全文获取类型
收费全文 | 1201篇 |
免费 | 81篇 |
国内免费 | 1篇 |
出版年
2023年 | 6篇 |
2022年 | 16篇 |
2021年 | 17篇 |
2020年 | 11篇 |
2019年 | 18篇 |
2018年 | 14篇 |
2017年 | 16篇 |
2016年 | 21篇 |
2015年 | 53篇 |
2014年 | 60篇 |
2013年 | 61篇 |
2012年 | 95篇 |
2011年 | 96篇 |
2010年 | 57篇 |
2009年 | 38篇 |
2008年 | 67篇 |
2007年 | 87篇 |
2006年 | 70篇 |
2005年 | 63篇 |
2004年 | 45篇 |
2003年 | 55篇 |
2002年 | 60篇 |
2001年 | 23篇 |
2000年 | 14篇 |
1999年 | 19篇 |
1998年 | 25篇 |
1997年 | 18篇 |
1996年 | 14篇 |
1995年 | 23篇 |
1994年 | 12篇 |
1993年 | 15篇 |
1992年 | 12篇 |
1991年 | 8篇 |
1990年 | 7篇 |
1989年 | 13篇 |
1988年 | 2篇 |
1987年 | 4篇 |
1986年 | 6篇 |
1984年 | 5篇 |
1983年 | 2篇 |
1982年 | 5篇 |
1981年 | 4篇 |
1980年 | 4篇 |
1979年 | 5篇 |
1978年 | 3篇 |
1977年 | 7篇 |
1971年 | 1篇 |
1970年 | 1篇 |
1969年 | 1篇 |
1968年 | 2篇 |
排序方式: 共有1283条查询结果,搜索用时 15 毫秒
991.
992.
Christian Otten Stefania De Benedetti Ahmed Gaballah Henrike Bühl Anna Kl?ckner Jarryd Brauner Hans-Georg Sahl Beate Henrichfreise 《PloS one》2015,10(4)
Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the “chlamydial anomaly.” 相似文献
993.
Tiago H.S. Souza Camila G. Andrade Fernanda V. Cabral José F. Sarmento-Neto Júlio S. Rebouças Beate S. Santos Martha S. Ribeiro Regina C.B.Q. Figueiredo Adriana Fontes 《Biochimica et Biophysica Acta (BBA)/General Subjects》2021,1865(7):129897
BackgroundPhotodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP4+ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites.MethodsZnTnHex-2-PyP4+ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed.ResultsZnTnHex-2-PyP4+, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells.ConclusionsZnTnHex-2-PyP4+ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP4+, indicating readiness for evaluation in in vivo studies as an alternative approach to CL.General significanceThis report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms. 相似文献
994.
Pallavi Agarwal Jan-Niklas Schulz Katrin Blumbach Kristofer Andreasson Dick Heinegård Mats Paulsson Cornelia Mauch Sabine A. Eming Beate Eckes Thomas Krieg 《Matrix biology》2013,32(6):325-331
Skin fibrosis is characterized by activated fibroblasts and an altered architecture of the extracellular matrix. Excessive deposition of extracellular matrix proteins and altered cytokine levels in the dermal collagen matrix are common to several pathological situations such as localized scleroderma and systemic sclerosis, keloids, dermatosclerosis associated with venous ulcers and the fibroproliferative tissue surrounding invasively growing tumors. Which factors contribute to altered organization of dermal collagen matrix in skin fibrosis is not well understood. We recently demonstrated that cartilage oligomeric matrix protein (COMP) functions as organizer of the dermal collagen I network in healthy human skin (Agarwal et al., 2012). Here we show that COMP deposition is enhanced in the dermis in various fibrotic conditions. COMP levels were significantly increased in fibrotic lesions derived from patients with localized scleroderma, in wound tissue and exudates of patients with venous leg ulcers and in the fibrotic stroma of biopsies from patients with basal cell carcinoma. We postulate enhanced deposition of COMP as one of the common factors altering the supramolecular architecture of collagen matrix in fibrotic skin pathologies. Interestingly, COMP remained nearly undetectable in normally healing wounds where myofibroblasts transiently accumulate in the granulation tissue. We conclude that COMP expression is restricted to a fibroblast differentiation state not identical to myofibroblasts which is induced by TGFβ and biomechanical forces. 相似文献
995.
Bianca Schulte Isabel John Bernd Simon Christoph Brockmann Stefan A. Oelmeier Beate Jahraus Henning Kirchgessner Selina Riplinger Teresa Carlomagno Guido H. Wabnitz Yvonne Samstag 《The Journal of biological chemistry》2013,288(41):29430-29439
Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics. 相似文献
996.
997.
