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81.
82.
Schibli R La Bella R Alberto R Garcia-Garayoa E Ortner K Abram U Schubiger PA 《Bioconjugate chemistry》2000,11(3):345-351
Functionalization of biologically relevant molecules for the labeling with the novel fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) precursor has gained considerable attention recently. Therefore, we tested seven different tridentate (histidine L(1)(), iminodiacetic acid L(2)(), N-2-picolylamineacetic acid L(3)(), N, N-2-picolylaminediacetic acid L(4)()) and bidentate (histamine L(5)(), 2-picolinic acid L(6)(), 2,4-dipicolinic acid L(7)()) ligand systems, with the potential to be bifunctionalized and attached to a biomolecule. The ligands allowed mild radiolabeling conditions with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) (30 min, 75 degrees C). The ligand concentrations necessary to obtain yields of >95% of the corresponding organometallic complexes 1-7 ranged from 10(-)(6) to 10(-)(4) M. Complexes of the general formula "fac-[(99m)TcL(CO)(3)]" (L = tridentate ligand) and "fac-[(99m)Tc(OH(2))L'(CO)(3)]" (L' = bidentate ligand), respectively, were produced. Challenge studies with cysteine and histidine revealed significant displacement of the ligands in complexes 5-7 but only little exchange with complexes 1-4 after 24 h at 37 degrees C in PBS buffer. However, no decomposition to (99m)TcO(4)(-) was observed under these conditions. All complexes showed a hydrophilic character (log P(o/w) values ranging from -2.12 to 0.32). Time-dependent FPLC analyses of compounds 1-7 incubated in human plasma at 37 degrees C showed again no decomposition to (99m)TcO(4)(-) after 24 h at 37 degrees C. However, the complexes with bidentate ligands (5-7) became almost completely protein bound after 60 min, whereas the complexes with tridentate coordinated ligands (1-4) showed no reaction with serum proteins. The compounds were tested for their in vivo stability and the biodistribution characteristics in BALB/c mice. The complexes with tridentate coordinated ligand systems (1-4) revealed generally a good and fast clearance from all organs and tissues. On the other hand, the complexes with only bidentate coordinated ligands (5-7) showed a significantly higher retention of activity in the liver, the kidneys, and the blood pool. Detailed radiometric analyses of murine plasma samples, 30 min p.i. of complex fac-[(99m)TcL(1)(CO)(3)], 1, revealed almost no reaction of the radioactive complex with the plasma proteins. By contrast, in plasma samples of mice, which were injected with complex fac-[(99m)Tc(OH(2))L(5)(CO)(3)](+), 5, the entire radioactivity coeluded with the proteins. On the basis of these in vitro and in vivo experiments, it appears that functionalization of biomolecules with tridentate-chelating ligand systems is preferable for the labeling with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+), since this will presumably result in radioactive bioconjugates with better pharmacokinetic profiles. 相似文献
83.
Albert M Repetschnigg W Ortner J Gomes J Paul BJ Illaszewicz C Weber H Steiner W Dax K 《Carbohydrate research》2000,327(4):395-400
A fast method for the simultaneous detection of different glycosidolytic activities in commercially available enzyme preparations and crude culture filtrates was found in using, as substrate, a mixture of different glycosyl fluorides and 19F NMR spectroscopy as a screening technique. Accompanying studies regarding the hydrolytic stability of these fluorides in various buffer systems, as well as conditions of their long-term storage, were carried out. A simple procedure for the preparation of beta-D-mannopyranosyl fluoride in gram quantities is given. 相似文献
84.
Schuster M Einhauer A Wasserbauer E Süssenbacher F Ortner C Paumann M Werner G Jungbauer A 《Journal of biotechnology》2000,84(3):237-248
The driving force for the modification of existing, or the development of new, protein expression systems lies in the identification of a tremendous number of potential novel drug targets through recent genomics approaches. Saccharomyces cerevisiae as a host for recombinant protein expression, offers many advantages, as its biosynthetic pathways resemble higher eukaryotic cells in many aspects. Two yeast vectors were compared to evaluate the versatility of this organism for expression of recombinant proteins. One expression vector enables the secretion of the recombinant protein into the culture medium through fusion with the leader sequence of the mating-type pheromone alpha; the other directs the expression product into the cytoplasm of the yeast cell through fusion with ubiquitin. To facilitate immunological detection and purification, proteins were expressed as fusions to an octapeptide, the so-called Flag-tag, which is recognised by a monoclonal antibody in the presence of Ca2+. We chose 20 functionally different cDNAs to compare the efficiency of both expression systems. All cDNAs could be expressed at the correct size but at varying yields and purity. Both expression systems differed greatly in the degree of glycosylation and other, not further analysed, post-translational modifications. Secretion of all model proteins into the cell culture supernatant could be accomplished if membrane domains or signal sequences were absent, but many proteins were heavily glycosylated as demonstrated by lectin mapping or enzymatical deglycosylation. Some proteins, however, were expressed as homogenous products, and could be easily purified for further functional studies. Further investigations on the expression biology of yeast are required, in order to optimise the conditions of fermentation which may finally lead to more homogeneous expression products. 相似文献
85.
