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41.
Robert S. Stelzer Damion R. Drover Susan L. Eggert Maureen A. Muldoon 《Biogeochemistry》2011,103(1-3):91-107
We measured net nitrate retention by mass balance in a 700-m upwelling reach of a third-order sand plains stream, Emmons Creek, from January 2007 to November 2008. Surface water and groundwater fluxes of nitrate were determined from continuous records of discharge and from nitrate concentrations based on weekly and biweekly sampling at three surface water stations and in 23 in-stream piezometers, respectively. Surface water nitrate concentration in Emmons Creek was relatively high (mean of 2.25 mg NO3?CN l?1) and exhibited strong seasonal variation. Net nitrate retention averaged 429 mg NO3?CN m?2 d?1 and about 2% of nitrate inputs to the reach. Net nitrate retention was highest during the spring and autumn when groundwater discharge was elevated. Groundwater discharge explained 57?C65% of the variation in areal net nitrate retention. Specific discharge and groundwater nitrate concentration varied spatially. Weighting groundwater solute concentrations by specific discharge improved the water balance and resulted in higher estimates of nitrate retention. Our results suggest that groundwater inputs of nitrate can drive nitrate retention in streams with high groundwater discharge. 相似文献
42.
Janine Walter Sascha Hausmann Thomas Drepper Michael Puls Thorsten Eggert Marcel Dihn�� 《PloS one》2012,7(9)
Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions. 相似文献
43.
Valentina Buttani Aba Losi Thorsten Eggert Ulrich Krauss Karl-Erich Jaeger Zhen Cao Wolfgang G?rtner 《Photochemical & photobiological sciences》2007,6(1):41-49
The Bacillus subtilis protein YtvA is related to plant phototropins in that it senses UVA-blue-light by means of the flavin binding LOV domain, linked to a nucleotide-binding STAS domain. The structural basis for interdomain interactions and functional regulation are not known. Here we report the conformational analysis of three YtvA constructs, by means of size exclusion chromatography, circular dichroism (CD) and molecular docking simulations. The isolated YtvA-LOV domain (YLOV, aa 25-126) has a strong tendency to dimerize, prevented in full-length YtvA, but still observed in YLOV carrying the N-terminal extension (N-YLOV, aa 1-126). The analysis of CD data shows that both the N-terminal cap and the linker region (aa 127-147) between the LOV and the STAS domain are helical and that the central beta-scaffold is distorted in the LOV domains dimers. The involvement of the central beta-scaffold in dimerization is supported by docking simulation of the YLOV dimer and the importance of this region is highlighted by light-induced conformational changes, emerging from the CD data analysis. In YtvA, the beta-strand fraction is notably less distorted and distinct light-driven changes in the loops/turn fraction are detected. The data uncover a common surface for LOV-LOV and intraprotein interaction, involving the central beta-scaffold, and offer hints to investigate the molecular basis of light-activation and regulation in LOV proteins. 相似文献
44.
Bruno Eggert 《Development genes and evolution》1936,135(2):269-281
Zusammenfassung Die Epithelkörper der einheimischen Salamandriden — untersucht wurdenTriton alpestris undSalamandra maculosa — entstehen gegen Ende der Metamorphose aus den Resten der 3. und 4. Kiemenspalte. Sie sind wohl ektodermaler Herkunft und gehen zunächst kontinuierlich in die Epidermis über. Bei der Weiterdifferenzierung lösen sie sich unter Rückbildung der Zellbrücke von der Epidermis, rücken in die Tiefe und verschmelzen gewöhnlich auf jeder Seite zu einem einheitlichen Organ. Die zwischen den Parenchymzellen liegenden epidermalen Drüsenzellen degenerieren oder lagern sich teilweise zu einer größeren Cyste zusammen, die ein mit Azan sich blau färbendes Sekret enthält. Diese Cysten gehen bei der Weiterentwicklung gewöhnlich zugrunde, da sie bisher bei erwachsenen Tieren nicht nachgewiesen worden sind. Nach Entfernung der Anlagen der Epithelkörper treten unter normalen Bedingungen keine äußerlich erkennbaren Ausfallserscheinungen auf. Derartige Tiere verhalten sich wie normale gleichaltrige und weisen ein diesen ähnliches Wachstum auf. 相似文献
45.
