
Two full-length (or nearly so) cDNA clones containing information for the protease inhibitors PI IV and C-II from soybean seeds were identified by means of a synthetic probe. DNA sequencing revealed that the two protease inhibitors are synthesized as precursors with a short peptide leader. The coding regions of the two clones show 80% homology, wheraes the 5 non-coding regions are 90% homologous. Homology of 75% is found in the region extending beyond the stop codons. 相似文献
60.
Don M. Wojchowski Stuart H. Orkin Arthur J. Sytkowski 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1987,910(3)
Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower Mr (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this Mr 23000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development. 相似文献