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31.
Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State 下载免费PDF全文
Mnica Ordax Ester Marco-Noales María M. Lpez Elena G. Biosca 《Applied microbiology》2006,72(5):3482-3488
Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper. 相似文献
32.
Ordax M Marco-Noales E López MM Biosca EG 《Applied and environmental microbiology》2006,72(5):3482-3488
Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu(2+) was inoculated with 10(7) CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper. 相似文献
33.
Pujadas M Pichini S Poudevida S Menoyo E Zuccaro P Farré M de la Torre R 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,798(2):249-255
A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), 3,4-methylenedioxyethylamphetamine (MDE or MDEA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair. Hair samples were digested with 1 M sodium sulfide at 37 degrees C (by shaking for 3 h and was kept at room temperature overnight), and extracted with two sequential extraction procedures: liquid-liquid extraction with tert-butyl methyl ether and solid-phase extraction with Bond-Elut Certify columns. Extracted analytes were derivatised with N-methyl-bis(trifluoroacetamide), separated by a 5% phenylmethylsilicone column and determined by a mass spectrometer detector in selected ion monitoring mode. A good reproducibility (intra-assay R.S.D.=1.5-15.7%), accuracy (intra-assay error = 2.0-11.7%) and sensitivity (LOD=0.03-0.08 ng/mg hair) were attained. The method was successfully applied to the analysis of the proximal (1 cm) hair segment to assess recent self-reported use in "ecstasy" consumers. Otherwise, further studies are needed to validate methodology developed in case of amphetamine consumption. 相似文献
34.
Heba Diab Masashi Ohira Mali Liu Ester Cobb Patricia M. Kane 《The Journal of biological chemistry》2009,284(20):13316-13325
Disassembly of the yeast V-ATPase into cytosolic V1 and membrane
V0 sectors inactivates MgATPase activity of the
V1-ATPase. This inactivation requires the V1 H subunit
(Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem.
275, 21761–21767), but its mechanism is not fully understood. The H
subunit has two domains. Interactions of each domain with V1 and
V0 subunits were identified by two-hybrid assay. The B subunit of
the V1 catalytic headgroup interacted with the H subunit N-terminal
domain (H-NT), and the C-terminal domain (H-CT) interacted with V1
subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic
N-terminal domain of V0 subunit Vph1p. V1-ATPase
complexes from yeast expressing H-NT are partially inhibited, exhibiting 26%
the MgATPase activity of complexes with no H subunit. The H-CT domain does not
copurify with V1 when expressed in yeast, but the bacterially
expressed and purified H-CT domain inhibits MgATPase activity in V1
lacking H almost as well as the full-length H subunit. Binding of full-length
H subunit to V1 was more stable than binding of either H-NT or
H-CT, suggesting that both domains contribute to binding and inhibition.
Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but
this fragment of Vph1p does not bind to V1 complexes containing
subunit H. We propose that upon disassembly, the H subunit undergoes a
conformational change that inhibits V1-ATPase activity and
precludes V0 interactions.V-ATPases are ubiquitous proton pumps responsible for compartment
acidification in all eukaryotic cells
(1,
2). These pumps couple
hydrolysis of cytosolic ATP to proton transport into the lysosome/vacuole,
endosomes, Golgi apparatus, clathrin-coated vesicles, and synaptic vesicles.
Through their role in organelle acidification, V-ATPases are linked to
cellular functions as diverse as protein sorting and targeting, zymogen
activation, cytosolic pH homeostasis, and resistance to multiple types of
stress (3). They are also
recruited to the plasma membrane of certain cells, where they catalyze proton
export (4,
5).V-ATPases are evolutionarily related to ATP synthases of bacteria and
mitochondria and consist of two multisubunit complexes, V1 and
V0, which contain the sites for ATP hydrolysis and proton
transport, respectively. Like the ATP synthase (F-ATPase), V-ATPases utilize a
rotational catalytic mechanism. ATP binding and hydrolysis in the three
catalytic subunits of the V1 sector generate sequential
conformational changes that drive rotation of a central stalk
(6–8).
The central stalk subunits are connected to a ring of proteolipid subunits in
the V0 sector that bind protons to be transported. The actual
transport is believed to occur at the interface of the proteolipids and
V0 subunit a. Rotational catalysis will be productive in proton
transport only if V0 subunit a is held stationary, whereas the
proteolipid ring rotates (8).
