全文获取类型
收费全文 | 1768篇 |
免费 | 193篇 |
国内免费 | 9篇 |
出版年
2021年 | 17篇 |
2020年 | 20篇 |
2019年 | 22篇 |
2018年 | 30篇 |
2017年 | 28篇 |
2016年 | 23篇 |
2015年 | 58篇 |
2014年 | 56篇 |
2013年 | 84篇 |
2012年 | 99篇 |
2011年 | 97篇 |
2010年 | 76篇 |
2009年 | 63篇 |
2008年 | 74篇 |
2007年 | 70篇 |
2006年 | 80篇 |
2005年 | 68篇 |
2004年 | 58篇 |
2003年 | 59篇 |
2002年 | 45篇 |
2001年 | 53篇 |
2000年 | 44篇 |
1999年 | 49篇 |
1998年 | 25篇 |
1997年 | 35篇 |
1996年 | 26篇 |
1995年 | 27篇 |
1994年 | 22篇 |
1993年 | 21篇 |
1992年 | 32篇 |
1991年 | 35篇 |
1990年 | 33篇 |
1989年 | 28篇 |
1988年 | 28篇 |
1987年 | 21篇 |
1986年 | 19篇 |
1985年 | 31篇 |
1984年 | 21篇 |
1983年 | 21篇 |
1982年 | 19篇 |
1981年 | 17篇 |
1980年 | 19篇 |
1979年 | 27篇 |
1978年 | 15篇 |
1977年 | 19篇 |
1976年 | 12篇 |
1975年 | 11篇 |
1974年 | 27篇 |
1973年 | 14篇 |
1969年 | 11篇 |
排序方式: 共有1970条查询结果,搜索用时 15 毫秒
31.
S. Schmidt D. Havekost K. Kaiser J. Kauling H.‐J. Henzler 《Engineering in Life Science》2005,5(3):273-276
Protein crystallization offers great potential in downstream processing of pharmaceutical protein active ingredients. The advantages, which are well known and widely utilized in low‐molecular weight crystallization, can also be expected to be found to some extent in protein crystallization. However, there is still a marked need for improvement in two main areas of protein processing, namely, in crystallization from impure solutions and scale‐up. 相似文献
32.
Benjamin Zipser Sylvia Kaiser Norbert Sachser 《Ethology : formerly Zeitschrift fur Tierpsychologie》2013,119(11):970-982
Behavioural phenotypes can be studied from a variety of perspectives. Recent developments have focused on the individual, seeking patterns of behaviour that are stable over time and/or across different contexts (animal personalities). This study applied this method of understanding individual behavioural variability to domestic guinea pigs. Two behavioural domains were investigated: emotionality and social behaviour. Additionally, individual cortisol–stress reactivity and dominance status were examined. Adult male domestic guinea pigs living in large mixed‐sex colonies were subjected to a series of behavioural and physiological tests twice with an intertest interval of 8 wk. Individual consistency over time was found regarding social behaviour, cortisol reactivity and dominance status, whereas no stability regarding emotional behaviour was detected. Furthermore, no stability over contexts was found. Our results suggest that the concept of animal personality is applicable to domestic guinea pigs. The ecological relevance of these data is underscored by the fact that they were obtained in animals from a very rich, socially complex scenario. Moreover, our study highlights that behaviour alone is not sufficient to describe individual phenotypic consistency. Physiological parameters such as stress reactivity should be included in animal personality research. Furthermore dominance – a relative measure which is not an absolute attribute of individuals – proved to be stable over time and thus also shed light on individuality. 相似文献
33.
Jens T. Kaiser Douglas C. Rees 《Protein science : a publication of the Protein Society》2013,22(4):502-509
The mechanosensitive channel of small conductance (MscS) contributes to the survival of bacteria during osmotic downshock by transiently opening large diameter pores for the efflux of cellular contents before the membrane ruptures. Two crystal structures of the Escherichia coli MscS are currently available, the wild type protein in a nonconducting state at 3.7 Å resolution (Bass et al., Science 2002; 298:1582–1587) and the Ala106Val variant in an open state at 3.45 Å resolution (Wang et al., Science 2008; 321:1179–1183). Both structures used protein solubilized in the detergent fos‐choline‐14. We report here crystal structures of MscS from E. coli and Helicobacter pylori solubilized in the detergent β‐dodecylmaltoside at resolutions of 4.4 and 4.2 Å, respectively. While the cytoplasmic domains are unchanged in these structures, distinct conformations of the transmembrane domains are observed. Intriguingly, β‐dodecylmaltoside solubilized wild type E. coli MscS adopts the open state structure of A106V E. coli MscS, while H. pylori MscS resembles the nonconducting state structure observed for fos‐choline‐14 solubilized E. coli MscS. These results highlight the sensitivity of membrane protein conformational equilibria to variations in detergent, crystallization conditions, and protein sequence. 相似文献
34.
Stefanie Zimmermann Mouhssin Oufir Alejandro Leroux R. Luise Krauth-Siegel Katja Becker Marcel Kaiser Reto Brun Matthias Hamburger Michael Adams 《Bioorganic & medicinal chemistry》2013,21(22):7202-7209
In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei—the causative agent of Human African Trypanosomiasis—by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC–MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism. 相似文献
35.
The taxonomic status of two southern African coastal pipefish species, Syngnathus temminckii and Syngnathus watermeyeri, was investigated using a combination of morphological and genetic data. Morphological data showed that S. temminckii is distinct from the broadly distributed European pipefish Syngnathus acus, and a molecular phylogeny reconstructed using mitochondrial DNA recovered S. temminckii and S. watermeyeri as sister taxa. The southern African species share an evolutionary origin with north‐eastern Atlantic Ocean and Mediterranean Sea species, including S. acus. These data support the existence of a distinct southern African clade of Syngnathus pipefishes that has diverged in situ to form the two species present in the region today. 相似文献
36.
