全文获取类型
收费全文 | 2856篇 |
免费 | 262篇 |
国内免费 | 22篇 |
专业分类
3140篇 |
出版年
2024年 | 7篇 |
2023年 | 15篇 |
2022年 | 40篇 |
2021年 | 62篇 |
2020年 | 40篇 |
2019年 | 58篇 |
2018年 | 58篇 |
2017年 | 44篇 |
2016年 | 82篇 |
2015年 | 160篇 |
2014年 | 183篇 |
2013年 | 218篇 |
2012年 | 251篇 |
2011年 | 235篇 |
2010年 | 148篇 |
2009年 | 147篇 |
2008年 | 158篇 |
2007年 | 149篇 |
2006年 | 145篇 |
2005年 | 125篇 |
2004年 | 124篇 |
2003年 | 79篇 |
2002年 | 94篇 |
2001年 | 63篇 |
2000年 | 58篇 |
1999年 | 52篇 |
1998年 | 33篇 |
1997年 | 12篇 |
1996年 | 13篇 |
1995年 | 9篇 |
1994年 | 8篇 |
1993年 | 7篇 |
1992年 | 29篇 |
1991年 | 23篇 |
1990年 | 17篇 |
1989年 | 19篇 |
1988年 | 21篇 |
1987年 | 13篇 |
1986年 | 15篇 |
1985年 | 11篇 |
1984年 | 10篇 |
1983年 | 13篇 |
1982年 | 10篇 |
1981年 | 7篇 |
1980年 | 7篇 |
1979年 | 10篇 |
1978年 | 6篇 |
1975年 | 7篇 |
1974年 | 9篇 |
1972年 | 5篇 |
排序方式: 共有3140条查询结果,搜索用时 0 毫秒
41.
Yong Chao Lu Hui Chen Kin Lam Fok Lai Ling Tsang Mei Kuen Yu Xiao Hu Zhang Jing Chen Xiaohua Jiang Yiu Wa Chung Alvin Chun Hang Ma Anskar Yu Hung Leung He Feng Huang Hsiao Chang Chan 《Cell research》2012,22(10):1453-1466
Although HCO3− is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO3− acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO3− on preimplantation embryo development can be suppressed by interfering the function of a HCO3−-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO3− or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO3− removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO3− to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs. 相似文献
42.
Daniil M. Prigozhin Inna V. Krieger John P. Huizar Daniela Mavrici Geoffrey S. Waldo Li-Wei Hung James C. Sacchettini Thomas C. Terwilliger Tom Alber 《PloS one》2014,9(12)
Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis. 相似文献
43.
The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers. 相似文献
44.
Introduction
Patients who require prolonged mechanical ventilation (PMV) are increasing and producing financial burdens worldwide. This study determines the cost per QALY (quality-adjusted life year), out-of-pocket expenses, and lifetime costs for PMV patients stratified by underlying diseases and cognition levels.Methods
A nationwide sample of 50,481 patients with continual mechanical ventilation for more than 21 days was collected during 1997–2007. After stratifying the patients according to specific diagnoses, a latent class analysis (LCA) was performed to categorise PMV patients with multiple co-morbidities into several homogeneous groups. The survival functions were estimated for individual groups using the Kaplan-Meier method and extrapolated to 300 months through a semi-parametric method. The survival functions were adjusted using an EQ-5D utility value derived from a convenience sample of 142 PMV patients to estimate quality-adjusted life expectancies (QALE). Another convenience sample of 165 patients was used to estimate the out-of-pocket expenses. The lifetime expenditures paid by the single-payer National Health Insurance (NHI) system and patients'' families were estimated by multiplying average monthly expenditures by the survival probabilities and summing the values over lifetime.Results
PMV therapy costs more than 100,000 U.S. dollars (USD) per QALY for all patients with poor cognition. For patients with partial cognition, PMV therapy costs less than 56,000 USD per QALY for those with liver cirrhosis, intracranial or spinal cord injuries, and 57,000–69,000 USD for patients with multiple co-morbidities under age of 65. The average lifetime cost of PMV was usually below 56,000 USD. The out-of-pocket expenses were often more than one-third of the total cost of treatment.Conclusions
PMV treatment for patients with poor cognition would cost more than 5 times Taiwan''s GDP (gross domestic products), or less cost-effective. The out-of-pocket expenses for PMV provision should also be considered in policy decision. 相似文献45.
46.