Stomatal density (SD) and stomatal conductance ( g s ) can be affected by an increase of atmospheric CO2 concentration. This study was conducted on 17 species growing in a naturally enriched CO2 spring and belonging to three plant communities. Stomatal conductance, stomatal density and stomatal index (SI) of plants from the spring, which were assumed to have been exposed for generations to elevated [CO2 ], and of plants of the same species collected in a nearby control site, were compared. Stomatal conductance was significantly lower in most of the species collected in the CO2 spring and this indicated that CO2 effects on g s are not of a transitory nature but persist in the long term and through plant generations. Such a decrease was, however, not associated with changes in the anatomy of leaves: SD was unaffected in the majority of species (the decrease was only significant in three out of the 17 species examined), and also SI values did not vary between the two sites with the exception of two species that showed increased SI in plants grown in the CO2 -enriched area. These results did not support the hypothesis that long-term exposure to elevated [CO2 ] may cause adaptive modification in stomatal number and in their distribution. 相似文献
86.
Isabella Di Lernia Alessandra Morana Antonio Ottombrino Stefania Fusco Mosè Rossi M. De Rosa 《Extremophiles : life under extreme conditions》1998,2(4):409-416
Enzymes that convert starch and dextrins to α,α-trehalose and glucose were found in cell homogenates of the hyperthermophilic
acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with
an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from α-1,4 to α-1,1.
The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl dextrins, forming
trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively,
and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70°C for SsTDFE and 85°C for
SsTFE. The third enzyme identified was an α-glycosidase (SsαGly), which catalyzed the hydrolysis of the α-1,4 glucosidic linkages
in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme
had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85°C.
Received: October 29, 1997 / Accepted: April 29, 1998 相似文献
87.
Rose Monnerat Erica Martins Cristina Macedo Paulo Queiroz Lilian Pra?a Carlos Marcelo Soares Helio Moreira Isabella Grisi Joseane Silva Mario Soberon Alejandra Bravo 《PloS one》2015,10(4)
Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda. 相似文献
88.
Daniel Yakubovich Shai Berlin Uri Kahanovitch Moran Rubinstein Isabella Farhy-Tselnicker Boaz Styr Tal Keren-Raifman Carmen W. Dessauer Nathan Dascal 《PLoS computational biology》2015,11(11)
G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2''s density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα. 相似文献
89.
Roman Gebauer Daniel Volaík Josef Urban Isabella Brja Nina Elisabeth Nagy Toril Drabls Eldhuset Paal Krokene 《Ecology and evolution》2015,5(21):4989-4998
Predicted increases in the frequency and duration of drought are expected to negatively affect tree vitality, but we know little about how water shortage will influence needle anatomy and thereby the trees’ photosynthetic and hydraulic capacity. In this study, we evaluated anatomical changes in sun and shade needles of 20‐year‐old Norway spruce trees exposed to artificial drought stress. Canopy position was found to be important for needle structure, as sun needles had significantly higher values than shade needles for all anatomical traits (i.e., cross‐sectional needle area, number of tracheids in needle, needle hydraulic conductivity, and tracheid lumen area), except proportion of xylem area per cross‐sectional needle area. In sun needles, drought reduced all trait values by 10–40%, whereas in shade needles, only tracheid maximum diameter was reduced by drought. Due to the relatively weaker response of shade needles than sun needles in drought‐stressed trees, the difference between the two needle types was reduced by 25% in the drought‐stressed trees compared to the control trees. The observed changes in needle anatomy provide new understanding of how Norway spruce adapts to drought stress and may improve predictions of how forests will respond to global climate change. 相似文献
90.
Nermina Buljubasic K. Martijn Akkerhuis Sanneke P. M. de Boer Jin M. Cheng Hector M. Garcia-Garcia Mattie J. Lenzen Rohit M. Oemrawsingh Linda C. Battes Melissa Rijndertse Evelyn Regar Patrick W. Serruys Robert-Jan van Geuns Eric Boersma Isabella Kardys 《PloS one》2015,10(10)