首页 | 本学科首页   官方微博 | 高级检索  
文章检索
  按 检索   检索词:      
出版年份:   被引次数:   他引次数: 提示:输入*表示无穷大
  收费全文   2729篇
  免费   200篇
  国内免费   121篇
  2023年   23篇
  2022年   57篇
  2021年   103篇
  2020年   72篇
  2019年   90篇
  2018年   106篇
  2017年   73篇
  2016年   88篇
  2015年   160篇
  2014年   159篇
  2013年   218篇
  2012年   225篇
  2011年   180篇
  2010年   128篇
  2009年   103篇
  2008年   95篇
  2007年   112篇
  2006年   105篇
  2005年   107篇
  2004年   105篇
  2003年   80篇
  2002年   55篇
  2001年   64篇
  2000年   55篇
  1999年   55篇
  1998年   28篇
  1997年   26篇
  1996年   23篇
  1995年   28篇
  1994年   27篇
  1993年   19篇
  1992年   39篇
  1991年   30篇
  1990年   18篇
  1989年   22篇
  1988年   15篇
  1987年   11篇
  1986年   13篇
  1985年   15篇
  1984年   17篇
  1983年   17篇
  1982年   14篇
  1981年   8篇
  1980年   6篇
  1979年   7篇
  1977年   6篇
  1976年   8篇
  1975年   8篇
  1974年   7篇
  1971年   8篇
排序方式: 共有3050条查询结果,搜索用时 31 毫秒
991.
992.
Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2 × 104 to 0.2 pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8 ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.  相似文献   
993.
994.
Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a β-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr353 may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.  相似文献   
995.
996.
Wolbachia are intracellular bacteria that infect numerous and diverse arthropod species including economic pests of crops and disease vectors affecting livestock and humans. Research on these symbionts has identified profound effects of Wolbachia on their hosts with possible application in pest control. Such research often requires methods to cure infections. To facilitate future research on these bacteria, we reviewed the Wolbachia literature to summarize results of 110 studies spanning 62 taxa that report on treatment methods and outcomes. Application of tetracycline in diet is the most common method and is typically successful. Rifampicin is secondarily used, and may be successful when tetracycline is not. Elevated temperatures can be used to eliminate infections, but is not often used. Rearing hosts under crowded conditions or starvation has been shown to reduce Wolbachia titre which affects maternal transmission. Application of treatment methods has a number of considerations with possible implications for the interpretation of data. This review is intended to alert the reader to treatment options and potential non-target effects.  相似文献   
997.
To report clinical outcomes of kidney transplantation from pediatric brain and cardiac death donors (DBCD) in a single Chinese center and to investigate its feasibility to expand organ donor pool. 18 recipients, transplanted between August 2011 and October 2013 in the First Affiliated Hospital of Zhengzhou University, receive a single graft from DBCD donors age ranged from 1.5 to 13 years old. Renal function expressed as serum creatinine, blood urea nitrogen as well as eGFR values at 1, 2 weeks as well as 1-, 3-, 6-, and 12-months post-transplantation was evaluated. Graft size was also monitored at the same time by ultrasonography. In addition, delayed graft function, acute rejection, surgical complication as well as patient and graft survival were also assessed. The primary causes of DBCD donors included six cases of severe brain trauma and three cases of cerebral hemorrhage. The mean age of DBCD donors was (7.2 ± 3.4) years (range 1.5–13). The mean weight of DBCD donors was (29.8 ± 15.3) kilogram (range 13–67). The mean height of DBCD donors was (118.3 ± 27.8) centimeter (range 70–173). ECMO was applied to DBCD donors to avoid warm ischemia time and the applicating time was (79.8 ± 44.5) (range 32–180) minutes.There were seven males and 11 females recipients. Among which, 16 recipients were pediatrics and two recipients were adults. The mean age of the recipients was (14.6 ± 9.7) years (range 4–47). The mean weight of recipients was (31.9 ± 12.4) kilogram (range 11–54). The mean height of recipients was (138.0 ± 23.7) centimeter (range 84–172). Renal function recovered to normal within the first-week post-operation except one recipient which occurred acute rejection. Two cases of renal artery stenosis were found 2-week and 3-month post-transplantation, respectively. They subsequently underwent ballon angioplasty and followed up for 8 and 12 months, respectively, and no recurrence was found. One recipient developed ureteral leak. Five weeks later, the ureter leak healed after adequate drainage and prolongation of indwelling catheter. Graft size significantly and continuously increased during the first year, especially in the first 3-month post-transplantation. All the 18 recipients are alive at the last follow-up. Among which, 16 recipients are followed up for 12 months and 1-year recipient/graft survival rate is 100 %. The use of single kidney graft from pediatric DBCD could yield good short-term results.  相似文献   
998.
Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.  相似文献   
999.

Background

Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.

Result

We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.

Conclusion

The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.  相似文献   
1000.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号