全文获取类型
收费全文 | 37426篇 |
免费 | 16027篇 |
国内免费 | 10篇 |
专业分类
53463篇 |
出版年
2024年 | 22篇 |
2023年 | 63篇 |
2022年 | 242篇 |
2021年 | 631篇 |
2020年 | 2282篇 |
2019年 | 3866篇 |
2018年 | 4051篇 |
2017年 | 4281篇 |
2016年 | 4457篇 |
2015年 | 4576篇 |
2014年 | 4347篇 |
2013年 | 4803篇 |
2012年 | 2789篇 |
2011年 | 2500篇 |
2010年 | 3621篇 |
2009年 | 2249篇 |
2008年 | 1449篇 |
2007年 | 923篇 |
2006年 | 854篇 |
2005年 | 856篇 |
2004年 | 842篇 |
2003年 | 731篇 |
2002年 | 614篇 |
2001年 | 609篇 |
2000年 | 511篇 |
1999年 | 365篇 |
1998年 | 105篇 |
1997年 | 86篇 |
1996年 | 58篇 |
1995年 | 58篇 |
1994年 | 51篇 |
1993年 | 35篇 |
1992年 | 96篇 |
1991年 | 63篇 |
1990年 | 48篇 |
1989年 | 54篇 |
1988年 | 30篇 |
1987年 | 29篇 |
1986年 | 22篇 |
1985年 | 24篇 |
1984年 | 16篇 |
1983年 | 16篇 |
1982年 | 13篇 |
1981年 | 12篇 |
1980年 | 11篇 |
1979年 | 9篇 |
1978年 | 16篇 |
1975年 | 13篇 |
1974年 | 13篇 |
1966年 | 6篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
211.
Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67 下载免费PDF全文
Jhon H. S. Pires Felipe Gazos‐Lopes Milene C. Vallejo Tiago J. P. Sobreira Igor C. Almeida Rosana Puccia 《The Journal of eukaryotic microbiology》2015,62(5):591-604
Cryptococcus neoformans is an opportunistic human pathogen that causes life‐threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal–host relationship is discussed. 相似文献
212.
213.
Jing‐Yuan Chuang Wei‐Hung Yang Hsien‐Te Chen Chun‐Yin Huang Tzu‐Wei Tan Yuh‐Tzy Lin Chin‐Jung Hsu Yi‐Chin Fong Chih‐Hsin Tang 《Journal of cellular physiology》2009,220(2):418-426
CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
214.
Hyang Choi Hyun-Ho Kyeong Jung Min Choi Hak-Sung Kim 《Applied microbiology and biotechnology》2014,98(17):7483-7490
Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes. 相似文献
215.
Inside Back Cover: Development of a 3‐dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen—Laser‐detector position considerations (J. Biophotonics 3/2018) 下载免费PDF全文
Jim Larsson Peilang Liao Patrik Lundin Emilie Krite Svanberg Johannes Swartling Märta Lewander Xu Joakim Bood Stefan Andersson‐Engels 《Journal of biophotonics》2018,11(3)
The picture depicts the different 3d‐printed organs, thorax, lungs, heart and bone. Assembled it is used as an optical phantom of a preterm infant for performing percutaneous optical measurements of the gas content in the lungs. In order to simulate the optical properties of the tissue, the heart and thorax can be filled with liquid phantoms, a mixture of Intralipid and Indian Ink. Further details can be found in the article by Jim Larsson et al. ( e201700097 ).
216.
Stable nitrogen isotope analysis of dentine serial sections elucidate sex differences in weaning patterns of wild chimpanzees (Pan troglodytes) 下载免费PDF全文
Geraldine E. Fahy Michael P. Richards Benjamin T. Fuller Tobias Deschner Jean‐Jacques Hublin Christophe Boesch 《American journal of physical anthropology》2014,153(4):635-642
Offspring provisioning is one of the most energetically demanding aspects of reproduction for female mammals. Variation in lactation length and weaning strategies between chimpanzees (Pan troglodytes), our closest living relative, and modern human societies have been reported. When and why these changes occurred is frequently debated. Our study used stable nitrogen isotope data of tooth root dentine from wild Western chimpanzees (Pan troglodytes verus) in Taï National Park, Côte d'Ivoire, to quantify weaning in these chimpanzees and explore if infant sex plays a role in maternal investment. We analyzed serial sections of deciduous lateral incisor root dentine from four Taï chimpanzees to establish the δ15N signal of nursing infants; we then analyzed serial sections of first permanent mandibular molar root dentine from 12 Taï chimpanzees to provide quantitative δ15N data on weaning in this population. Up to 2 years of age both sexes exhibited dentine δ15N values ≈2–3‰ higher than adult female Taï chimpanzees, consistent with a nursing signal. Thereafter a steady decrease in δ15N values consistent with the onset, and progression, of weaning, was visible. Sex differences were also evident, where male δ15N values decreased at a significantly slower rate compared to females. Confirmation of sex differences in maternal investment among Taï chimpanzees, demonstrates the viability of using isotope analysis to investigate weaning in non‐human primates. Additionally, assuming that behaviors observed in the Taï chimpanzees are illustrative of the ancestral pattern, our results provide a platform to enable the trajectory of weaning in human evolution to be further explored. Am J Phys Anthropol 153:635–642, 2014. © 2013 Wiley Periodicals, Inc. 相似文献
217.
Jeong KJ Choi JH Yoo WM Keum KC Yoo NC Lee SY Sung MH 《Protein expression and purification》2004,36(1):150-156
High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner. 相似文献
218.
Ronny Martinez Felix Jakob Ran Tu Petra Siegert Karl‐Heinz Maurer Ulrich Schwaneberg 《Biotechnology and bioengineering》2013,110(3):711-720
Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (Kcat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5‐fold increased specific activity (15°C) and over 100 times prolonged half‐life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity‐altering amino acid substitutions were from non‐charged to non‐charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc. 相似文献
219.
Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8 approximately 6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72 approximately 5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes. 相似文献
220.