全文获取类型
收费全文 | 1181篇 |
免费 | 123篇 |
国内免费 | 56篇 |
出版年
2024年 | 2篇 |
2023年 | 16篇 |
2022年 | 31篇 |
2021年 | 57篇 |
2020年 | 43篇 |
2019年 | 51篇 |
2018年 | 66篇 |
2017年 | 36篇 |
2016年 | 63篇 |
2015年 | 73篇 |
2014年 | 76篇 |
2013年 | 82篇 |
2012年 | 100篇 |
2011年 | 99篇 |
2010年 | 57篇 |
2009年 | 61篇 |
2008年 | 52篇 |
2007年 | 51篇 |
2006年 | 48篇 |
2005年 | 24篇 |
2004年 | 23篇 |
2003年 | 28篇 |
2002年 | 28篇 |
2001年 | 28篇 |
2000年 | 14篇 |
1999年 | 10篇 |
1998年 | 21篇 |
1997年 | 9篇 |
1996年 | 7篇 |
1995年 | 5篇 |
1994年 | 7篇 |
1993年 | 3篇 |
1992年 | 8篇 |
1991年 | 13篇 |
1990年 | 4篇 |
1989年 | 7篇 |
1988年 | 8篇 |
1987年 | 8篇 |
1986年 | 3篇 |
1985年 | 3篇 |
1984年 | 4篇 |
1982年 | 3篇 |
1981年 | 2篇 |
1980年 | 5篇 |
1979年 | 4篇 |
1977年 | 4篇 |
1976年 | 6篇 |
1974年 | 3篇 |
1972年 | 2篇 |
1971年 | 1篇 |
排序方式: 共有1360条查询结果,搜索用时 281 毫秒
981.
Sandra Stefanovic‐Barrett Anna S Dickson Stephen P Burr James C Williamson Ian T Lobb Dick JH van den Boomen Paul J Lehner James A Nathan 《EMBO reports》2018,19(5)
Misfolded or damaged proteins are typically targeted for destruction by proteasome‐mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome‐mediated degradation of the soluble misfolded reporter, mCherry‐CL1, involves two ER‐resident E3 ligases, MARCH6 and TRC8. mCherry‐CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail‐anchored protein heme oxygenase‐1 (HO‐1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO‐1 following intramembrane proteolysis. Our results highlight how ER‐resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail‐anchored proteins. 相似文献
982.
Bradykinin‐mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c‐Kit+ progenitor cells 下载免费PDF全文
Gang Li Hui Che Wei‐Yin Wu Ling‐Jun Jie Guo‐Sheng Xiao Yan Wang Gui‐Rong Li 《Journal of cellular and molecular medicine》2018,22(10):4688-4699
Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin‐mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ () by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store‐operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free via ER‐IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit+ progenitor cells. 相似文献
983.
Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration 下载免费PDF全文
Choun‐Sea Lin Chen‐Tran Hsu Ling‐Hung Yang Lan‐Ying Lee Jin‐Yuan Fu Qiao‐Wei Cheng Fu‐Hui Wu Han C.‐W. Hsiao Yesheng Zhang Ru Zhang Wan‐Jung Chang Chen‐Ting Yu Wen Wang Li‐Jen Liao Stanton B. Gelvin Ming‐Che Shih 《Plant biotechnology journal》2018,16(7):1295-1310
Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants. 相似文献
984.
Developing a flexible,high‐efficiency Agrobacterium‐mediated sorghum transformation system with broad application 下载免费PDF全文
Ping Che Ajith Anand Emily Wu Jeffry D. Sander Marissa K. Simon Weiwei Zhu Amy L. Sigmund Gina Zastrow‐Hayes Michael Miller Donglong Liu Shai J. Lawit Zuo‐Yu Zhao Marc C. Albertsen Todd J. Jones 《Plant biotechnology journal》2018,16(7):1388-1395
Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi‐arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium‐mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free ‘quality events’ ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84‐7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9‐mediated gene knockouts in Tx430. 相似文献
985.
