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81.
为解决豫北地区没有鱼腥草栽培,缺乏耐低温能越冬材料的需求,本实验以鱼腥草茎段为外植体进行不同植物生长调节剂及其浓度的组织培养诱导侧芽发生,优化了鱼腥草侧芽组织培养体系;使用不同浓度的甲基磺酸乙酯(EMs)诱变侧芽,诱导耐低温突变体,通过测定诱变材料可溶性糖含量、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性变化鉴定材料耐寒性。结果表明茎段侧芽诱导的最佳植物生长调节剂组合培养基MS+6-BA2.0mg·L-1+NAA0.2mg·L-1;诱变处理的最佳组合为0.4%EMS处理6h,获得8株耐低温突变体;侧芽生根的最佳浓度组合为1/2MS+NAA0.8mg·L-1。 相似文献
82.
Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate
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Xiaoqi Tao Wenjun Wang Zhanhui Wang Xingyuan Cao Jinghui Zhu Lanlan Niu Xiaoping Wu Haiyang Jiang Jianzhong Shen 《Luminescence》2014,29(4):301-306
In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP‐C) in the presence of 3‐(10'‐phenothiazinyl) propane‐1‐sulfonate (SPTZ) and 4‐morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP‐catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N‐azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
83.
Xiaoming Yang Jinjiang Fan Alexander A. Ishchenko Devang Patel Murat K. Saparbaev Dindial Ramotar 《DNA Repair》2012,11(10):811-822
Caenorhabditis elegans possesses two distinct DNA repair enzymes EXO-3 and APN-1 that have been identified by cross-specie complementation analysis of the Saccharomyces cerevisiae apn1Δ apn2Δ tpp1Δ triple mutant deficient in the ability to repair apurinic/apyrimidinc (AP) sites and DNA strand breaks with blocked 3′-ends. While purified EXO-3 directly incises AP sites and removes 3′-blocking groups, such characterization has not been previously reported for APN-1. We recently documented that C. elegans knockdown for apn-1 is unable to maintain integrity of the genome. Despite the presence of EXO-3, the apn-1 knockdown animals are also defective in the division of the P1 blastomere, an observation consistent with the accumulation of unrepaired DNA lesions suggesting a unique role for APN-1 DNA repair functions. Herein, we show that C. elegans APN-1 is stably expressed as GST-fusion protein in S. cerevisiae only when it carries a nuclear localization signal, and with this requirement rescued the DNA repair defects of the S. cerevisiae apn1Δ apn2Δ tpp1Δ triple mutant. We purified the APN-1 from the yeast expression system and established that it displays AP endonuclease and 3′-diesterase activities. In addition, we showed that APN-1 also possesses a 3′- to 5′-exonuclease and the nucleotide incision repair activity. This latter activity is capable of directly incising DNA at the 5′-side of various oxidatively damaged bases, as previously observed for Escherichia coli endonuclease IV and S. cerevisiae Apn1, underscoring the importance of this family of enzymes in removing these types of lesions. Glycine substitution of the conserved amino acid residue Glu261 of APN-1, corresponding to Glu145 involved in coordinating Zn2+ ions in the active site pocket of E. coli endonuclease IV, resulted in an inactive variant that lose the ability to rescue the DNA repair defects of S. cerevisiae apn1Δ apn2Δ tpp1Δ mutant. Interestingly, the Glu261Gly variant did not sustain purification and yielded a truncated polypeptide. These data suggest that the Glu261 residue of APN-1 may have a broader role in maintaining the structure of the protein. 相似文献
84.
Zahra Amini-BayatSaman Hosseinkhani Rahim JafariKhosro Khajeh 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(2):350-358
Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D474 and K445 accompanying with some H-bond deletion may be involved in destabilization of D474K mutant. 相似文献
85.
Ramautar R Heemskerk AA Hensbergen PJ Deelder AM Busnel JM Mayboroda OA 《Journal of Proteomics》2012,75(13):3814-3828
Capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a powerful technique for the analysis of proteins and peptides. Over the past few years, significant progress has been made in the development of novel and more effective interfaces for hyphenating CE to MS. This review provides an overview of these new interfacing techniques for coupling CE to MS, covering the scientific literature from January 2007 to December 2011. The potential of these new CE-MS interfacing techniques is demonstrated within the field of (clinical) proteomics, more specifically "bottom-up" proteomics, by showing examples of the analysis of various biological samples. The relevant papers on CE-MS for proteomics are comprehensively summarized in tables, including, e.g. information on sample type and pretreatment, interfacing and MS detection mode. Finally, general conclusions and future perspectives are provided. 相似文献
86.
