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1.
The dehydration of human and bovine methemoglobins was monitored using ESR spectroscopy of the iron signal. The interconversion of the Fe(III) signal between the high spin form (at g
6) in solution and low spin form (at g
2) was quantitatively studied as a function of hydration. The dehydration process leads also to a loss of paramagnetism resulting in the appearance of about 40% Fe(II) below 0.40 grH2O/grHb. The remaining 60% of Fe(III) ESR signal is distributed as the residual high spin form (at g
6, 5%) and low spin form (hemichromes H and P, 55%). The formation of hemichrome P was explained as resulting from the coordination of the cysteine residue at β93 with the iron atom which follows the rupture of the proximal histidine bond. Experiments with hemoglobins where the sulphur atom of cysteine β was blocked (N-ethylmaleimide) did not showed the hemichrome P, confirming the involvement of the sulphur atom. This implies that the dehydration process induces displacements and torsion of the F helix, drastically changing the iron coordination at proximal site. In agreement with this proposition the Fe(II) symmetry is pentacoordinated with the disrupted bond to the proximal histidine at fifth coordination. This is also supported by ESR experiments with nitrosyl complex at low hydrations. All conformational changes were reversibly modulated by hydration degree and partially by lyophilization rate. A one-cycle dehydration of bovine hemoglobin followed by solubilization shows 100% reversibility of hemichrome P. Increasing the number of cycles of dehydration-hydration reduces the reversibility degree. With three cycles a reversibility of 70%–75% is observed. The level of 0.40 grH2O/grHb was the critical hydration for the molecules to return to aquo met form and correspond also to a minimal water content necessary to cover all protein surface as obtained from other techniques. 相似文献
2.
Fausto Almeida Aline Sardinha-Silva Thiago Aparecido da Silva André Moreira Pessoni Camila Figueiredo Pinzan Ana Claudia Paiva Alegre-Maller Nerry Tatiana Cecílio Nilmar Silvio Moretti André Ricardo Lima Damásio Wellington Ramos Pedersoli José Roberto Mineo Roberto Nascimento Silva Maria Cristina Roque-Barreira 《PloS one》2015,10(12)
Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50°C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection. 相似文献
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Reverse enzyme synthesis in microemulsion-based organo-gels. 总被引:8,自引:0,他引:8
G D Rees M da Graca Nascimento T R Jenta B H Robinson 《Biochimica et biophysica acta》1991,1073(3):493-501
Lipase from three different sources has been immobilised in microemulsion-based gels (MBGs) with retention of catalytic activity. Such lipase-containing MBGs prove to be novel solid-phase catalysts for use in apolar organic solvents such as n-heptane. Using these systems, preparative-scale synthesis of a wide variety of esters under mild conditions was possible with products easily isolated and obtained in high yield. Stereoselective esterification of octan-2-ol was observed for all three lipases with Chromobacterium viscosum (CV) lipase yielding product with an enantiomeric excess of 92%. Repeated usage of a CV lipase-containing MBG resulted in a visually unchanged gel whose activity was 75% of the initial value after 30 days. The sectioned MBGs were well suited for use in column flow reactors and were also found to be effective esterification catalysts at temperatures as low as -20 degrees C. 相似文献
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Carolina G. dos Santos André L. Silva Flavio L. Souza Alexandre J. C. Lanfredi Paolo Di Mascio Otaciro R. Nascimento Tiago Rodrigues Iseli L. Nantes 《PloS one》2013,8(10)
The present study shows the factors that modulate the photodamage promoted by phenothiazines. Cytochrome c was irradiated with UV light for 120 min, over a pH range from 4.0 to 8.0, in the absence and in the presence of different concentrations of thioridazine (TR) and fluphenazine (FP). In the absence of phenothiazines, the maximal rate of a Soret band blue shift (nm/min) from 409 to 406 nm was obtained at pH 4.0 (0.028 nm/min). The presence of phenothiazines at the concentration range 10-25 µmol/L amplified and accelerated a cytochrome c blue shift (409 to 405 nm, at a rate = 0.041 nm/min). Above 25 µmol/L, crescent concentrations of phenothiazines contributed to cytochrome c protection with (maximal at 2500 µmol/L). Scanning electronic microscopy revealed the formation of nanostructures. The pH also influenced the effect of low phenothiazine concentrations on cytochrome c. Thus, the predominance of phenothiazine-promoted cytochrome c damage or protection depends on a balance of the following factors: the yield of photo-generated drug cation radicals, which is favored by acidic pH; the stability of the cation radicals, which is favored by the drug aggregation; and the cytochrome c structure, modulated by the pH. 相似文献
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In this ESR work we have studied the pentacoordinate symmetry in horse, whale and sperm-whale myoglobin (Mb) in different physical states such as solution and powder. Experiments were performed in which the following parameters were varied: the sample temperature, pH, reaction time with NO, and NO concentration. The results enabled us to explain the NO reaction mechanism in the oxy and met forms of myoglobin. The study of powder samples at different degrees of hydration allowed us to identify the diamagnetic intermediate species existent in the reaction of NO with met-Mb proposed in the literature. The results presented explain adequately the pH effect and temperature dependence observed in the ESR spectra obtained using the met-Mb sample solutions from Sigma Chemical Co., which consist of a mixture (13%) of Mb-O2. 相似文献
9.
A quantitative determination of maleimide spin label (MAL) binding in oxi and met hemoglobin (Hb) and bovine serum albumin are investigated using double integration to the ESR signal. This determination permitted the observation that a considerable fraction of MAL is reduced, losing its paramagnetism. Experiments using the same spin label with myoglobin and Hb with blocked-SH groups, where reduction was not observed, indicate the involvement of SH groups in the process. The 4-hydroxy-2,2,6,6-tetramethylpiperidino-1-oxyl spin label (which is not able to bind in the SH group) is reduced too, but the dependence on the molar ratio is different in comparison with the MAL case. In both cases the reduction percentage depends on the molar ratio spin label to protein and to the protein concentration. In order to obtain the total SH groups labeled (two in the Hb case) it is necessary to use an excessive amount of label (around 18:1) in the 0.5 mM Hb concentration. 相似文献
10.