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71.
72.
Elisabetta Catalani Silvia Tomassini Massimo Dal Monte Luigi Bosco Giovanni Casini 《Cell and tissue research》2009,336(3):423-438
Fibroblast growth factors (FGFs) exert basic functions both during embryonic development and in the adult. The expression
of FGFs and their receptors has been reported in mammalian retinas, although information on the organization of the FGF system
is still incomplete. Here, we report a detailed double-label immunohistochemical investigation of the localization patterns
of FGF1 and its receptors FGFR1 and FGFR2 in adult and early postnatal mouse retinas. In adult retinas, FGF1 is localized
to ganglion cells, horizontal cells, and photoreceptor inner and outer segments. FGFR1 is found in ganglion cells and Müller
cells, whereas FGFR2 is primarily located in ganglion cells, the nuclei of Müller cells, and glycine-containing amacrine cells.
During postnatal development, the patterns of FGF1, FGFR1, and FGFR2 immunostaining are similar to those in the adult, although
transient FGF1-expressing cells have been detected in the proximal inner nuclear layer before eye opening. These patterns
are consistent with a major involvement of FGF1, FGFR1, and FGFR2 in ganglion cell maturation (during development) and survival
(in the adult). Moreover, FGF1 may affect amacrine cell development, whereas Müller cells appear to be regulated via both
FGFR1 and FGFR2 throughout postnatal life. In immature retinas, large numbers of amacrine cells, including those containing
calbindin and glycine, display both FGF1 and FGFR2 immunoreactivities in their nuclei, suggesting an action of FGF1 on FGFR2
leading to the maturation of these amacrine cells during a restricted period of postnatal development.
This work was supported by funding from the Italian Ministry of Education. 相似文献
73.
Pietrovski EF Otuki MF Regoli D Bader M Pesquero JB Cabrini DA Zampronio AR 《Regulatory peptides》2009,152(1-3):67-72
Peptide and non-peptide kinin receptor antagonists were evaluated in cutaneous inflammation models in mice. Topical and i.p. application of kinin B(1) and B(2) receptor antagonists caused a significant inhibition of the capsaicin-induced cutaneous neurogenic inflammatory response. The calculated mean ID(50) for Hoe140 and SSR240612 were 23.83 (9.14-62.14) nmol/kg and 0.23 (0.15-0.36) mg/ear, respectively. The I(max) observed for Hoe140, SSR240612, R-715, FR173657, and FR plus SSR were 61+/-5%, 56+/-3%, 65+/-10%, 48+/-8%, and 52+/-4%, respectively. Supporting these results, double B(1) and B(2) kinin receptors knockout mice showed a significant inhibition of capsaicin-induced ear oedema (42+/-7%). However, mice with a single deletion of either B(1) or B(2) receptors exhibited no change in their capsaicin responses. In contrast, all of the examined kinin receptor antagonists were unable to inhibit the oedema induced by TPA and the results from knockout mice confirmed the lack of kinin receptor signaling in this model. These findings show that kinin receptors are present in the skin and that both kinin receptors seem to be important in the neurogenic inflammatory response. Moreover, non-peptide antagonists were very effective in reducing skin inflammation when topically applied, thereby suggesting that they could be useful tools in the treatment of some skin inflammatory diseases. 相似文献
74.
Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools. 相似文献
75.
Probing the Flexibility of Large Conformational Changes in Protein Structures through Local Perturbations
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Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states. 相似文献
76.
Schwenkert S Umate P Dal Bosco C Volz S Mlçochová L Zoryan M Eichacker LA Ohad I Herrmann RG Meurer J 《The Journal of biological chemistry》2006,281(45):34227-34238
Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently. 相似文献
77.
Bosco Christiano Maciel da Silva Maria Fernanda Rios Grassi Raimundo Coutinho Rita Elizabeth Moreira Mascarenhas Viviana Nilla Olavarria Adriana Coutinho-Borgo Jorge Kalil Edecio Cunha Neto Simone Gon?alves Fonseca 《Memórias do Instituto Oswaldo Cruz》2014,109(8):999-1004
The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of
Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and
potentially protective CD4+ T-cell epitopes from the most conserved
regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2,
phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm.
Seven peptide sequences predicted to bind to multiple human leukocyte antigen
(HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot
(ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux
tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight
percent of TST-positive donors responded to at least one of the peptides, compared to
25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs
from at least 31% of the TST-positive donors. The magnitude of the response against
all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among
TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p =
0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group.
This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort
of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose
latent TB and it may be included in ELISPOT-based IFN-γ assays to identify
individuals with this condition. 相似文献
78.
Yu B Yang M Wong HY Watt RM Song E Zheng BJ Yuen KY Huang JD 《Applied microbiology and biotechnology》2011,91(1):177-188
Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector
for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved
strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red
homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate
replacement of regions of chromosomal DNA with PCR-generated ‘targeting cassettes’ that contain flanking regions of shared
homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method
involves the use of linear DNA-targeting cassettes that contain relatively long flanking ‘arms’ of sequence (ca. 1,000 bp)
homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome
in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes
as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency
and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four
heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be
used to construct recombinant Ty21a antigen-expressing strains. 相似文献
79.
Ognibene M Vanni C Segalerba D Mancini P Merello E Torrisi MR Bosco MC Varesio L Eva A 《The Journal of biological chemistry》2011,286(34):29973-29983
The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between β1 and β2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin. 相似文献
80.
Anderson Messias Rodrigues Eduardo Bagagli Zoilo Pires de Camargo Sandra de Moraes Gimenes Bosco 《Mycopathologia》2014,177(3-4):199-206
Sporotrichosis is a polymorphic disease of man and animals caused by traumatic implantation of propagules into the skin and subcutaneous tissue. Pathogenic species includes S. brasiliensis, S. schenckii, S. globosa and S. luriei. The disease is remarkable for its occurrence as sapronoses and/or zoonosis outbreaks in tropical and subtropical areas; although, the ecology of the clinical clade is still puzzling. Here, we describe an anamorphic Sporothrix strain isolated from soil in an armadillo’s burrow, which was located in a hyper endemic area of Paracoccidioidomycosis in Brazil. This isolate was identified as S. schenckii sensu stricto (Clade IIa) based on morphological and physiological characteristics and phylogenetic analyses of calmodulin sequences. We then discuss the role of the nine-banded armadillo Dasypus novemcinctus as a natural carrier of Sporothrix propagules to better understand Sporothrix sources in nature and reveal essential aspects about the pathogen’s eco-epidemiology. 相似文献