全文获取类型
收费全文 | 2549篇 |
免费 | 205篇 |
国内免费 | 1篇 |
出版年
2022年 | 26篇 |
2021年 | 44篇 |
2020年 | 24篇 |
2019年 | 33篇 |
2018年 | 42篇 |
2017年 | 32篇 |
2016年 | 58篇 |
2015年 | 149篇 |
2014年 | 120篇 |
2013年 | 211篇 |
2012年 | 209篇 |
2011年 | 214篇 |
2010年 | 115篇 |
2009年 | 115篇 |
2008年 | 144篇 |
2007年 | 196篇 |
2006年 | 156篇 |
2005年 | 134篇 |
2004年 | 122篇 |
2003年 | 121篇 |
2002年 | 111篇 |
2001年 | 19篇 |
2000年 | 9篇 |
1999年 | 28篇 |
1998年 | 32篇 |
1997年 | 25篇 |
1996年 | 21篇 |
1995年 | 16篇 |
1994年 | 24篇 |
1993年 | 19篇 |
1992年 | 7篇 |
1991年 | 13篇 |
1990年 | 10篇 |
1988年 | 5篇 |
1987年 | 4篇 |
1986年 | 7篇 |
1985年 | 4篇 |
1984年 | 8篇 |
1982年 | 6篇 |
1981年 | 5篇 |
1980年 | 5篇 |
1979年 | 4篇 |
1978年 | 6篇 |
1976年 | 4篇 |
1975年 | 5篇 |
1974年 | 4篇 |
1973年 | 7篇 |
1950年 | 3篇 |
1943年 | 3篇 |
1937年 | 3篇 |
排序方式: 共有2755条查询结果,搜索用时 15 毫秒
191.
Wilna J. Moree Florence Jovic Timothy Coon Jinghua Yu Bin-Feng Li Fabio C. Tucci Dragan Marinkovic Raymond S. Gross Siobhan Malany Margaret J. Bradbury Lisa M. Hernandez Zhihong O’Brien Jianyun Wen Hua Wang Samuel R.J. Hoare Robert E. Petroski Aida Sacaan Ajay Madan Paul D. Crowe Graham Beaton 《Bioorganic & medicinal chemistry letters》2010,20(7):2316-2320
SAR of lead benzothiophene H1-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H1-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound. 相似文献
192.
Fiona P. Brennan Florence Abram Fabio A. Chinalia Karl G. Richards Vincent O'Flaherty 《Applied and environmental microbiology》2010,76(7):2175-2180
Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.Escherichia coli is a well-established indicator of fecal contamination in the environment. The organism''s validity as an indicator of water pollution is dependent, among other factors, on its fecal specificity and its inability to multiply outside the primary host, the gastrointestinal tracts of humans and warm-blooded animals (9). While many pathogens and indicator organisms are considered to be poorly adapted for long-term survival, or proliferation, outside their primary hosts (24), there is increasing evidence that this view needs to be reconsidered with respect to E. coli (17, 38). In particular, questions remain about its fate and survival capacity in environmental matrices, such as soil. While the habitat within the primary host is characterized by constant warm temperature conditions and a ready availability of nutrients and carbon, that of soil is often characterized by oligotrophic and highly dynamic conditions, temperature and pH variation, predatory populations, and competition with environmentally adapted indigenous microflora (39). Soils are thus typically considered to be suboptimal environments for enteric organisms, and growth is thought to be negligible, with die-off of organisms at rates reported to be a function of the interaction of numerous factors, including the type and physiological state of the microorganism, the physical, chemical, and biological properties of the soil, atmospheric conditions (including sunlight, moisture, and temperature), and organism application method (10).In recent years, the growth of E. coli in soils, sediments, and water in tropical and subtropical regions has been widely documented, and the organism is considered to be an established part of the soil biota within these regions (4, 5, 7, 12, 14, 19, 25, 32). The integration of E. coli as a component of the indigenous microflora in soils of tropical and subtropical regions may be attributable to the nutrient-rich nature and warm temperatures of these habitats (21, 39), combined with the metabolic versatility of the organism and its simple nutritional requirements (21). In addition to tropical and subtropical regions, the presence of autochthonous E. coli populations in the cooler soils of temperate and northern temperate regions has also been reported (6, 20, 22, 37), with one report on an alpine soil (34) and, most recently, a report on a maritime temperate grassland soil (3). The growth of E. coli within soils can act as a reservoir for the further contamination of bodies of water (20, 31, 32), compromising the indicator status of E. coli within these regions. As such, an understanding of the ecological characteristics of E. coli in soil is critical to its validation as an indicator organism. With respect to the input of pathogenic E. coli into the environment, this knowledge becomes essential for assessing the potential health risk to human and animal hosts from agricultural activities such as landspreading of manures and slurries (24).It has been suggested that E. coli can sustain autochthonous populations within soils in temperate regions, wherever favorable conditions exist (21). The phenotypic traits of the organism (including