Jürgen Hartleb York Damm Rüdiger Arndt Enno Christophers Eggert Stockfleth 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,727(1-2)
5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S-
-cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l−1 and 25 to 5000 μg l−1 for plasma and urine samples, respectively, a limit of detection of 0.17 μg l−1, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons. 相似文献
46.
A survey of academics in Germany shows a lack of and a great demand for training in leadership skills. Subject Categories: Careers, Science Policy & PublishingSuccess and productivity in science is measured largely by the number of publications in scientific journals and the acquisition of third‐party funding to finance further research (Detsky, 2011). Consequently, as young researchers advance in their careers, they become highly trained in directly related skills, such as scientific writing, so as to increase their chances in securing publications and grants. Acquiring leadership skills, however, is often neglected as these do not contribute to the evaluation of scientific success (Detsky, 2011). Therefore, an early‐career researcher may become leader of a research group based on publication record and solicitation of third‐party funding, but without any training of leadership or team management skills (Lashuel, 2020). Leadership, in the context of academic research, requires a unique list of competencies, knowledge and skills in addition to “traditional” leadership skills (Anthony & Antony, 2017), such as managing change, adaptability, empathy, motivating individuals, and setting direction and vision among others. Academic leadership also requires promoting the research group’s reputation, networking, protecting staff autonomy, promoting academic credibility, and managing complexity (Anthony & Antony, 2017). 相似文献
47.
Sequence specific DNA binding proteins in eukaryotic cells must efficiently locate their binding sites in chromosomes. Restriction enzymes provide a simple model system with which to investigate the factors which influence this process. We have used P element mediated transformation to introduce a DNA fragment containing a set of characterized restriction sites into the Drosophila germline. Embryonic nuclei prepared from these transgenic animals were treated with restriction enzymes to probe the accessibility of the target restriction sites. The results show that the insert is within an accessible region of the chromosome and that restriction sites within the inserted sequence can be cut. However, the rate of cutting is biphasic. At each restriction site, a fraction of the chromosomes is cut rapidly after which the remainder is refractory. Similar levels of incomplete cutting are obtained when the same P element construct is examined at a different chromosomal location, when different sequence elements are introduced into the P element vector or when the experiment is carried out on nuclei from different embryonic stages. These results are discussed in terms of how sequence specific DNA binding proteins may locate their genomic targets in vivo. 相似文献
48.
49.
