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31.
N6‐Methyladenosine (m6A) is one of the most important RNA modifications in epigenetics. The development of detection method for m6A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m6A modification and target RNAs without m6A modification. We verified the fact that the presence of m6A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m6A detection methods in the future.  相似文献   
32.
袁福香  刘实  胡艳全  张玉书  戴勇  曹蕊 《生态学报》2014,34(22):6711-6721
基于气候资料和日本松干蚧传播资料,根据传播扩散范围及入侵地的气候特征,分析了日本松干蚧主要影响因子的年代变化对日本松干蚧在东北地区扩散的影响。结果表明:东北地区最冷月各旬及月平均最低气温总的呈升高趋势(r=0.86,P0.05),冬季极端最低气温也有缓慢上升趋势(r=0.93,P0.01),其年代间的冷暖变化与日本松干蚧在东北地区扩散有明显的相关性。1月平均最低气温和冬季极端最低气温明显升高的20世纪70年代和90年代,日本松干蚧快速扩散、危害地虫口密度大、危害程度重。日本松干蚧大范围扩散和爆发都发生在1月份最低气温较高的年份。1月最低气温和冬季极端最低气温升高是日本松干蚧在东北地区传播扩散的重要因素。复苏后的降水量、卵孵化期的空气相对湿度和夏季最高气温的年代变化对日本松干蚧扩散的影响不显著。  相似文献   
33.
A phage display library of exendin-4 mutants was screened with the extracellular domain of rat glucagon-like peptide 1 receptor as the target. A novel variant of exendin-4 with higher affinity for the receptor fraction than that of the wild type was identified. The increased affinity was attributed to the substitution of Glu(16) by Val(16). Although the substitution probably caused an increased entropic cost to the helix region, the linker around Val(16) is more flexible resulting in the increase of affinity for the receptor.  相似文献   
34.
本研究以赶黄草地上部分为材料,研究大孔树脂纯化赶黄草黄酮的工艺,并评价体外抗氧化活性。根据大孔树脂对赶黄草黄酮的吸附和解吸性能,从7种不同类型的大孔树脂中筛选出适宜的树脂,进一步优化其纯化工艺,并比较纯化前后黄酮的体外抗氧化活性。试验结果表明,DM130大孔树脂对赶黄草黄酮有较好的吸附和解吸效果,其最佳纯化工艺参数:上样液黄酮浓度为1.0 mg/mL、pH为5、上样速度为1.0 mL/min、上样量为110 mL、洗脱液为70%乙醇、洗脱速度为1.0 mL/min和洗脱体积为40 mL。该工艺条件下,黄酮的纯度由20.04%提高至43.93%,提高了23.89%,表明DM130树脂对赶黄草黄酮的纯化效果较好。另外,纯化后赶黄草黄酮的DPPH自由基清除能力和还原力均显著提高。  相似文献   
35.
An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30 min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods.Methods for the analysis of cellular protein synthesis should be quantitative and fast. In 2006, Dieterich and coworkers introduced a proteomics discovery tool called bioorthogonal noncanonical amino acid tagging (BONCAT),1 in which noncanonical amino acids (ncAAs) with bioorthogonal functional groups (e.g. azides or alkynes) are used as metabolic labels to distinguish new proteins from old (1, 2). Labeled proteins can be conjugated to fluorescent reporters for visualization or affinity tags for purification and subsequent identification by mass spectrometry (3). Because the ncAA probe can be introduced to cells in a well-defined “pulse,” affinity purification removes pre-existing proteins and provides both reduced sample complexity and excellent time resolution.The methionine (Met) surrogate l-azidohomoalanine (Aha) has become standard in the application of BONCAT methodologies. Using Aha and fluorescent tagging, Tcherkezian et al. observed co-localization of the DCC receptor with sites of protein synthesis, providing support for the role of netrin as a stimulant of extranuclear protein production in neurons (4). Combining Aha labeling and 2D gel electrophoresis, Yoon et al. discovered that the protein lamin B2 is synthesized in axons and crucial to mitochondrial function and axon maintenance in Xenopus retinal glial cells (5). Aha has also been used to study histone turnover (6), protein palmitoylation (7), pathogen amino acid uptake (8), inflammatory response (9), and local translation in neuronal dendrites and axons (10). These labeling techniques have been expanded to tissue and animal culture, where Aha has been used to profile protein synthesis in rat hippocampal brain slices (11, 12) and zebrafish embryos (13).The development of fast, reliable, quantitative BONCAT methods will enable new insights into proteome dynamics in response to biological stimuli. Recent work by Eichelbaum et al. combined Aha labeling with stable isotope labeling to measure lipopolysaccharide-stimulated protein secretion by macrophages (14). Using similar approaches, Somasekharan et al. identified a set of proteins that are translationally regulated by the Y-box binding protein-1 (YB-1) in TC-32 Ewing sarcoma cells (15), and Howden et al. monitored changes in protein expression following stimulation of primary T cells with phorbol 12-myristate 13-acetate and ionomycin (16).A concern that arises in the use of Aha (as it does for all chemical probes of biological processes) is that the protocols used for Aha labeling might perturb cellular protein synthesis. The development of ncAAs as reliable analytic tools hinges on our ability to understand and minimize such unintended effects. For Aha, previous work has shown that protein labeling does not visibly alter cellular morphology in dissociated hippocampal neurons or HEK293 cells, and 1D gels reveal no discrepancies between the proteomes of Aha- and Met-treated cells (1). These experiments, however, offer only coarse measures of effects on protein synthesis, and as Aha labeling is frequently coupled to mass spectrometry-based proteomic analysis, the biological effects of Aha treatment must be investigated with equivalent sensitivity and resolution.Here we report sound methods for fast, reliable measurement of proteome dynamics via noncanonical amino acid tagging. First, we use the quantitative proteomics technique pulsed stable isotope labeling with amino acids in cell culture (pSILAC) to investigate potential unintended effects of Aha labeling on protein abundance in HeLa cell cultures, and we develop a strategy for minimizing these effects. Second, we show that a combined BONCAT-pSILAC approach, capable of both enriching and quantifying newly synthesized proteins, yields detailed proteomic information on time scales that are inaccessible to isotope labeling techniques alone.  相似文献   
36.
