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91.
Spa47 is an oligomerization‐activated type three secretion system (T3SS) ATPase from Shigella flexneri
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Jamie L. Burgess Heather B. Jones Prashant Kumar Ronald T. Toth IV C. Russell Middaugh Edwin Antony Nicholas E. Dickenson 《Protein science : a publication of the Protein Society》2016,25(5):1037-1048
Gram‐negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti‐infective agents. 相似文献
92.
Vertical polyacrylamide gel electrophoresis was used to separate enzyme proteins at 73 putative loci in natural house fly
populations sampled in central Iowa. Thirty-nine of the loci were polymorphic (53%). The mean effective number of alleles
per polymorphic locus was 1.93 and 1.47 alleles among 68 scored loci. Observed and expected heterozygosities at 34 house fly
loci were 0.1628 and 0.1834, respectively. No statistically significant differentiation was detected among nine central Iowa
fly populations in 1989 or among nine Iowa and three Minnesota populations in 1990.
Journal Paper No. J-14125 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project No. 2949. 相似文献
93.
Henry Hoppe IV Katherine M. Call Phaik-Mooi Leong William G. Thilly 《Mutation research》1991,250(1-2):411-421
Cell of the human lymphoblast line WI-L2 and its derivative TK-6 were synchronized by centrifugal elutriation and cell-cycle dependent mutation to 6TGR (HPRT) and OUAR (Na+, K+ ATPase) measured. Bromodeoxyuridine induced 6TGR and OUAR mutations within S phase while butylmethylsulfonate induced mutation displayed no cell-cycle dependence. The data indicate that centrifugal elutriation is a facile means to obtain a useful degree of synchrony for these cell lines. 相似文献
94.
Richard W. Joy IV Edward C. Yeung Lisheng Kong Trevor A. Thorpe 《In vitro cellular & developmental biology. Plant》1991,27(1):32-41
Summary The growth and development of white spruce somatic embryos was followed from the filamentous immature to the mature cotyledonary
embryo stage. Histochemical examination of the various stages of embryo development showed that lipids, proteins, and polysaccharides
were produced to varying degrees during the process. During early stages (1 to 2 wk on ABA), mostly polysaccharide was produced,
whereas during later stages, polysaccharides, lipids, and protein accumulated. Electron microscopy indicated that lipid deposition
in somatic embryos started during the first week after transfer to ABA-containing medium. Deposition of the storage products
began at the basal end of the embryonal mass and within the proximal zone of the suspensors. Accumulation continued to the
peripheral regions and then inward toward the cortex of the developing embryo. In all cases, polysaccharide accumulated first,
followed by lipid and lastly, protein. Quantitatively, cotyledonary stage somatic embryos had less lipid and protein and more
starch when compared to zygotic embryos at the same developmental stage. Total protein profiles elucidated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis indicated that the majority of proteins were similar in zygotic and somatic embryos.
Prominent protein bands were found at 30, 20, 19.5, 15, 14.4, 12, and 10 Kd. However, protein bands at 40, 15, and 12 Kd in
total protein from somatic embryos were either absent or highly underexpressed. 相似文献
95.
96.
Erik A Karlsson Christopher T Small Pamela Freiden MM Feeroz Frederick A Matsen IV Sorn San M Kamrul Hasan David Wang Lisa Jones-Engel Stacey Schultz-Cherry 《PLoS pathogens》2015,11(11)
Astroviruses (AstVs) are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP) can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific. 相似文献
97.
98.
Grace S. Tan Barry G. Garchow Xuhang Liu Jennifer Yeung John P. Morris IV Trinna L. Cuellar Michael T. McManus Marianthi Kiriakidou 《Nucleic acids research》2009,37(22):7533-7545
Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells. 相似文献
99.
Roman Aranda IV Shauna M. Dineen Rhonda L. Craig James M. Robertson 《Analytical biochemistry》2009,387(1):122-127
Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng μl−1 range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-μl sample in the 500 to 5-ng μl−1 range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng μl−1 range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng μl−1 range. However, the Agilent kits require 1 μl of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful. 相似文献
100.