its metabolic diversity and its ability to grow both aerobically and anaerobically in a broad temperature range) may assist the persistence, colonization, and growth of E. coli when conditions permit. The challenging nature of the soil environment and the disparity of conditions between the primary host and the secondary habitat raises the question of how these E. coli populations survive and compete for niche space among the highly competitive and diverse coexisting populations of the indigenous microflora (15, 21). There is some evidence that naturalized E. coli may form genetically distinct populations in the environment (17, 20, 34, 36). This suggests that autochthonous E. coli populations in soil may have increased environmental fitness, facilitating their residence in soil (20, 34, 38). Little is known, however, of the physiology of these organisms, and their capacity for survival in soil remains poorly understood (21).Previous work (3) recorded continuous low-level leaching of viable E. coli from lysimeters of a poorly drained Luvic Stagnosol soil type, more than 9 years after the last application of fecal material. This finding was indicative of the growth of E. coli within the soil and suggested the presence of autochthonous E. coli populations within the soil that could be leached subsequently. To our knowledge, prior to this report, naturalized autochthonous E. coli populations persisting under the relatively oligotrophic, low-temperature conditions of maritime temperate soil environments had not been described previously. Growth within this soil was attributed chiefly to favorable characteristics of the soil, which include high clay and moisture contents, nutrient retention, and the presence of anaerobic zones. The objective of this work was to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached. In particular, we were interested in determining if the isolates possessed phenotypic characteristics that may enhance their capacity to survive and occupy niche space within the soil. This study tested the hypothesis that E. coli clones persisting in lysimeters of this soil form a genetically distinct grouping and possess a physiology tailored to the soil environment. 相似文献
193.
Yakhya Dieye Virginie Oxaran Florence Ledue-Clier Walid Alkhalaf Girbe Buist Vincent Juillard Chang Won Lee Jean-Christophe Piard 《Applied and environmental microbiology》2010,76(21):7332-7337
Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.The GRAS (generally recognized as safe) status of lactic acid bacteria (LAB) has catalyzed a myriad of promising applications using these bacteria as a vehicle for in situ delivery of bioactive proteins such as antigens or digestive enzymes in the gastrointestinal tract of the human host (4, 26). In the context of therapeutic applications of LAB, a major fundamental goal is to determine whether they can be engineered to deliver bioactive proteins to the right bacterial and host locations. We previously designed a protein-targeting system in LAB that addressed proteins to the desired bacterial site (i.e., cytoplasm, cell wall, or external medium), as validated using a model protein reporter and various antigens (14, 15). Studies investigating the use of LAB as vaccine delivery vehicles suggested that the cell-wall-anchored protein form may possess superior ability to induce a strong immune response (3, 14). Among the various surface display systems described in Gram-positive bacteria (13), a dedicated surface protein anchoring system catalyzed by sortases was first described and characterized in Staphylococcus aureus (29). It covalently anchors proteins via their C-terminal cell wall anchor (CWA) domain to the bacterial peptidoglycan. SrtA-like sortases process proteins bearing an LPXTG C-terminal motif and are considered to be the housekeeping sortase that anchors most proteins harboring a sorting signal (32). Other sortases were subsequently shown to anchor proteins bearing the same or other motifs (11, 16).Surprisingly, while the roles of sortases and LPXTG proteins are well documented in pathogens, few reports have examined these functions in other bacteria. A report suggests a relationship between sortase activity and adhesion of the LAB Lactobacillus salivarius, although direct involvement of sortase was not demonstrated (47). Recently, sortase activity was correlated to assembly of pili and adhesion properties in Lactobacillus rhamnosus (21). To further characterize sortase in LAB, we chose an industrially important member of this bacterial group, Lactococcus lactis, to study sortase A functionality in anchoring its putative substrates on the cell wall. 相似文献
194.
