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101.
Wolfgang Schmitt Stefan Odenbreit Dorothee Heuermann Rainer Haas 《Molecular genetics and genomics : MGG》1995,248(5):563-572
The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ70 promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ?) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein. 相似文献
102.
103.
Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b 总被引:12,自引:0,他引:12
Jürgen Kreft Dorothee Funke Albert Haas Friedrich Lottspeich Werner Goebel 《FEMS microbiology letters》1989,57(2):197-202
In culture supernatants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as listeriolysin O (LLO). In the case of L. ivanovii a second major supernatant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supernatants of L. ivanovii a sphingomyelinase and a lecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-terminal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown. 相似文献
104.
High density lipoprotein modulates platelet function. 总被引:1,自引:0,他引:1
Stefan Barlage Dorothee Boettcher Alfred Boettcher Ashraf Dada Gerd Schmitz 《Cytometry. Part A》2006,69(3):196-199
BACKGROUND: Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP-binding cassette transporter A1 (ABCA1). METHODS: ABCA1 expression and its association with cholesterol rich membrane domains was analyzed by mRNA and Western blot analysis. HDL effects on platelet receptor clustering were analyzed by flow cytometric analysis of fluorescence resonance energy transfer between fluorochrome-labeled antibodies. RESULTS: ABCA1 expression increased upon megakaryocytic differentiation of human stem cells and ABCA1 protein partially associated to LubroIWX-resistant membrane domains. Plasma HDL-cholesterol in healthy donors negatively correlated to the platelet membrane cholesterol content. Receptor cluster analysis revealed a decrease in the association of Gplb and FcgammaRII upon incubation of platelets with HDL3. CONCLUSION: Our results suggest that HDL modulates platelet reactivity by altering lipid raft associated receptor clustering. 相似文献
105.
Jan N Hansen Fabian Kaiser Philipp Leyendecker Birthe Stüven JensHenning Krause Fatemeh Derakhshandeh Jaazba Irfan Tommy J Sroka Kenley M Preval Paurav B Desai Michael Kraut Heidi Theis AnnaDorothee Drews Elena DeDomenico Kristian Hndler Gregory J Pazour David J P Henderson David U Mick Dagmar Wachten 《EMBO reports》2022,23(8)
The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase‐4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease. 相似文献
106.
Bühler P Wolf P Gierschner D Schaber I Katzenwadel A Schultze-Seemann W Wetterauer U Tacke M Swamy M Schamel WW Elsässer-Beile U 《Cancer immunology, immunotherapy : CII》2008,57(1):43-52
Background Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists
after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking
of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific
cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy
with bispecific diabodies could be a promising novel treatment option for prostate cancer.
Methods A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA
single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv VHCD3-VLPSMA and VHPSMA-VLCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography
(IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as
on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation
the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model.
Results By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both
to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to
be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor
xenografts with the diabody and PBL efficiently inhibited tumor growth.
Conclusions The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal
residual disease.
P. Bühler and P. Wolf equally contributed to the work. 相似文献
107.
Atzler D Mieth M Maas R Böger RH Schwedhelm E 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(23):2294-2298
Nitric oxide (NO), the endogenous modulator of vascular tone and structure, originates from oxidation of L-arginine catalysed by NO synthase (NOS). The L-arginine derivative L-homoarginine serves as an alternative NOS substrate releasing NO, competing with L-arginine for NOS, arginase, and arginine transport. In the present article we report a liquid chromatography-tandem mass spectrometric (LC-tandem MS) method for the determination of L-homoarginine in human plasma by stable-isotope dilution. L-[(13)C(6)]-Homoarginine was used as internal standard. This method provides high sample throughput of 25-μl aliquots of plasma with an analysis time of 4 min using LC-tandem MS electrospray ionisation in the positive mode (ESI+). Specific transitions for L-homoarginine and L-[(13)C(6)]-homoarginine were m/z 245 → m/z 211 and m/z 251 → m/z 217, respectively. The mean intra- and interassay CVs were 7.4 ± 4.5% (±SD) for 0.1-50 μmol/L and 7.5 ± 2.0% for 2 and 5 μmol/L, respectively. Applying this method, a mean plasma concentration of L-homoarginine of 2.5 ± 1.0 μmol/L was determined in 136 healthy humans. 相似文献
108.