A first approach to discover new antimalarials has been recently performed in a combined approach with data from GlaxoSmithKline Tres Cantos Antimalarial Set, Novartis-GNF Malaria Box Data set and St. Jude Children’s Research Hospital. These data are assembled in the Malaria Box. In a first phenotypic forward chemical genetic approach, 400 chemicals were employed to eradicate the parasite in the erythrocytic stages. The advantage of phenotypic screens for the identification of novel chemotypes is that no a priori assumptions are made concerning a fixed target and that active compounds inherently have cellular bioavailability. In a first screen 40 mostly heterocyclic, highly active compounds (in nmol range of growth inhibition) were identified with EC50 values ≤2 μM against chloroquine-resistant Plasmodium falciparum strains and a therapeutic window ≥10 against two mammalian cell lines. 78 % of the compounds had no violations with the Lipinski Rule of 5 and only 1 % of the compounds showed cytotoxicity when applied at concentrations of 10 μM. This pre-selective step of parasitic eradication will be used further for a test of the Malaria Box with a potential in iron chelating capacity to inhibit deoxyhypusine hydroxylase (DOHH) from P. falciparum and vivax. DOHH, a metalloprotein which consists of ferrous iron and catalyzes the second step of the posttranslational modification at a specific lysine in eukaryotic initiation factor 5A (EIF-5A) to hypusine. Hypusine is a novel, non-proteinogenic amino acid, which is essential in eukaryotes and for parasitic proliferation. DOHH seems to be a “druggable” target, since it has only 26 % amino acid identity to its human orthologue. For a High-throughput Screening (HTS) of DOOH inhibitors, rapid and robust analytical tools are a prerequisite. A proteomic platform for the detection of hypusine metabolites is currently established. Ultra performance Liquid Chromatography enables the detection of hypusine metabolites with retention times of 7.4 min for deoxyhypusine and 7.3 min for hypusine. Alternatively, the analytes can be detected by their masses with gas chromatography/mass spectrometry or one-dimensional chromatography coupled to mass spectrometry. Moreover, the identified hits will be tracked further to test their efficacy in novel “in vitro assays”. Subsequently in vivo inhibition in a humanized mouse model will be tested. 相似文献
37.
38.
Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation
Jue Wang Lisa Wagner-Britz Anna Bogdanova Sandra Ruppenthal Kathrina Wiesen Elisabeth Kaiser Qinghai Tian Elmar Krause Ingolf Bernhardt Peter Lipp Stephan E. Philipp Lars Kaestner 《PloS one》2013,8(6)
Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca2+ sensor Fluo-4. Additionally, we developed an approach for analysing the Ca2+ responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca2+ influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca2+ response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca2+ revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca2+ influx and the associated pro-thrombotic activity. 相似文献
39.
Jurgen Vercauteren Gertjan Beheydt Mattia Prosperi Pieter Libin Stijn Imbrechts Ricardo Camacho Bonaventura Clotet Andrea De Luca Zehava Grossman Rolf Kaiser Anders S?nnerborg Carlo Torti Eric Van Wijngaerden Jean-Claude Schmit Maurizio Zazzi Anna-Maria Geretti Anne-Mieke Vandamme Kristel Van Laethem 《PloS one》2013,8(4)
Introduction
Clinically evaluating genotypic interpretation systems is essential to provide optimal guidance in designing potent individualized HIV-regimens. This study aimed at investigating the ability of the latest Rega algorithm to predict virological response on a short and longer period.Materials & Methods
9231 treatment changes episodes were extracted from an integrated patient database. The virological response after 8, 24 and 48 weeks was dichotomized to success and failure. Success was defined as a viral load below 50 copies/ml or alternatively, a 2 log decrease from the baseline viral load at 8 weeks. The predictive ability of Rega version 8 was analysed in comparison with that of previous evaluated version Rega 5 and two other algorithms (ANRS v2011.05 and Stanford HIVdb v6.0.11). A logistic model based on the genotypic susceptibility score was used to predict virological response, and additionally, confounding factors were added to the model. Performance of the models was compared using the area under the ROC curve (AUC) and a Wilcoxon signed-rank test.Results
Per unit increase of the GSS reported by Rega 8, the odds on having a successful therapy response on week 8 increased significantly by 81% (OR = 1.81, CI = [1.76–1.86]), on week 24 by 73% (OR = 1.73, CI = [1.69–1.78]) and on week 48 by 85% (OR = 1.85, CI = [1.80–1.91]). No significant differences in AUC were found between the performance of Rega 8 and Rega 5, ANRS v2011.05 and Stanford HIVdb v6.0.11, however Rega 8 had the highest sensitivity: 76.9%, 76.5% and 77.2% on 8, 24 and 48 weeks respectively. Inclusion of additional factors increased the performance significantly.Conclusion
Rega 8 is a significant predictor for virological response with a better sensitivity than previously, and with rules for recently approved drugs. Additional variables should be taken into account to ensure an effective regimen. 相似文献40.
Fabrice E. Graf Philipp Ludin Tanja Wenzler Marcel Kaiser Reto Brun Patient Pati Pyana Philippe Büscher Harry P. de Koning David Horn Pascal M?ser 《PLoS neglected tropical diseases》2013,7(10)
The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol. 相似文献