195.
Alexis Samba Mialoundama Dimitri Heintz Nurul Jadid Paul Nkeng Alain Rahier Jozsef Deli Bilal Camara Florence Bouvier 《Plant physiology》2010,153(3):970-979
The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.Later steps of carotenoid biosynthesis involve the formation of diverse cyclic carotenoids. For example, β-carotene, the vitamin A precursor, is synthesized de novo by photosynthetic organisms, limited nonphototrophic bacteria and fungi, and also by aphids (Moran and Jarvik, 2010) according to a multistep pathway that ends with the cyclization of lycopene by lycopene β-cyclase (LCY-B). Similarly, in pepper (Capsicum annuum) chromoplasts, antheraxanthin and violaxanthin are converted into the κ-cyclic carotenoids capsanthin and capsorubin, respectively, by capsanthin-capsorubin synthase (CCS). In both cases, the proposed mechanism involves a concerted protic attack and stabilization of a transient carbocation without any net redox change (Camara, 1980; Bouvier et al., 1994; Britton, 1998). Several cDNAs for LCY-B have been cloned from bacteria (Misawa et al., 1990; Cunningham et al., 1994; Armstrong, 1997; Cunningham and Gantt, 2001), fungi (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001), and plants (Hugueney et al., 1995; Ronen et al., 2000) using functional complementation. Information available from primary structures suggest that the cyclization of lycopene is catalyzed by holomeric proteins in photosynthetic organisms (Cunningham et al., 1994; Maresca et al., 2007), by holomeric (Misawa et al., 1990) or heteromeric (Krubasik and Sandmann, 2000; Viveiros et al., 2000) proteins in nonphotosynthetic bacteria, and by holomeric, bifunctional proteins in fungi that combine the activities of phytoene synthase and lycopene cyclase (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001). This structural diversity of LCY-Bs coupled to a lack of significant amino acid sequence identity between the lycopene cyclases from bacteria, fungi, and plants hinder our understanding of the catalytic mechanism of LCY-Bs and CCS. In addition, the N terminus of plant LCY-B and CCS contains an amino sequence motif characteristic of a polypeptide predicted to adopt a Rossmann fold (Rossmann et al., 1974) and suggests the binding of an as yet unknown dinucleotide prosthetic ligand. It has been shown using recombinant bacterial enzyme that the cyclization of lycopene into β-carotene strictly requires NADPH but proceeds without any net redox change (Schnurr et al., 1996; Hornero-Mendez and Britton, 2002). Under the same conditions, FAD alone could not sustain bacterial LCY-B activity (Schnurr et al., 1996). Much less is known about the dinucleotide requirements of plant carotenoid cyclases, which are highly conserved within plants but are extremely divergent in nonplant organisms. Previously, a crucial acidic domain for lycopene cyclase activity was identified using an affinity-labeling strategy followed by site-directed mutagenesis (Bouvier et al., 1997) in the absence of any crystal structures. This so-called 293-FLEET-297 motif of LCY-B and CCS contained two tandem Glu-295-Glu-296 residues that were essential for LCY-B- and κ-cyclase activities (Bouvier et al., 1997). However, it still remains unclear how the protic mechanism is compatible with the requirement of dinucleotide cofactors.To further explore the mechanism of plant carotenoid cyclases, we first choose pepper CCS as a prototypic enzyme because it displays a strong identity (52%) to pepper LCY-B, and we have shown previously that CCS could also catalyze the cyclization of lycopene into β-carotene (up to 25% of activity compared with LCY-B; Hugueney et al., 1995). Herein, we have shown that monomeric CCS purified to homogeneity from plant chromoplasts or recombinant