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991.
Udo zur Stadt Jan Rohr Florian Koch Julia Pagel Brigitte Kasper Christian Becker Karin Beutel Gillian Griffiths Hans Christian Hennies 《American journal of human genetics》2009,85(4):482-492
Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis. 相似文献
992.
Acid-sensitive outwardly rectifying anion channels (ASOR) have been described in several mammalian cell types. The present
whole-cell patch-clamp study elucidated whether those channels are expressed in erythrocytes. To this end whole-cell recordings
were made in human erythrocytes from healthy donors treated with low pH and high osmotic pressure. When the pipette solution
had a reduced Cl− concentration, treatment of the cells with Cl−-containing normal and hyperosmotic (addition of sucrose and polyethelene glycol 1000 [PEG-1000] to the Ringer) media with
low pH significantly increased the conductance of the cells at positive voltages. Channel activity was highest in the PEG-1000
media (95 and 300 mM PEG-1000, pH 4.5 and 4.3, respectively) where the current–voltage curves demonstrated strong outward
rectification and reversed at −40 mV. Substitution of the Cl−-containing medium with Cl−-free medium resulted in a decrease of the conductance at hyperpolarizing voltages, a shift in reversal potential (to 0 mV) and loss of outward rectification. The chloride currents were inhibited by chloride channels blockers DIDS and NPPB (IC50 for both was ~1 mM) but not with niflumic acid and amiloride. The observations reveal expression of ASOR in erythrocytes. 相似文献
993.
994.
Florian Brodhun Cornelia G?bel Ellen Hornung Ivo Feussner 《The Journal of biological chemistry》2009,284(18):11792-11805
The homothallic ascomycete Aspergillus nidulans serves as model
organism for filamentous fungi because of its ability to propagate with both
asexual and sexual life cycles, and fatty acid-derived substances regulate the
balance between both cycles. These so-called psi (precocious
sexual inducer) factors are produced by psi
factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted
the presence of two different heme domains in Ppo proteins: in the N-terminal
region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas
in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze
the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia
coli as an active enzyme. The protein was purified by 62-fold and
identified as a homotetrameric ferric heme protein that metabolizes mono- as
well as polyunsaturated C16 and C18 fatty acids at pH
∼7.25. The presence of thiolate-ligated heme was confirmed on the basis of
sequence alignments and the appearance of a characteristic 450 nm CO-binding
spectrum. Studies on its reaction mechanism revealed that PpoA uses different
heme domains to catalyze two separate reactions. Within the heme peroxidase
domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic
acid by abstracting a H-atom from C-8 of the fatty acid, yielding a
carbon-centered radical that reacts with molecular dioxygen. In the second
reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450
heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as
a bifunctional P450 fusion protein that uses a previously unknown reaction
mechanism for forming psi factors.The fungus Aspergillus nidulans (teleomorph Emericella
nidulans) is a homothallic ascomycete that has a defined sexual and
asexual developmental cycle. Therefore, it serves as a model system for the
understanding of fungal development
(1). Oxidized unsaturated fatty
acids, so-called oxylipins, derived from endogenous fatty acids were found to
influence the development of the asexual conidiophores and sexual
cleistothecia
(2–6).
Moreover, they seem to regulate the secondary metabolism of the fungus
(7). These substances were
collectively named psi factors and are primarily a mixture of hydroxylated
oleic (18:1Δ9Z;
x:yΔz denotes a fatty
acid with x carbons and y double bonds in position
z counting from the carboxyl end), linoleic
(18:2Δ9Z,12Z),
and α-linolenic
(18:3Δ9Z,12Z,15Z)
acids. They are termed psiβ, psiα, and psiγ, respectively.
Psi factors can be further classified by the number and positioning of hydroxy
groups on the fatty acid backbone: psiB (OH at C-8, e.g.