37.
Ma X  Zheng W  Wei D  Ma Y  Wang T  Wang J  Liu Q  Yang S 《Journal of biotechnology》2006,123(3):367-378
Survivin, a novel member of the IAP family, was observed to express in the most common human cancers. Anti-cancer therapy targeting survivin had drawn considerable attention. This study focused on high-level expression of recombinant protein TAT-survivin (T34A) mutant in E. coli, purification and bioactivity of pro-apoptosis to various cell lines in vitro. The cDNA encoding survivin was cloned by RT-PCR from breast cancer cell lines B-cap37. After PCR site-directed mutagenesis and construction of expression vector pRSET-B-TAT-survivin (T34A), targeted TAT-survivin (T34A) protein was expressed highly in E. coli BL21 (DE3) by 0.5mM IPTG induction and its yield could reach 650 mg/l in fermentation culture. The fusion protein in a form of inclusion body was then solubilized, refolded and purified to a purity of 98% by cation exchange chromatography and size-exclusion chromatography. Four hundred and eighty milligrams protein of interest was obtained in per liter fermentation culture. This showed that the efficient procedures of large-scale expression and purification were successful for the mass production of the recombinant protein. Pro-apoptosis effects of target protein on four cancer cell lines and one normal cell line from human were confirmed by the change of morphology, and pro-apoptosis activity was evaluated by MTT, fluorescent staining of nuclei and flow cytometry assay. Results indicated that B-cap37 and SW1990 were very sensitive to TAT-survivin (T34A) protein. This finding revealed the recombinant protein was promising as an anti-cancer drug.  相似文献   
38.
Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35°C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.  相似文献   
39.
Survivin gene and its two alternatively spliced variants, survivin-2 B and survivin- Delta Ex3 gene were cloned from human breast cancer cell lines B-cap37 firstly. A new gene designated as survivin-image (SI) was cloned from above cell lines, which has not been reported yet to clone from any cell lines. It was found that the novel gene 507 bp comprises partial survivin gene (345 bp), partial image gene (155 bp) of eye cancer and other insertion of 7 bp by analyzing with a series of recent bioinformatics software at the level of nucleotide and protein deduced. Predicted 3-D structures of the new molecule showed greatly similar to that of survivin in N-terminal containing BIR by homology modeling. These results suggested SI gene (GenBank accession No.AY830084) might be a novel alternatively spliced isoforms of the survivin gene involved in other functional significances related to tumorigenesis.  相似文献   
40.
Iodine excess may lead to thyroid diseases. Our previous 5-year prospective survey showed that the prevalence and incidence of hypothyroidism or autoimmune thyroiditis increased with iodine intake. The aim of the present study was to investigate the optimal range of iodine intake by comparing the prevalence of thyroid diseases in three areas with slightly different levels of iodine intake. In 2005, 778 unselected women subjects from three areas with different iodine intake levels were enrolled. Levels of serum thyroid hormones, thyroid autoantibodies, and urinary iodine were measured, and thyroid B ultrasounds were performed. Among the subjects with mildly deficient iodine intake, those with adequate intake, and those with more than adequate intake, the prevalence of clinical and subclinical hypothyroidism was 0, 1.13, and 2.84%, respectively (P = 0.014); that of thyroid goiter was 24.88, 5.65, and 11.37%, respectively (P < 0.001); that of serum thyrotropin values was1.01, 1.25, and 1.39 mIU/l, respectively; and that of serum thyrotropin/thyroglobulin ratio was 7.98, 6.84, and 5.11, respectively (P < 0.001). In conclusion, median urinary iodine 100~200 μg/l may reflect the safe range of iodine intake levels. Serum thyrotropin/thyroglobulin ratio might be a better index of evaluating iodine status.  相似文献   
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