CCS purified from Escherichia coli-transformed cells are typical flavoproteins containing one noncovalently bound FAD. We also observed that CCS-bound FAD is required for enzyme activity in the presence of NADPH, which functions as a reductant of FAD. During this process, no hydrogen is transferred to β-carotene or κ-cyclic carotenoids. In addition to this cofactor requirement, we also show from extensive site-directed mutagenesis using pepper CCS and LCY-B and Erwinia herbicola LCY-B (Mialoundama, 2009) that Glu-295 of pepper CCS and LCY-B plays a key role in the formation of β-carotene and κ-cyclic carotenoids, and we demonstrate that a similar role is played in structurally divergent bacterial LCY-Bs by Glu-196. These characteristics suggest that plant CCS and LCY-Bs are mechanistically similar to non-metal-assisted terpene cyclases, such as squalene:hopene cyclase and oxidosqualene cyclase, and additionally represent a new subfamily of flavoproteins like isopentenyl diphosphate isomerase type II, which catalyze carotenoid cyclization without any net redox modification of the substrate. 相似文献
196.
Soline Vigneau Florence Levillayer Herv Crespeau Laurence Cattolico Bernard Caudron Franck Bihl Catherine Robert Michel Brahic Jean Weissenbach Jean-Franois Bureau 《Genomics》2001,78(3):206-213
We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow). 相似文献
197.
van Duivenvoorde LM Han WG Bakker AM Louis-Plence P Charbonnier LM Apparailly F van der Voort EI Jorgensen C Huizinga TW Toes RE 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(3):1506-1515
Dendritic cells (DCs) are professional APCs which have the unique ability to present both foreign and self-Ags to T cells and steer the outcome of immune responses. Because of these characteristics, DCs are attractive vehicles for the delivery of therapeutic vaccines. Fully matured DCs are relatively well-defined and even used in clinical trials in cancer. DCs also have the potential to influence the outcome of autoimmunity by modulating the underlying autoimmune response. To gain a better appreciation of the abilities and mechanisms by which immunomodulatory DCs influence the outcome of T cell responses, we studied several immunomodulatory DCs (TNF-, IL-10-, or dexamethasone-stimulated bone marrow-derived DCs) side by side for their ability to modulate T cell responses and autoimmune diseases. Our data show that these differentially modulated DCs display a different composition of molecules involved in T cell activation. Although, all DC subsets analyzed were able to inhibit the induction of collagen-induced arthritis, the modulation of the underlying immune response was different. Vaccination with TNF- or IL-10-modulated DCs altered the Th1/Th2 balance as evidenced by the induction of IL-5- and IL-10-secreting T cells and the concomitant reduction of the IgG2a-IgG1 ratio against the immunizing Ag. In contrast, DCs modulated with dexamethasone did not affect the ratio of IL-5-producing vs IFN-gamma-producing T cells and tended to affect the Ab response in a nonspecific manner. These data indicate that distinct mechanisms can be used by distinct DC subsets to change the outcome of autoimmunity. 相似文献
198.
Teillet F Lacroix M Thiel S Weilguny D Agger T Arlaud GJ Thielens NM 《Journal of immunology (Baltimore, Md. : 1950)》2007,178(9):5710-5716
Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical. 相似文献
199.
CD8 T cell help for innate antitumor immunity 总被引:1,自引:0,他引:1
Shanker A Verdeil G Buferne M Inderberg-Suso EM Puthier D Joly F Nguyen C Leserman L Auphan-Anezin N Schmitt-Verhulst AM 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(10):6651-6662
Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis. 相似文献
200.
NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions. 相似文献