全文获取类型
收费全文 | 4283篇 |
免费 | 303篇 |
国内免费 | 1篇 |
专业分类
4587篇 |
出版年
2023年 | 12篇 |
2022年 | 41篇 |
2021年 | 58篇 |
2020年 | 39篇 |
2019年 | 53篇 |
2018年 | 77篇 |
2017年 | 75篇 |
2016年 | 122篇 |
2015年 | 184篇 |
2014年 | 198篇 |
2013年 | 304篇 |
2012年 | 332篇 |
2011年 | 287篇 |
2010年 | 213篇 |
2009年 | 174篇 |
2008年 | 274篇 |
2007年 | 236篇 |
2006年 | 231篇 |
2005年 | 195篇 |
2004年 | 204篇 |
2003年 | 203篇 |
2002年 | 182篇 |
2001年 | 49篇 |
2000年 | 59篇 |
1999年 | 46篇 |
1998年 | 54篇 |
1997年 | 40篇 |
1996年 | 36篇 |
1995年 | 44篇 |
1994年 | 38篇 |
1993年 | 32篇 |
1992年 | 30篇 |
1991年 | 32篇 |
1990年 | 41篇 |
1989年 | 28篇 |
1988年 | 24篇 |
1987年 | 21篇 |
1986年 | 12篇 |
1985年 | 30篇 |
1984年 | 34篇 |
1983年 | 30篇 |
1982年 | 20篇 |
1981年 | 31篇 |
1980年 | 12篇 |
1979年 | 14篇 |
1977年 | 13篇 |
1976年 | 12篇 |
1975年 | 18篇 |
1974年 | 12篇 |
1971年 | 11篇 |
排序方式: 共有4587条查询结果,搜索用时 15 毫秒
41.
42.
Pozzolini M Sturla L Cerrano C Bavestrello G Camardella L Parodi AM Raheli F Benatti U Müller WE Giovine M 《Marine biotechnology (New York, N.Y.)》2004,6(6):594-603
In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies. 相似文献
43.
Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient 总被引:4,自引:0,他引:4
Mandruzzato S Rossi E Bernardi F Tosello V Macino B Basso G Chiarion-Sileni V Rossi CR Montesco C Zanovello P 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(7):4017-4024
It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3. 相似文献
44.
Margalith I Suter C Ballmer B Schwarz P Tiberi C Sonati T Falsig J Nyström S Hammarström P Aslund A Nilsson KP Yam A Whitters E Hornemann S Aguzzi A 《The Journal of biological chemistry》2012,287(23):18872-18887
Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits. 相似文献
45.
Understanding food web interactions in native or agricultural ecosystems is an important step towards establishing sustainable
pest management strategies. While the role of generalist predators as biological control agents is increasingly appreciated,
the study of trophic interactions between individual predator species and their prey provides practical difficulties. Recently,
different approaches have been suggested to determine prey items from predator guts using molecular methods. Macrolophus caliginosus is a generalist predator active in herbaceous agro-ecosystems. We developed a system to identify the DNA of its prey after
ingestion, using Myzus persicae as a model. Esterase (MpEST) and cytochrome oxidase I (MpCOI) genes were targeted in the aphid, while M. caliginosus COI gene was used as control for predator DNA. Real time PCR proved to be specific and sensitive enough to detect prey DNA upon
ingestion after feeding experiments. The system provided a linear amplification response with only 10 fg of prey genomic DNA
as template. The detection system of MpCOI gene was more sensitive than MpEST, while the detection period was similar for both genes. Possibilities for using the system in ecological and biosafety studies
with regard to sustainable pest management are discussed.
相似文献
Salvatore ArpaiaEmail: |
46.
Gil-Agusti MT Campostrini N Zolla L Ciambella C Invernizzi C Righetti PG 《Proteomics》2005,5(3):710-718
Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two-dimensional (2-D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and beta-mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 microL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3-(3-cholamidopropyldimethyl-amino)-1-propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2-D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up-regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down-regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix-assisted laser desorption/ionisation-time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase-high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed. 相似文献
47.
Mastroberardino PG Farrace MG Viti I Pavone F Fimia GM Melino G Rodolfo C Piacentini M 《Biochimica et biophysica acta》2006,1757(9-10):1357-1365
In this study we provide the first in vivo evidences showing that, under physiological conditions, "tissue" transglutaminase (TG2) might acts as a protein disulphide isomerase (PDI) and through this activity contributes to the correct assembly of the respiratory chain complexes. Mice lacking TG2 exhibit mitochondrial energy production impairment, evidenced by decreased ATP levels after physical challenge. This defect is phenotypically reflected in a dramatic decrease of motor behaviour of the animals. We propose that the molecular mechanism, underlying such a phenotype, resides in a defective disulphide bonds formation in ATP synthase (complex V), NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II) and cytochrome c oxidase (complex IV). In addition, TG2-PDI might control the respiratory chain by modulating the formation of the prohibitin complexes. These data elucidate a new pathway that directly links the TG2-PDI enzymatic activity with the regulation of mitochondrial respiratory chain function. 相似文献
48.
Leone M Di Lello P Ohlenschläger O Pedone EM Bartolucci S Rossi M Di Blasio B Pedone C Saviano M Isernia C Fattorusso R 《Biochemistry》2004,43(20):6043-6058
No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature. 相似文献
49.
Cervellati C Franzoni L Squerzanti M Bergamini CM Spinozzi F Mariani P Lanzara V Spisni A 《Amino acids》2009,36(4):633-641
Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site
to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP
counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible
with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is
reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine
concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In
contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium.
DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken
in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for
the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme
may acquire non-native conformations relevant to its patho-physiological functions. 相似文献
50.