Khanitta Somtrakoon Sudarat Suanjit Prayad Pokethitiyook Maleeya Kruatrachue Hung Lee Suchart Upatham 《World journal of microbiology & biotechnology》2008,24(4):523-531
Pyrene and fluoranthene, when supplied as the sole carbon source, were not degraded by Burkholderia sp. VUN10013. However, when added in a mixture with phenanthrene, both pyrene and fluoranthene were degraded in liquid broth
and soil. The amounts of pyrene and fluoranthene in liquid media (initial concentrations of 50 mg l−1 each) decreased to 42.1% and 41.1%, respectively, after 21 days. The amounts of pyrene and fluoranthene in soil (initial
concentrations of 75 mg kg−1 dry soil each) decreased to 25.8% and 12.1%, respectively, after 60 days. None of the high molecular weight (HMW) polycylic
aromatic hydrocarbons (PAHs) tested adversely affected phenanthrene degradation by this bacterial strain and the amount of
phenanthrene decreased rapidly within 3 and 15 days of incubation in liquid broth and soil, respectively. Anthracene also
stimulated the degradation of pyrene or fluoranthene by Burkholderia sp. VUN10013, but to a lesser extent than phenanthrene. The extent of anthracene degradation decreased in the presence of
these HMW PAHs. 相似文献
47.
KCNQ1 variants associate with hypertension in type 2 diabetes and affect smooth muscle contractility in vitro 下载免费PDF全文
Kuo‐Chin Huang Te‐Mao Li Xiang Liu Jin‐Hua Chen Wen‐Kuei Chien Yi‐Tzone Shiao Hsinyi Tsang Ting‐Hsu Lin Chiu‐Chu Liao Shao‐Mei Huang Ju‐Pi Li Cheng‐Wen Lin Jung‐Chun Lin Chih‐Chien Lin Chih‐Ho Lai Chi‐Fung Cheng Wen‐Miin Liang Chien‐Hui Hung Ching‐Chu Chen Ying‐Ju Lin Fuu‐Jen Tsai 《Journal of cellular physiology》2017,232(12):3309-3316
48.
Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes
meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that
allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid
and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development
of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount
of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h.
We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a
number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The
second LLO construct contained an N-terminus His tag; it gave a yield of 3.5–8 mg l−1. The third contained a C-terminus His tag; it gave a yield of 2.5 mg l−1 LLO. The purified LLO from the latter two constructs retained its activity at 4°C for over a year as determined by bovine
red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant
LLO, and is the first to provide evidence of long-term stability of the toxin for further applications. 相似文献
49.
ShuChun Kuo ChungChing Chio ChaoHung Yeh JuiTi Ma WenPin Liu MaoTsun Lin KaoChang Lin ChingPing Chang 《Aging cell》2021,20(5)
Amyloid‐beta (Aβ) oligomer is known to contribute to the pathophysiology of age‐related macular degeneration. Herein, we aimed to elucidate the in vivo and in vitro effects of Aβ1‐42 application on retinal morphology in rats. Our in vivo studies revealed that intracerebroventricular administration of Aβ1‐42 oligomer caused dysmorphological changes in both retinal ganglion cells and retinal pigment epithelium. In addition, in vitro studies revealed that ARPE‐19 cells following Aβ1‐42 oligomer application had decreased viability along with apoptosis and decreased expression of the tight junction proteins, increased expression of both phosphor‐AKT and phosphor‐GSK3β and decreased expression of both SIRT1 and β‐catenin. Application of conditioned medium (CM) obtained from mesenchymal stem cells (MSC) protected against Aβ1‐42 oligomer‐induced retinal pathology in both rats and ARPE‐19 cells. In order to explore the potential role of peptides secreted from the MSCs, we applied mass spectrometry to compare the peptidomics profiles of the MSC‐CM. Gene ontology enrichment analysis and String analysis were performed to explore the differentially expressed peptides by predicting the functions of their precursor proteins. Bioinformatics analysis showed that 3‐8 out of 155–163 proteins in the MSC‐CM maybe associated with SIRT1/pAKT/pGSK3β/β‐catenin, tight junction proteins, and apoptosis pathway. In particular, the secretomes information on the MSC‐CM may be helpful for the prevention and treatment of retinal pathology in age‐related macular degeneration. 相似文献
50.
Antitumor agents. 258. Syntheses and evaluation of dietary antioxidant--taxoid conjugates as novel cytotoxic agents 总被引:1,自引:0,他引:1
Nakagawa-Goto K Yamada K Nakamura S Chen TH Chiang PC Bastow KF Wang SC Spohn B Hung MC Lee FY Lee FC Lee KH 《Bioorganic & medicinal chemistry letters》2007,17(18):5204-5209
Various dietary antioxidants, including vitamins, flavonoids, curcumin, and a coumarin, were conjugated with paclitaxel (1) through an ester linkage. The newly synthesized compounds were evaluated for cytotoxic activity against several human tumor cell lines as well as the corresponding normal cell lines. Interestingly, most tested conjugates selectively inhibited the growth of 1A9 (ovarian) and KB (nasopharyngeal) tumor cells without activity against other cell lines. Particularly, conjugates 16 and 20 were highly active against 1A9 (ED(50) value of 0.005 microg/mL) as well as KB (ED(50) values of 0.005 and 0.14 microg/mL, respectively) cells. Compound 22b, the glycinate ester salt of vitamin E conjugated with 1, appears to be a promising lead for further development as a clinical trial candidate as it exhibited strong inhibitory activity against Panc-1 (pancreatic cancer) with less effect on the related E6E7 (normal) cell line. 相似文献