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81.
羟胺切割Neurturin融合蛋白表达载体的构建、表达产物纯化及生物活性 总被引:4,自引:0,他引:4
Neurturin (NTN)是新近发现的一种神经营养因子 ,是 GDNF家族的成员之一 .将 5′端引入了羟胺切割位点的 h NTN基因克隆到硫氧还蛋白融合表达载体 p Thio His A,在宿主菌 BL2 1中获得了稳定、高效表达 ,表达产物以包涵体形式存在 .在变性条件下经羟胺切割、柱层析纯化后复性 ,获得纯度达 90 %以上的 rh NTN.经鸡胚背根神经节 (DRG)培养法测定具有生物学活性 . 相似文献
82.
A. R. Simonyan J. Vepsalainen A. R. Khomutov 《Russian Journal of Bioorganic Chemistry》2006,32(6):578-585
Convenient methods of synthesis of 1-aminooxy-3,8-diaza-11-aminoundecane, its earlier unknown N 1-and N 11-acetyl derivatives, and also 1,10-bis(aminooxy)-3,8-diazadecane are suggested. It is shown that it is possible to selectively delete the acid-labile ethoxyethylidene protection of aminooxy group by hydrosulfates in the presence of N-tert-butyloxycarbonyl group. 相似文献
83.
In 15 sites in the T4rII gene, mutation from the ochre (UAA) codon to amber (UAG), opal (UGA) and the wild-type was measured with and without 2-aminopurine treatment. It is shown that a particular base pair in the DNA may show variable mutability, depending on its nearest neighbors. Also, similar base pairs at different sites in the gene can vary in their mutability despite the fact that they are flanked by similar neighbors. 相似文献
84.
N(4)-amino-and N(4)-hydroxycytosines as base analogue mutagens 总被引:2,自引:0,他引:2
N(4)-Aminocytosine [N(4)NH2C] and N(4)-amino-2′-deoxycytidine [N(4)NH2dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T4. There is some evidence that they are incorporated into the φ 80 DNA but could not be detected in the bacterial DNA.N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect. 相似文献
85.
The reactivity of hydroxylamine with HO2/O2- radicals was studied by pulse radiolysis and stopped-flow photolysis over a pH range of 1.1-10.5. Upper limits for the rate of reaction indicate that hydroxylamine, if it reacts at all, reacts at a very slow rate. Its use as an indicator for O-2 and an assay for superoxide dismutase is, therefore, inappropriate. 相似文献
86.
The presoaked seeds of a rice cultivar, Tellakattera, were treated with three different concentrations of hydrazine (HZ) or hydroxylamine (HA) in combination with diethylsulphate (dES) (0.05%). In the M1 generation more than additive effects were observed for increase in chlorophyll chimeras and decrease in seed fertility. A synergistic effect was also observed for both chlorophyll mutation and mutant frequencies, in the M2 generation, in sequential treatments. However, the degree of synergism, based on M2 chlorophyll mutant frequency, was greater in dES posttreatment combinations with HA or HZ, compared with dES pretreatments. These differences in reciprocal treatments may be due to more efficient fixation of premutational events by dES than HZ or HA. 相似文献
87.
Hydroxylamine cleavage of proteins in polyacrylamide gels 总被引:7,自引:0,他引:7
A modification of the hydroxylamine cleavage of proteins is presented in which proteins were cleaved while immobilized in the matrix of a polyacrylamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier, facilitates simultaneous treatment of many samples, and contributes to a high recovery efficiency (60-90%) of the cleavage products. The cleavage is performed with individual protein bands excised from dried slab gels after detection by staining, autoradiography, or fluorography. The procedure can be easily combined with other techniques to further characterize the cleavage fragments. Also a two-dimensional version of the cleavage method was developed, which allows rapid recognition of interrelationships between proteins in a complicated mixture. The versatility of the procedure is demonstrated in a number of applications. Highly related strains of murine leukemia viruses were easily distinguished from one another by the unique cleavage patterns of their gag- and env-precursor polypeptides. Comparing the env-precursor gPr82env synthesized in the presence or absence of tunicamycin with its cell-free synthesized counterpart, revealed the presence of an amino-terminal signal sequence. Cleavage patterns of pro-opiomelanocortin (POMC) from three different species revealed a high degree of homology between rat and mouse POMC, whereas Xenopus POMC was very different. Regions to which carbohydrates are attached could be identified by comparing glycosylated and unglycosylated forms of POMC. Combining the hydroxylamine cleavage procedure with immunological characterization of the fragments showed a small but significant difference between the amino-terminal sequences of the recombinant transforming protein P120 of Abelson murine leukemia virus and of its parent molecule Pr65gag of Moloney murine leukemia virus. 相似文献
88.
Novel principles in the microbial conversion of nitrogen compounds 总被引:24,自引:0,他引:24
Mike S.M. Jetten Susanne Logemann Gerard Muyzer Lesley A. Robertson Simon de Vries Mark C.M. van Loosdrecht J. Gijs Kuenen 《Antonie van Leeuwenhoek》1997,71(1-2):75-93
Some aspects of inorganic nitrogen conversion by microorganisms like N2O emission and hydroxylamine metabolism studied by Beijerinck and Kluyver, founders of the Delft School of Microbiology, are still actual today. In the Kluyver Laboratory for Biotechnology, microbial conversion of nitrogen compounds is still a central research theme. In recent years a range of new microbial processes and process technological applications have been studied. This paper gives a review of these developments including, aerobic denitrification, anaerobic ammonium oxidation, heterotrophic nitrification, and formation of intermediates (NO2-, NO, N2O), as well as the way these processes are controlled at the genetic and enzyme level. 相似文献
89.
Latypov RF Harvey TS Liu D Bondarenko PV Kohno T Fachini RA Rosenfeld RD Ketchem RR Brems DN Raibekas AA 《Journal of molecular biology》2007,368(4):1187-1201
Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes. 相似文献
90.
Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S0 S1, S1 S2, S2 S3, S3 (S4) S0. State S1 is most stable in the dark. Oxygen is released during S3 (S4) S0. Hydroxylamine and hydrazine interact with S1. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S1 S2 : S2 S3 : S3 (S4) S0 : S0 S1 :... in the absence of the agents, to S1
* S0 : S0 S1 : S1 S2 : S2 S3 :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH+(S1 S2) : 1H+(S2 S3) : 2H(S3 S0) : 1H+(S0 S1) :... to 2H+(S1
* S0) : 1H+(S0 S1) : OH+(S1 S2) : 1H+(S2 S3) : 2H+(S3 S0) :... The 2H+ were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53–57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n3 molecules of hydroxylamine and of n2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl- or OH-, which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S1.Abbreviations DNP-INT
dinitrophenylether of iodonitrothymol
- FWHM
full width at half maximum
- NR
neutral red (3-amino-7-dimethylamino-2-methylphenazine-HCI)
- PS II
photosystem II
- WOC or (in formulas:) W
water-oxidizing complex
Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement. 相似文献