Weiling Wang Hao Xu Yang Shi Sandhya Nandedkar Hao Zhang Haiqing Gao Thom Feroah Dorothee Weihrauch Marie L. Schulte Deron W. Jones Jason Jarzembowski Mary Sorci-Thomas Kirkwood A. Pritchard Jr. 《Journal of lipid research》2010,51(9):2560-2570
The relationship between high-density lipoprotein and pulmonary function is unclear. To determine mechanistic relationships we investigated the effects of genetic deletion of apolipoprotein A-I (apoA-I) on plasma lipids, paraoxonase (PON1), pro-inflammatory HDL (p-HDL), vasodilatation, airway hyperresponsiveness and pulmonary oxidative stress, and inflammation. ApoA-I null (apoA-I−/−) mice had reduced total and HDL cholesterol but increased pro-inflammatory HDL compared with C57BL/6J mice. Although PON1 protein was increased in apoA-I−/− mice, PON1 activity was decreased. ApoA-I deficiency did not alter vasodilatation of facialis arteries, but it did alter relaxation responses of pulmonary arteries. Central airway resistance was unaltered. However, airway resistance mediated by tissue dampening and elastance were increased in apoA-I−/− mice, a finding also confirmed by positive end-expiratory pressure (PEEP) studies. Inflammatory cells, collagen deposition, 3-nitrotyrosine, and 4-hydroxy-2-nonenal were increased in apoA-I−/− lungs but not oxidized phospholipids. Colocalization of 4-hydroxy-2-nonenal with transforming growth factor β-1 (TGFβ-1 was increased in apoA-I−/− lungs. Xanthine oxidase, myeloperoxidase and endothelial nitric oxide synthase were increased in apoA-I−/− lungs. Dichlorodihydrofluorescein-detectable oxidants were increased in bronchoalveolar lavage fluid (BALF) in apoA-I−/− mice. In contrast, BALF nitrite+nitrate levels were decreased in apoA-I−/− mice. These data demonstrate that apoA-I plays important roles in limiting pulmonary inflammation and oxidative stress, which if not prevented, will decrease pulmonary artery vasodilatation and increase airway hyperresponsiveness. 相似文献
109.
Two new oribatid mite species of the genus Truncozetes (Oribatida, Epactozetidae), Truncozetes ecuadoriensis
sp. n. and Truncozetes monodactylus
sp. n., are described from the Ecuadorian soils. The morphology of the gnathosoma and the legs is presented in detail for the first time for the species of Truncozetes. An identification key to all known species of the family Epactozetidae is given. 相似文献
110.
MDHM, a macrophage-activating product of Mycoplasma fermentans, stimulates murine macrophages to synthesize nitric oxide and become tumoricidal 总被引:1,自引:0,他引:1
Dorothee Ruschmeyer Hansjörg Thude Peter F. Mühlradt 《FEMS immunology and medical microbiology》1993,7(3):223-230
Abstract In continuation of previous work on macrophage activation by a Mycoplasma fermentans -derived product, originally named “mycoplasma-derived high mol. wt. material” (MDHM), we have investigated whether MDHM was capable of inducing synthesis of the reactive nitrogen intermediate nitric oxide (NO), thus rendering macrophages cytocidal. Mycoplasmas were first delipidated with acetone, and MDHM activity was then extracted with 50 mM 1-O-octyl-β- d -glucopyranoside to yield a particularly active new preparation of MDHM which we have named MDHM-D (D for detergent). In combination with IFN-γ, MDHM-D activated macrophages to produce reactive nitrogen intermediates and kill P815 mastocytoma cells in co-culture. P815 target cells were chosen because they are TNF-resistant. Macrophages from the LPS-low responder strain C3H/HeJ were used to minimize interference from possible LPS contamination. MDHM-D activity in this system was strictly IFN-γ-dependent. In the presence of 25 U/ml IFN-γ MDHM-D gave a half maximal response at a dilution of 1/100 000, showing a parallel concentration dependency for nitrite production and cytocidal activity. 相似文献