This “stator function” resides in a single peripheral stalk in
F-ATPases (9,
10), but is distributed among
up to three peripheral stalks in V-ATPases
(11–13).
The peripheral stator stalks link V0 subunit a to the catalytic
headgroup and ensures that there is rotation of the central stalk complex
relative to the V0 a subunit and catalytic headgroup.Eukaryotic V-ATPases are highly conserved in both their overall structure
and the sequences of individual subunits. Although homologs of most subunits
of eukaryotic V-ATPases are present in archaebacterial V-ATPases (also known
as A-ATPases), the C and H subunits are unique to eukaryotes. Both subunits
have been localized at the interface of the V1 and V0
sectors, suggesting that they are positioned to play a critical role in
structural and functional interaction between the two sectors
(14–16).
The yeast C and H subunits are the only eukaryotic V-ATPase subunits for which
x-ray crystal structures are available
(17,
18). The structure of the C
subunit revealed an elongated “dumbbell-shaped” molecule, with
foot, head, and neck domains
(18). The structure of the H
subunit indicated two domains. The N-terminal 348 amino acids fold into a
series of HEAT repeats and are connected by a 4-amino acid linker to a
C-terminal domain containing amino acids 352–478
(17). These two domains have
partially separable functions in the context of the assembled V-ATPase
(19). Complexes containing
only the N-terminal domain of the H subunit
(H-NT)2 supported some
ATP hydrolysis but little or no proton pumping in isolated vacuolar vesicles
(19,
20). The C-terminal domain
(H-CT) assembled with the rest of the V-ATPase in the absence of intact
subunit H, but supported neither ATPase nor proton pumping activity
(19). However, co-expression
of the H-NT and H-CT domains results in assembly of both sectors with the
V-ATPase and allows increased ATP-driven proton pumping in isolated vacuolar
vesicles. These results suggest that the H-NT and H-CT domains play distinct
and complementary roles even when the two domains are not covalently
attached.In addition to their role as dedicated proton pumps, eukaryotic V-ATPases
are also distinguished from F-ATPases and archaeal V-ATPases in their
regulation. Eukaryotic V-ATPases are regulated in part by reversible
disassembly of the V1 complex from the V0 complex
(1,
21,
22). In yeast, disassembly of
previously assembled complexes occurs in response to glucose deprivation, and
reassembly is rapidly induced by glucose readdition to glucose-deprived cells.
Disassembly down-regulates pump activity, and both the disassembled sectors
are inactivated. Inhibition of ATP hydrolysis in free V1 sectors is
particularly critical, because release of an active ATPase into the cytosol
could deplete cytosolic ATP stores. This inhibition is dependent in part on
the H subunit. V1 complexes isolated from vma13Δ
mutants, which lack the H subunit gene (V1(-H) complexes) have
MgATPase activity. Consistent with a physiological role for H subunit
inhibition of V1, heterozygous diploids containing elevated levels
of free V1 complexes without subunit H have severe growth defects
(23). V1 complexes
containing subunit H have no MgATPase activity, but retain some CaATPase
activity, suggesting a role for nucleotides in inhibition
(24,
25). Consistent with such a
role, both the CaATPase activity of native V1 and the MgATPase
activity of V1(-H) complexes are lost within a few minutes of
nucleotide addition (24).A number of points of interaction between the H subunit and the
V1 and V0 complexes have been identified through
two-hybrid assays, binding of expressed proteins, and cross-linking
experiments. These experiments have indicated that the H subunit binds to
V1 subunits E and G of the V-ATPase peripheral stalks
(26,
27), the catalytic subunit
(V1 subunit A)
(28), regulatory V1
subunit B (15), and the
N-terminal domain of subunit a
(28). Recently, Jeffries and
Forgac (29) have found that
cysteines introduced into the C-terminal domain of subunit H can be
cross-linked to subunit F in isolated V1 sectors via a 10-Å
cross-linking reagent.In this work, we examine both the subunit-subunit interactions and
functional roles of the H-NT and H-CT domains in inhibition of
V1-ATPase activity. When expressed in yeast cells lacking subunit
H, H-NT can be isolated with cytosolic V1 complexes, but H-CT
cannot. We find that both of these domains contribute to inhibition of ATPase
activity, but that stable binding to V1 and full inhibition of
activity requires both domains. We also find that the H-CT can bind to the
cytosolic N-terminal domain of V0 subunit Vph1p (Vph1-NT) in
isolation, but does not support tight binding of Vph1-NT to isolated
V1 complexes. 相似文献
35.
