全文获取类型
收费全文 | 2120篇 |
免费 | 84篇 |
专业分类
2204篇 |
出版年
2022年 | 6篇 |
2021年 | 28篇 |
2020年 | 13篇 |
2019年 | 24篇 |
2018年 | 33篇 |
2017年 | 35篇 |
2016年 | 50篇 |
2015年 | 68篇 |
2014年 | 86篇 |
2013年 | 151篇 |
2012年 | 151篇 |
2011年 | 159篇 |
2010年 | 108篇 |
2009年 | 71篇 |
2008年 | 125篇 |
2007年 | 115篇 |
2006年 | 110篇 |
2005年 | 104篇 |
2004年 | 111篇 |
2003年 | 89篇 |
2002年 | 102篇 |
2001年 | 19篇 |
2000年 | 24篇 |
1999年 | 22篇 |
1998年 | 23篇 |
1997年 | 20篇 |
1996年 | 11篇 |
1995年 | 17篇 |
1994年 | 19篇 |
1993年 | 21篇 |
1992年 | 13篇 |
1991年 | 18篇 |
1990年 | 15篇 |
1989年 | 17篇 |
1988年 | 14篇 |
1987年 | 13篇 |
1986年 | 12篇 |
1985年 | 11篇 |
1984年 | 12篇 |
1983年 | 7篇 |
1982年 | 11篇 |
1981年 | 12篇 |
1980年 | 6篇 |
1979年 | 6篇 |
1977年 | 7篇 |
1976年 | 8篇 |
1974年 | 7篇 |
1970年 | 6篇 |
1969年 | 13篇 |
1937年 | 5篇 |
排序方式: 共有2204条查询结果,搜索用时 15 毫秒
111.
Hirokazu Kariyazono Ryo Nadai Rin Miyajima Yuki Takechi‐Haraya Teruhiko Baba Akira Shigenaga Keiichiro Okuhira Akira Otaka Hiroyuki Saito 《Journal of peptide science》2016,22(2):116-122
Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A‐I (apoA‐I) and phospholipids. Although peptide‐based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA‐I‐based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline‐punctuated bihelical amphipathic structure based on apoA‐I mimetic peptides. NSP formed α‐helical structure on 1‐palmitoyl‐2‐oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA‐I‐POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA‐I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
112.
Kyo Seong Seo Kwang Ho Choo Ho Nam Chang Joong Kon Park 《Applied microbiology and biotechnology》2009,83(2):217-223
The biochemical oxygen demand (BOD) determination was studied using a novel flow injection analysis (FIA) system with encapsulated
Saccharomyces cerevisiae cells and an oxygen electrode and was compared with conventional 5-day BOD tests. S. cerevisiae cells were packed in a calcium alginate capsule at a dry cell weight of 250 g/l of capsule core. The level of dissolved oxygen
(DO) was reduced due to the enhanced respiratory activity of the microbial cells when the injected nutrient passed through
the bioreactor. The decrease in DO (ΔDO) was intensified with the amount of microbial cells packed in the bioreactor. However,
the specific ΔDO decreased as the amount of cells loaded in the bioreactor increased. The ΔDO value was dependent on the pH
and temperature of the mobile phase and reached its maximum value at 35°C and pH 7–8. Also, ΔDO became larger at longer response
times as the flow rate of the mobile phase decreased. The measurement of ΔDO was repeated more than six times consecutively
using a 20-ppm standard glucose and glutamic acid solution, which confirmed the reproducibility with a standard deviation
of 0.95%. A strong linear correlation between ΔDO and BOD was also observed. The 5-day BOD values of actual water and wastewater
samples were in accordance with the BOD values obtained by this FIA method using encapsulated S. cerevisiae cells. Unlike the cell-immobilized bead system, there was no contamination of the bioreactor resulting from any leak of yeast
cells from the sensor capsules during BOD measurements. 相似文献
113.
Ippei Sato Koichiro Morihira Hiroshi Inami Hirokazu Kubota Tatsuaki Morokata Keiko Suzuki Kazuki Ohno Yosuke Iura Aiko Nitta Takayuki Imaoka Toshiya Takahashi Makoto Takeuchi Mitsuaki Ohta Shin-ichi Tsukamoto 《Bioorganic & medicinal chemistry》2009,17(16):5989-6002
Our laboratory has identified several acrylamide derivatives with potent CCR3 inhibitory activity. In the present study, we evaluated the in vitro metabolic stability (CLint; mL/min/kg) of these compounds in human liver microsomes (HLMs), and assessed the relationship between their structures and CLint values. Among the compounds identified, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-[1-(2-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30j) was found to be a potent inhibitor (IC50 = 8.4 nM) with a high metabolic stability against HLMs. 相似文献
114.
Ogata Makoto Nakajima Makoto Kato Tatsuya Obara Takakiyo Yagi Hirokazu Kato Koichi Usui Taichi Park Enoch Y 《BMC biotechnology》2009,9(1):1-13
Background
Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.Results
Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 μg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.Conclusion
Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein. 相似文献115.
Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose
Mitsubayashi K Ohgoshi T Okamoto T Wakabayashi Y Kozuka M Miyajima K Saito H Kudo H 《Biosensors & bioelectronics》2009,24(5):1518-1521
A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent. 相似文献
116.
Manami Miyai Shingo Eikawa Akihiro Hosoi Tamaki Iino Hirokazu Matsushita Midori Isobe Akiko Uenaka Heiichiro Udono Jun Nakajima Eiichi Nakayama Kazuhiro Kakimi 《PloS one》2015,10(8)
Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring. 相似文献
117.
A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl α-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the K(I) value with little change in the t(1/2) value of the enzyme activity relative to the basic skeleton (2). 相似文献
118.
Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade + lac+ pro+ toMycobacterium phlei PA adeStm r by means of sexduction is described. Frequency of the phenotype PAade + Stmr increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade + Stmr to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful. 相似文献
119.
Mani SK Shiraishi H Balasubramanian S Yamane K Chellaiah M Cooper G Banik N Zile MR Kuppuswamy D 《American journal of physiology. Heart and circulatory physiology》2008,295(1):H314-H326
Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy. 相似文献
120.
Jaideep Dhariwal Jeremy Kitson Reema E. Jones Grant Nicholson Tanushree Tunstall Ross P. Walton Grace Francombe Jane Gilbert Andrew J. Tan Robert Murdoch Onn Min Kon Peter J. Openshaw Trevor T. Hansel 《PloS one》2015,10(9)