Nuttee Suree Chu Kong Liew Valerie A. Villareal William Thieu Evgeny A. Fadeev Jeremy J. Clemens Michael E. Jung Robert T. Clubb 《The Journal of biological chemistry》2009,284(36):24465-24477
In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2–4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9–14, 72).The sortase A (SrtA)7 enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between the threonine and glycine residues, forming a thioacyl-linked protein-sortase intermediate (16). It then catalyzes the formation of an amide bond between the carboxyl group of the threonine and the cell wall precursor molecule lipid II (undecaprenyl-pyrophosphate-MurNAc(-l-Ala-d-iGln-l-Lys(NH2-Gly5)-d-Ala-d-Ala)-β1–4-GlcNAc)), creating a protein-lipid II-linked product that is incorporated into the peptidylglycan via the transglycosylation and transpeptidation reactions of bacterial cell wall synthesis (18–20). Over 900 sortase-attached proteins in 72 different strains of bacteria have thus far been identified (21, 22). The vast majority of these proteins contain a COOH-terminal sorting signal harboring an LPXTG motif and are anchored to the cell wall by enzymes closely related to SrtA.In vitro studies of SrtA have begun to define the mechanism of transpeptidation. SrtA consists of two parts: an unstructured amino-terminal tail that contains a stretch of nonpolar residues that embed it in the membrane and an autonomously folded catalytic domain that competently performs the transpeptidation reaction in vitro (SrtAΔN59, residues 60–206) (16, 23–25). Catalysis occurs through a ping-pong mechanism that is initiated when the thiol group of amino acid Cys184 nucleophilically attacks the carbonyl carbon of the threonine residue within the LPXTG sorting signal (16, 23–25). This forms a transient tetrahedral intermediate that, upon breakage of the threonine-glycine peptide bond, rearranges into a more stable thioacyl enzyme-substrate linkage. SrtA then joins the terminal amine group within the pentaglycine branch of lipid II to the carbonyl carbon of the threonine, creating a second tetrahedral intermediate that is resolved into the lipid II-linked protein product (23).Sortase enzymes contain three conserved residues within their active sites: His120, Cys184, and Arg197 (SrtA numbering). These residues play a critical role in catalysis, since their mutation in SrtA causes severe reductions in enzyme activity (16, 26–30). Although it is well established that Cys184 forms a covalent linkage to the sorting signal, the functions of His120 and Arg197 are controversial. A variety of disparate functions have been ascribed to Arg197. These include deprotonating Cys184 (28), deprotonating lipid II (31), or stabilizing the binding of either the LPXTG sorting signal (28, 32) or oxyanion intermediates (31, 32). Different functions have also been proposed for His120. Originally, it was suggested that it activated Cys184 by forming an imidazolium-thiolate ion pair (26). However, subsequent pKa measurements revealed that both His120 and Cys184 are predominantly uncharged at physiological pH values, leading to the suggestion that His120 functions as a general base during catalysis (33). Most recently, it has been proposed that the most active form of the enzyme contains His120 and Cys184 in their charged states but that only a small fraction of SrtA exists in this form (∼0.06%) prior to binding to the sorting signal (25).NMR and crystal structures of SrtAΔN59 have revealed that it adopts an eight-stranded β-barrel fold (31, 34). Other sortase enzymes have also been shown to possess a similar overall structure, including SrtB from S. aureus (27, 35), SrtB from Bacillus anthracis (27, 36), SrtA from S. pyogenes (37), and the SrtC-1 and SrtC-3 enzymes from S. pneumoniae (38). However, the molecular basis of substrate recognition remains poorly understood, because all of the structures reported to date have not contained a sorting signal bound to the enzyme. The lone exception is the crystal structure of SrtAΔN59 bound to an LPETG peptide (31). However, in this structure the peptide substrate is bound nonspecifically (see below) (32, 39).In this paper, we report the structure and dynamics of SrtA covalently bound to an analog of the LPXTG sorting signal. The structure of the complex resembles the thioacyl intermediate of catalysis, providing insights into the molecular basis of binding of the LPXTG sorting signal and the functions of key active site residues. Notably, the mechanism of substrate binding visualized in the NMR structure differs substantially from a previously reported crystal structure of SrtAΔN59 non-covalently bound to a LPETG peptide (31). We have also used NMR chemical shift mapping experiments to localize the binding site for a triglycine cell wall substrate analog. A mechanism of transpeptidation compatible with these new data is proposed. 相似文献