Aparicio E Parera M Franco S Pérez-Alvarez N Tural C Clotet B Martínez MA 《PloS one》2010,5(10):e13771
Recent genome-wide association studies report that the SNP rs8099917, located 8.9 kb upstream of the start codon of IL28B, is associated with both disease chronicity and therapeutic response to pegIFN-α and RBV in patients infected with genotype 1 HCV. To determine the effect of rs8099917 variation on the response of HCV to therapy, we genotyped this variant in a cohort of 160 HCV/HIV-1 coinfected patients in our clinic unit who received combined peg-IFN-α/RBV therapy. The rs8099917 T/G or G/G genotypes were observed in 56 patients (35%). Treatment failure occurred in 80% of G-allele carriers versus 48% of non-carriers (P<0.0001). This result reveals that the G allele was strongly associated with treatment failure in this patient cohort. Importantly, a highly significant association was found between the G-allele and response to therapy in HCV genotype 1-infected patients (P<0.0001) but not in HCV genotype 3-infected patients. Multivariate analysis (odds ratio; 95% confidence interval; P value) indicated that the rs8099917 TT genotype was a strong predictor of treatment success (5.83; 1.26-26.92; P = 0.021), independent of baseline plasma HCV-RNA load less than 500 000 IU/ml (4.85; 1.18-19.95; P = 0.025) and absence of advanced liver fibrosis (5.24; 1.20-22.91; P = 0.025). These results reveal the high prevalence of the rs8099917 G allele in HCV/HIV-1 coinfected patients as well as its strong association with treatment failure in HCV genotype 1-infected patients. rs8099917 SNP genotyping may be a valid pre-treatment predictor of which patients are likely to respond to treatment in this group of difficult-to-treat HCV/HIV-infected patients. 相似文献
36.
37.
38.
Pedrolli DB Matern A Wang J Ester M Siedler K Breaker R Mack M 《Nucleic acids research》2012,40(17):8662-8673
Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B(2)) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands. 相似文献
39.
Joseph M. Sherwood Ester Reina-Torres Jacques A. Bertrand Barnaby Rowe Darryl R. Overby 《PloS one》2016,11(3)
Elevated intraocular pressure (IOP) is the predominant risk factor for glaucoma, and reducing IOP is the only successful strategy to prevent further glaucomatous vision loss. IOP is determined by the balance between the rates of aqueous humour secretion and outflow, and a pathological reduction in the hydraulic conductance of outflow, known as outflow facility, is responsible for IOP elevation in glaucoma. Mouse models are often used to investigate the mechanisms controlling outflow facility, but the diminutive size of the mouse eye makes measurement of outflow technically challenging. In this study, we present a new approach to measure and analyse outflow facility using iPerfusion™, which incorporates an actuated pressure reservoir, thermal flow sensor, differential pressure measurement and an automated computerised interface. In enucleated eyes from C57BL/6J mice, the flow-pressure relationship is highly non-linear and is well represented by an empirical power law model that describes the pressure dependence of outflow facility. At zero pressure, the measured flow is indistinguishable from zero, confirming the absence of any significant pressure independent flow in enucleated eyes. Comparison with the commonly used 2-parameter linear outflow model reveals that inappropriate application of a linear fit to a non-linear flow-pressure relationship introduces considerable errors in the estimation of outflow facility and leads to the false impression of pressure-independent outflow. Data from a population of enucleated eyes from C57BL/6J mice show that outflow facility is best described by a lognormal distribution, with 6-fold variability between individuals, but with relatively tight correlation of facility between fellow eyes. iPerfusion represents a platform technology to accurately and robustly characterise the flow-pressure relationship in enucleated mouse eyes for the purpose of glaucoma research and with minor modifications, may be applied in vivo to mice, as well as to eyes from other species or different biofluidic systems. 相似文献
40.
Rafael Rodrigues Silva Rafael M. Mariante Andrea Alice Silva Ana Luiza Barbosa dos Santos Ester Roffê Helton Santiago Ricardo Tostes Gazzinelli Joseli Lannes-Vieira 《PloS one》2015,10(2)
The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD. 相似文献