Pierce A Mirzaei H Muller F De Waal E Taylor AB Leonard S Van Remmen H Regnier F Richardson A Chaudhuri A 《Journal of molecular biology》2008,382(5):1195-1210
It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS. 相似文献
87.
Duarte IC Oliveira LL Saavedra NK Fantinatti-Garboggini F Oliveira VM Varesche MB 《Biodegradation》2008,19(3):375-385
The purpose of this work was to assess the degradation of linear alkylbenzene sulfonate (LAS) in a horizontal-flow anaerobic
immobilized biomass (HAIB) reactor. The reactor was filled with polyurethane foam where the sludge from a sanitary sewage
treatment was immobilized. The hydraulic detention time (HDT) used in the experiments was of 12 h. The reactor was fed with
synthetic substrate (410 mg l−1 of meat extract, 115 mg l−1 of starch, 80 mg l−1 of saccharose, 320 mg l−1 of sodium bicarbonate and 5 ml l−1 of salt solution) in the following stages of operation: SI—synthetic substrate, SII—synthetic substrate with 7 mg l−1 of LAS, SIII—synthetic substrate with 14 mg l−1 of LAS and SIV—synthetic substrate containing yeast extract (substituting meat extract) and 14 mg l−1 of LAS, without starch. At the end of the experiment (313 days) a degradation of ∼35% of LAS was achieved. The higher the
concentration of LAS, the greater the amount of foam for its adsorption. This is necessary because the isotherm of LAS adsorption
in the foam is linear for the studied concentrations (2 to 50 mg l−1). Microscopic analyses of the biofilm revealed diverse microbial morphologies, while Denaturing Gradient Gel Eletrophoresis
(DGGE) profiling showed variations in the population of total bacteria and sulphate-reducing bacteria (SRB). The 16S rRNA
gene sequencing and phylogenetic analyses revealed that the members of the order Clostridiales were the major components of
the bacterial community in the last reactor operation step. 相似文献
88.
Peressutti SR Olivera NL Babay PA Costagliola M Alvarez HM 《Journal of applied microbiology》2008,105(2):476-484
Aims: To isolate and identify linear alkylbenzene sulfonate (LAS)-degrading bacteria from Río de la Plata and adjacent waters, and to assay their degradation capability as a consortium and as single organisms.
Methods and Results: A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO2 release (mineralization) than individual cultures, and degraded 86% of LAS (20 mg l−1 ), whereas pure strains degraded between 21% and 60%. Bacterial desulfonation from LAS was evidenced in the consortium and A. caviae strains. A complete disappearance of LAS (10 mg l−1 ) was accomplished, and LAS levels of 50 and 100 mg l−1 led to a pronounced decrease in the biodegradation extent and inhibition of culture growth.
Conclusions: A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study: The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems. 相似文献
Methods and Results: A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO
Conclusions: A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study: The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems. 相似文献
89.
Enhanced fluorescence of tetrasulfonated zinc phthalocyanine by graphene quantum dots and its application in molecular sensing/imaging
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When excited at 435 nm, tetra‐sulfonate zinc phthalocyanine (ZnPcS4) emitted dual fluorescence at 495 and 702 nm. The abnormal fluorescence at 495 nm was experimentally studied and analyzed in detail for the first time. The abnormal fluorescence at 495 nm was deduced to originate from triplet–triplet (T–T) energy transfer of excited phthalocyanine (3*ZnPcS4). Furthermore, graphene quantum dots (GQDs) enhanced the 495 nm fluorescence quantum yield (Q) of ZnPcS4. The fluorescence properties of ZnPcS4–GQDs conjugate were retained in a cellular environment. Based on the fluorescence of ZnPcS4–GQDs conjugate, we designed and prepared an Apt29/thrombin/Apt15 sandwich thrombin sensor with high specificity and affinity. This cost‐saving, simple operational sensing strategy can be extended to use in sensing/imaging of other biomolecules. 相似文献
90.
Eduardo Lissi Elsa Abuin María E. Lanio Carlos Alvarez 《Journal of biochemical and biophysical methods》2002,50(2-3):261-268
A new and simple method useful for the evaluation of the association of surfactants to proteins is proposed. The method is based on an analysis of the effect promoted by surfactant addition upon the fluorescence intensity of the intrinsic tryptophan chromophore and its dependence with protein concentration. The proposed methodology is applied to quantify the binding of an anionic (sodium dodecylsulfate), a zwitterionic (N-hexadecyl-N,N′-dimethyl-3-ammonio-1-propane-sulfonate) and a neutral (Triton X-100, reduced) surfactant to bovine serum albumin (BSA). 相似文献