R B Whitaker R S Kauffman M Che R Finberg 《Journal of immunology (Baltimore, Md. : 1950)》1982,129(2):900-903
By fusing primed murine lymphocytes with a syngeneic T cell lymphoma, we have been able to select for H-2-restricted, virus-specific cytotoxic T cell hybridomas (CTH). These T cell hybrids, which replicate in ordinary tissue culture medium or in ascites, are capable of lysing virally infected target cells, and their activity is facilitated by the presence of lectins in the assay medium. Unlike cells mediating lectin nonspecific lysis, these hybridomas are H-2 restricted and specific for single viral proteins. The ability to maintain these cells in culture for over 18 mo and to pass them in vivo without loss of activity or specificity indicates that they will provide sufficient material for the analysis of surface proteins and genetic information required for the recognition and lysis of virally infected cells by killer T cells. 相似文献
986.
The common methods for analyzing bioaerosols are based on maintaining organism viability and quantifying culturability which
may result in the underestimation of microbial concentrations. The present study employed a well-developed technique that
only requires cellular DNA to identify organisms. Polymerase chain reaction (PCR) was chosen to amplify specific DNA sequence
from an organism, to detect and semi-quantify organisms. Suspensions ofFrancisella tularensis were aerosolized in a chamber, and air samples were collected using impingers. Samples were analyzed using limiting dilution
PCR, and the results compared with those from a traditional plate counting. Results indicated that the limiting dilution PCR
provides a new way to identify and quantify bioaerosols that does not rely on viability and culturability. Therefore, the
method would provide a more reliable estimate of airborne bacterial concentrations compared to traditional plate counts. 相似文献
987.
Loss of rotational mobility of band 3 proteins in human erythrocyte membranes induced by antibodies to glycophorin A. 总被引:2,自引:1,他引:1 下载免费PDF全文
The effect of antibodies to glycophorin A on the rotational diffusion of band 3 in human erythrocyte membranes was investigated by transient dichrosim. Three antibodies that recognize different epitopes on the exofacial domain of glycophorin A all strongly reduce the rotational mobility of band 3. The effect is at most only weakly dependent on the distance of the epitope from the membrane surface. The degree of immobilization obtained with two of the antibodies, BRIC14 and R18, is very similar to that produced by antibodies to band 3 itself. Similar results were obtained with membranes stripped of skeletal proteins. Fab fragments and an antibody to glycophorin C had no effect on band 3 rotational mobility. These results rule out a mechanism whereby band 3 rotational immobilization results from enhanced interactions with the membrane skeleton that are mediated by a conformational change in glycophorin A. Rather, they strongly indicate that the antibodies to glycophorin A cross-link existing band 3-glycophorin A complexes that have lifetimes that are long compared with the millisecond time scale of the transient dichroism measurements. 相似文献
988.
Membrane specializations in the peripheral retina of the housefly Musca domestica L. 总被引:1,自引:0,他引:1
Membrane specializations of the peripheral retina of the housefly (Musca domestica) are revealed in thin sections and freeze fracture/etch replicas. Septate junctions are abundant in corner areas of the pseudocone enclosure bonding: between homologous corneal pigment cells (CPC); between homologous large pigment cells (LPC); between CPC-LPC; between Semper cells (SC); between SC-CPC. Spot desmosomes are present between Semper cells. It is likely that septate junctions function as strengthening adhesions in this area. A new membrane specialization similar to a continuous junction was observed between retinular cells of the same or adjacent ommatidium. This junction has indistinct septa in the 115 A intermembrane cleft and is intermittent in character. When this junction is absent, the apposed cells gape apart. In freeze fracture studies, this junction is characterized by bridges composed of fused membrane particles and randomly arranged particles on the P face, and noncorresponding grooves on the E face. The ridges are elongate and roughly parallel and sometimes they form enclosures. Mitochondria line up along these junctions, often within 90 A of the unit membrane. This membrane specialization has characteristics of tight and continuous junctions. In line with previous findings, we suggest that this junction assists in retinular cell orientation, possibly in enforcing the ommatidial twist and in maintaining localized ionic concentration gradients between retinular cells. 相似文献
989.