(8R)-HODE),2
psiA (OH at C-5 and C-8, e.g. (5S,8R)-DiHODE), and
psiC (OH at C-8 and the δ-lactone ring)
(8,
9).The psi factor (8R)-HODE was first discovered in the fungus
Laetisaria arvalis
(10,
11); it was later also found
in Gaeumannomyces graminis
(12,
13), where the first enzyme,
which is responsible for production of (8R)-HPODE, 7,8-LDS, was
detected (13). This
heme-containing enzyme is bifunctional because it oxidizes
18:2Δ9Z,12Z
in a first reaction step to (8R)-HPODE and subsequently isomerizes
this intermediate compound to (7S,8S)-DiHODE
(13–15).After the genome of A. nidulans was available, Keller and
co-workers (6,
16,
17) found three genes that
share a high homology with the sequence of 7,8-LDS, namely ppoA,
ppoB, and ppoC. They showed that the deletion of these genes had
a significant effect (i) on the developmental ratio between the asexual
conidiospores and sexual ascospores; (ii) on the production of psi factors;
and (iii) on the production of secondary metabolites, the mycotoxins
(6,
7,
16,
17). Furthermore, the encoded
proteins showed remarkable sequence homology to both mammalian PGHS isoforms,
enzymes that are responsible for the synthesis of prostaglandins
(18). Using the NCBI conserved
domain search analysis tool, it turned out that ppoA amino acid
residues 210–580 contain a domain similar to mammalian heme peroxidases,
whereas residues 650–1050 contain a CYPX domain, similar to
P450 heme thiolate enzymes
(16). However, for 7,8-LDS
from G. graminis, only the mammalian heme peroxidase domain is
predicted. The identity of conserved catalytic domains between Ppo enzymes and
mammalian PGHS ranges from 25 to 29% for PGHS-2 and from 25 to 26% for PGHS-1
(19). PpoA and 7,8-LDS show
42% amino acid identity.Oliw and co-workers (20)
observed that incubation of homogenates of mycelia of A. nidulans
with
18:2Δ9Z,12Z
converted the fatty acid to (8R)-HODE and
(5S,8R)-DiHODE as the major products. (8R)-HPODE,
(10R)-HODE, and (10R)-HPODE were detected as minor products.
Incubation of mycelia of Aspergillus fumigatus with deuterium-labeled
18:2Δ9Z,12Z
revealed that the synthesis of (8R)-HPODE is accomplished via
pro-S-hydrogen abstraction at C-8 and antarafacial dioxygen
insertion. (5S,8R)-DiHODE is generated via an additional
pro-S-hydrogen abstraction at C-5 of the substrate
(20,
21).Additional studies with fungal knock-out strains led to the hypothesis that
PpoA may be responsible for the synthesis of (8R)-hydroperoxides,
which are partially reduced to (8R)-hydroxides
(20). It was suggested that,
analogous with 7,8-LDS, (8R)-hydroperoxides are then converted to
5,8-dihydroxides by PpoA. Furthermore, it was concluded that ppoC may
code for linoleate (10R)-DOX
(20). Analysis of Ppo enzymes
from A. nidulans in studies published so far has been performed
either by using knock-out mutants to demonstrate the absence of a subset of
psi factors or by using crude mycelial extracts; both experimental setups have
the disadvantage of observing multiple enzymatic reactions in parallel.To characterize the biochemical properties of PpoA in more detail, we
cloned and expressed recombinant PpoA in Escherichia coli. After
purification of the enzyme by up to 62-fold, biochemical characterization was
performed. The studies revealed mechanistic as well as structural similarities
to and differences from 7,8-LDS from G. graminis. Both enzymes were
found to be homotetrameric ferric heme proteins that catalyze the synthesis of
(8R)-HPODE. Whereas G. graminis 7,8-LDS converts the
intermediate formed to (7S,8S)-DiHODE, PpoA produces
5,8-DiHODE.Using site-directed mutagenesis, we provide evidence that there are
striking differences between both enzymes regarding the catalytic reaction
cycle. Thus, we found that PpoA uses different domains to catalyze the two
reaction steps. We suggest that the DOX reaction, yielding 8-HPODE, takes
place in the N-terminal heme peroxidase domain. The isomerization of this
intermediate product to the end product, 5,8-DiHODE, is accomplished, however,
independently by the C-terminal P450 heme thiolate domain in an
8-hydroperoxide isomerase reaction.In addition, we are able to provide evidence that, during the catalysis,
PpoA generates a carbon-centered radical presumably at C-8, like G.
graminis 7,8-LDS. Furthermore, we determined the kinetic parameters for
the first reaction step. 相似文献
995.