Constanze S?nger Andrea Schenk Lars Ole Schwen Lei Wang Felix Gremse Sara Zafarnia Fabian Kiessling Chichi Xie Weiwei Wei Beate Richter Olaf Dirsch Uta Dahmen 《PloS one》2015,10(11)
Introduction
The intra-hepatic vascular anatomy in rodents, its variations and corresponding supplying and draining territories in respect to the lobar structure of the liver have not been described. We performed a detailed anatomical imaging study in rats and mice to allow for further refinement of experimental surgical approaches.Methods
LEWIS-Rats and C57Bl/6N-Mice were subjected to ex-vivo imaging using μCT. The image data were used for semi-automated segmentation to extract the hepatic vascular tree as prerequisite for 3D visualization. The underlying vascular anatomy was reconstructed, analysed and used for determining hepatic vascular territories.Results
The four major liver lobes have their own lobar portal supply and hepatic drainage territories. In contrast, the paracaval liver is supplied by various small branches from right and caudate portal veins and drains directly into the vena cava. Variations in hepatic vascular anatomy were observed in terms of branching pattern and distance of branches to each other. The portal vein anatomy is more variable than the hepatic vein anatomy. Surgically relevant variations were primarily observed in portal venous supply.Conclusions
For the first time the key variations of intrahepatic vascular anatomy in mice and rats and their surgical implications were described. We showed that lobar borders of the liver do not always match vascular territorial borders. These findings are of importance for the design of new surgical procedures and for understanding eventual complications following hepatic surgery. 相似文献998.
Progress toward forecasting product quality and quantity of mammalian cell culture processes by performance‐based modeling 下载免费PDF全文
Timo Schmidberger Christoph Posch Alexandra Sasse Carina Gülch Robert Huber 《Biotechnology progress》2015,31(4):1119-1127
The production of biopharmaceuticals requires highly sophisticated, complex cell based processes. Once a process has been developed, acceptable ranges for various control parameters are typically defined based on process characterization studies often comprising several dozens of small scale bioreactor cultivations. A lot of data is generated during these studies and usually only the information needed to define acceptable ranges is processed in more detail. Making use of the wealth of information contained in such data sets, we present here a methodology that uses performance data (such as metabolite profiles) to forecast the product quality and quantity of mammalian cell culture processes based on a toolbox of advanced statistical methods. With this performance based modeling (PBM) the final product concentration and 12 quality attributes (QAs) for two different biopharmaceutical products were predicted in daily intervals throughout the main stage process. The best forecast was achieved for product concentration in a very early phase of the process. Furthermore, some glycan isoforms were predicted with good accuracy several days before the bioreactor was harvested. Overall, PBM clearly demonstrated its capability of early process endpoint prediction by only using commonly available data, even though it was not possible to predict all QAs with the desired accuracy. Knowing the product quality prior to the harvest allows the manufacturer to take counter measures in case the forecasted quality or quantity deviates from what is expected. This would be a big step towards real‐time release, an important element of the FDA's PAT initiative. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1119–1127, 2015 相似文献
999.
Mutations in PINK1 and PARK2/Parkin are a main risk factor for familial Parkinson disease. While the physiological mechanism of their activation is unclear, these proteins have been shown in tissue culture cells to serve as a key trigger for autophagy of depolarized mitochondria. Here we show that ablation of the mitochondrial rhomboid protease PARL leads to retrograde translocation of an intermembrane space-bridging PINK1 import intermediate. Subsequently, it is rerouted to the outer membrane in order to recruit PARK2, which phenocopies mitophagy induction by uncoupling agents. Consistent with a role of this retrograde translocation mechanism in neurodegenerative disease, we show that pathogenic PINK1 mutants which are not cleaved by PARL affect PINK1 kinase activity and the ability to induce PARK2-mediated mitophagy. Altogether we suggest that PARL is an important intrinsic player in mitochondrial quality control, a system substantially impaired in Parkinson disease as indicated by reduced removal of damaged mitochondria in affected patients. 相似文献
1000.
Katrin Paul Thomas Hartmann Christoph Posch Dirk Behrens Christoph Herwig 《Engineering in Life Science》2020,20(9-10):412-421
With increasing bioreactor volumes, the mixing time of the reactor increases as well, which creates an inhomogeneous environment for the cells. This can result in impaired process performance in large‐scale production reactors. Particularly the addition of base through the reactor headspace can be problematic, since it creates an area, where cells are repeatedly exposed to an increased pH. The aim of this study is to simulate this large‐scale phenomenon at lab‐scale and investigate its impact. Two different cell lines were exposed to pH amplitudes of a maximal magnitude of 0.05 units (pH of 6.95). Both cell lines showed similar responses, like decreased viable cell counts, but unaffected lactate levels. However, cell line B showed an initially increased specific productivity in response to the introduced amplitudes, whereas cell line A showed a consistently lower specific productivity. Furthermore, the time point at which base addition is started influences the impact, which pH amplitudes have on process performance. When pH control was started earlier in the process, maximal viable cell counts decreased and the lactate metabolic shift was less pronounced. These results show that the potential negative impact of pH amplitudes can be minimized by strategic process design. 相似文献