Haijiao Jin Wei Fang Mingli Zhu Zanzhe Yu Yan Fang Hao Yan Minfang Zhang Qin Wang Xiajing Che Yuanyuan Xie Jiaying Huang Chunhua Hu Haifen Zhang Shan Mou Zhaohui Ni 《PloS one》2016,11(11)
BackgroundSeveral studies have suggested that urgent-start peritoneal dialysis (PD) is a feasible alternative to hemodialysis (HD) in patients with end-stage renal disease (ESRD), but the impact of the dialysis modality on outcome, especially on short-term complications, in urgent-start dialysis has not been directly evaluated. The aim of the current study was to compare the complications and outcomes of PD and HD in urgent-start dialysis ESRD patients.MethodsIn this retrospective study, ESRD patients who initiated dialysis urgently without a pre-established functional vascular access or PD catheter at a single center from January 2013 to December 2014 were included. Patients were grouped according to their dialysis modality (PD and HD). Each patient was followed for at least 30 days after catheter insertion (until January 2016). Dialysis-related complications and patient survival were compared between the two groups.ResultsOur study enrolled 178 patients (56.2% male), of whom 96 and 82 patients were in the PD and HD groups, respectively. Compared with HD patients, PD patients had more cardiovascular disease, less heart failure, higher levels of serum potassium, hemoglobin, serum albumin, serum pre-albumin, and lower levels of brain natriuretic peptide. There were no significant differences in gender, age, use of steroids, early referral to a nephrologist, prevalence of primary renal diseases, prevalence of co-morbidities, and other laboratory characteristics between the groups. The incidence of dialysis-related complications during the first 30 days was significantly higher in HD than PD patients. HD patients had a significantly higher probability of bacteremia compared to PD patients. HD was an independent predictor of short-term (30-day) dialysis-related complications. There was no significant difference between PD and HD patients with respect to patient survival rate.ConclusionIn an experienced center, PD is a safe and feasible dialysis alternative to HD for ESRD patients with an urgent need for dialysis. 相似文献
990.
Complex longitudinal diversification across South China and Vietnam in Stejneger's pit viper,Viridovipera stejnegeri (Schmidt, 1925) (Reptilia: Serpentes: Viperidae) 下载免费PDF全文
Fei Zhu Guang H. Zhong Xin Chen Edward A. Myers Jing Che Liang Zhang Thomas Ziegler Truong Q. Nguyen Frank T. Burbrink 《Molecular ecology》2016,25(12):2920-2936
Viridovipera stejnegeri is one of the most common pit vipers in Asia, with a wide distribution in southern China and Vietnam. We investigated historical demography and explored how the environment and climatic factors have shaped genetic diversity and the evolutionary history of this venomous snake. A total of 171 samples from 47 localities were sequenced and analysed for two mitochondrial gene fragments and three nuclear genes. Gene trees reveal the existence of two well‐supported clades (Southwest China and Southeast China) with seven distinct and strongly supported, geographically structured subclades within V. stejnegeri. Estimation of divergence time and ancestral area suggests that V. stejnegeri originated at ~6.0 Ma in the late Miocene on the Yunnan–Guizhou Plateau. The estimated date of origin and divergence of the island populations of Taiwan and Hainan closely matches the geological origin of the both islands. The mtDNA gene tree reveals the presence of west–east diversification in V. stejnegeri populations. Complex orogenesis and heterogeneous habitats, as well as climate‐mediated habitat differentiation including glacial cycles, all have influenced population structure and the distribution of this taxon. The validity of V. stejnegeri chenbihuii is questionable, and this subspecies most probably represents an invalid taxon. 相似文献