In mass spectrometry‐based proteomics, most conventional search engines match spectral data to sequence databases. These search databases thus play a crucial role in the identification process. While search engines can derive peptides in silico from protein sequences, this is usually limited to standard digestion algorithms. Customized search databases that provide detailed control over the search space can vastly outperform such standard strategies, especially in gel‐free proteomics experiments. Here we present Database on Demand, an easy‐to‐use web tool that can quickly produce a wide variety of customized search databases. 相似文献
996.
Grit Nebrich Marion Herrmann Daniela Hartl Madeleine Diedrich Thomas Kreitler Christoph Wierling Joachim Klose Patrick Giavalisco Claus Zabel Dr. Lei Mao 《Proteomics》2009,9(7):1795-1808
In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large‐scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2‐DE/MS‐based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2‐DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2‐DE experiments with MS‐data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our “PROTEOMER” database is its high cross‐referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross‐referencing can transform information into biological knowledge. 相似文献
997.
Gregor S Zimmermann Claus Neurohr Heidrun Villena-Hermoza Rudolf Hatz Juergen Behr 《Respiratory research》2009,10(1):89
Background
Human Bronchial epithelial cells (hu-BEC) have been claimed to play a significant role in the pathogenesis of chronic inflammatory airway diseases like COPD. In this context IL-8 and GM-CSF have been shown to be key cytokines. Some antibiotics which are routinely used to treat lower respiratory tract infections have been shown to exert additional immunomodulatory or anti-inflammatory effects. We investigated whether these effects can also be detected in hu-BEC.Methods
Hu-BEC obtained from patients undergoing lung resections were transferred to air-liquid-interface (ALI) culture. These cultures were incubated with cefuroxime (CXM, 10-62.5 mg/l), azithromycin (AZM, 0.1-1.5 mg/l), levofloxacin (LVX, 1-8 mg/l) and moxifloxacin (MXF, 1-16 mg/l). The spontaneous and TNF-α (10 ng/ml) induced expression and release of IL-8 and GM-CSF were measured using PCR and ELISA in the absence or presence of these antibiotics.Results
The spontaneous IL-8 and GM-CSF release was significantly reduced with MXF (8 mg/l) by 37 ± 20% and 45 ± 31%, respectively (both p < 0.01). IL-8 release in TNF-α stimulated hu-BEC decreased by 16 ± 8% (p < 0.05) with AZM (1.5 mg/l). With MXF a concentration dependent decrease of IL-8 release was noted up to 39 ± 7% (p < 0.05). GM-CSF release from TNF-α stimulated hu-BEC was maximally decreased by 35 ± 24% (p < 0.01) with MXF (4 mg/l).Conclusion
Using ALI cultures of hu-BEC we observed differential effects of antibiotics on spontaneous and TNF-α induced cytokine release. Our data suggest that MXF and AZM, beyond bactericidal effects, may attenuate the inflammatory process mediated by hu-BEC. 相似文献998.
Florian Hahne Nolwenn LeMeur Ryan R Brinkman Byron Ellis Perry Haaland Deepayan Sarkar Josef Spidlen Errol Strain Robert Gentleman 《BMC bioinformatics》2009,10(1):106-8
Background
Recent advances in automation technologies have enabled the use of flow cytometry for high throughput screening, generating large complex data sets often in clinical trials or drug discovery settings. However, data management and data analysis methods have not advanced sufficiently far from the initial small-scale studies to support modeling in the presence of multiple covariates. 相似文献999.
1000.
Yeast has two phosphate‐uptake systems that complement each other: the high‐affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low‐affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino‐terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate‐uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split‐ubiquitin assays and co‐immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low‐affinity phosphate transport through a physical interaction with Spl2. 相似文献