Simone Eggert Brea Midthune Barbara Cottrell Edward H. Koo 《The Journal of biological chemistry》2009,284(42):28943-28952
The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by β- and γ-secretase lead to the generation of the amyloid-β (Aβ) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Aβ generation. However, these studies have relied on APP mutations within the Aβ sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Aβ production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Aβ sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Aβ levels in transfected N2a cells. Similar results were obtained with the direct precursor of β-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Aβ peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect γ-secretase processing and that dimerization is not required for Aβ production.The mechanism of β-amyloid protein (Aβ)2 generation from the amyloid precursor protein is of major interest in Alzheimer disease research because Aβ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (1, 2). In the amyloidogenic pathway Aβ is released from the amyloid precursor protein (APP) (3) after sequential cleavages by β-secretase BACE1 (4–6) and by the γ-secretase complex (7, 8). BACE1 cleavage releases the large ectodomain of APP while generating the membrane-anchored C-terminal APP fragment (CTF) of 99 amino acids (C99). Cleavage of β-CTF by γ-secretase leads to the secretion of Aβ peptides of various lengths and the release of the APP intracellular domain (AICD) into the cytosol (9–11). The γ-secretase complex consists of at least four proteins: presenilin, nicastrin, Aph-I, and Pen-2 (12). Presenilin is thought to be the catalytic subunit of the enzyme complex (13), but how the intramembrane scission is carried out remains to be elucidated. Alternatively, APP can first be cleaved in the non-amyloidogenic pathway by α-secretase within the Aβ domain between Lys-16 and Leu-17 (14, 15). This cleavage releases the APP ectodomain (APPsα) while generating the membrane-bound C-terminal fragment (α-CTF) of 83 amino acids (C83). The latter can be further processed by the γ-secretase complex, resulting in the secretion of the small 3-kDa fragment p3 and the release of AICD.APP, a type I transmembrane protein (16) of unclear function, may act as a cell surface receptor (3). APP and its two homologues, APLP1 and APLP2, can dimerize in a homotypic or heterotypic manner and, in so doing, promote intercellular adhesion (17). In vivo interaction of APP, APLP1, and APLP2 was demonstrated by cross-linking studies from brain homogenates (18). To date at least four domains have been reported to promote APP dimerization; that is, the E1 domain containing the N-terminal growth factor-like domain and copper binding domain (17), the E2 domain containing the carbohydrate domain in the APP ectodomain (19), the APP juxtamembrane region (20), and the transmembrane domain (21, 22). In the latter domain the dimerization appears to be mediated by the GXXXG motif near the luminal face of the transmembrane region (21, 23). In addition to promoting cell adhesion, APP dimerization has been proposed to increase susceptibility to cell death (20, 24).Interestingly, by introducing cysteine mutations into the APP juxtamembrane region, it was shown that stable dimers through formation of these disulfide linkages result in significantly enhanced Aβ production (25). This finding is consistent with the observation that stable Aβ dimers are found intracellularly in neurons and in vivo in brain (26). Taken together, these results have led to the idea that APP dimerization can positively regulate Aβ production. However, other laboratories have not been able to confirm some of these observations using slightly different approaches (23, 27).To further address the question of how dimerization of APP affects cleavage by α-, β-, and γ-secretase, we chose to test this with a controlled dimerization system. Accordingly, we engineered a chimeric APP molecule by fusing a portion of the FK506-binding protein (FKBP) to the C terminus of APP such that the addition of the synthetic membrane-permeant bifunctional compound, AP20187, will induce dimerization of the APP-FKBP chimera in a controlled manner by binding to the FKBP domains. Using this system, efficient dimerization of APP up to 70% can be achieved in a time and concentration-dependent fashion. Our studies showed that controlled homodimerization of APP-FKBP leads to decreased total Aβ levels in transfected N2a cells. Homodimerization of the β-CTF/C99 fragment, the direct precursor of γ-secretase cleavage, showed comparable results. In addition, induced dimerization of APP did not lead to a modulation of different Aβ peptides as it was reported for GXXXG mutants within the transmembrane domain of APP (21). 相似文献
50.
Brockmeier U Caspers M Freudl R Jockwer A Noll T Eggert T 《Journal of molecular biology》2006,362(3):393-402
Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of biotechnology and protein production. In this study, the Gram-positive model bacterium Bacillus subtilis was optimized for heterologous protein secretion using a novel approach. Two lipolytic enzymes, cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin, were chosen as reporters for heterologous protein secretion. In a systematic screening approach, all naturally occurring (non-lipoprotein) Sec-type signal peptides (SPs) from B. subtilis were characterized for their potential in heterologous protein secretion. Surprisingly, optimal SPs in cutinase secretion were inefficient in esterase secretion and vice versa, indicating the importance of an optimal fit between the SP and the respective mature part of the desired secretion target proteins. These results highlight the need for individually optimal signal peptides for every heterologous secretion target. Therefore, the SP library generated in this study represents a powerful tool for secretion optimization in Gram-positive expression hosts. 相似文献