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1.
J W Wray W A Baase J D Lindstrom L H Weaver A R Poteete B W Matthews 《Journal of molecular biology》1999,292(5):1111-1120
The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability. 相似文献
2.
The chromosomes of two patients with ring 13 (r13) were studied using high-resolution RBG banding of prometaphase cells. The rings of the two patients differ slightly in breakpoints. Cell with multiple single, double-sized rings, quadruple-sized rings, rod- and ring-shaped fragments, and fragments showing varied states of condensation were seen, as were cells monosomic for chromosome 13. The evolution of these cell lines as a result of sister chromatid exchange, nondisjunction, ring breakage, and premature chromosome condensation is discussed. Clinical features of these patients reflect the heterogeneity of phenotype for r13 patients. Each case includes a feature of trisomy 13. The significance of mosaicism of cell lines in patients bearing ring chromosomes is considered with respect to variation in clinical findings. 相似文献
3.
Aurin tricarboxylic acid, the anti-AIDS compound, prevents the binding of interferon-alpha to its receptor 总被引:2,自引:0,他引:2
Y X Gan J L Weaver P S Pine K C Zoon A Aszalos 《Biochemical and biophysical research communications》1990,172(3):1298-1303
Binding of HIV to its receptor, the CD4 molecule of lymphocytes, can be prevented by chemical agents. These agents could be considered as potential anti-AIDS drugs. We have shown that aurin tricarboxylic acid (ATA, 3 microM) specifically blocks the binding of gp120, the HIV coat protein, to the CD4 molecule. We have also found that ATA prevents the binding of interferon-alpha to its receptor in a dose-dependent manner (12-50 microM range). Membrane potential shift, associated with binding of interferon-alpha to its receptor, was also blocked by ATA in a dose-dependent fashion. Our results indicate that potential anti-AIDS drugs should be screened for such undesired side effects. 相似文献
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6.
S Porasuphatana J Weaver T A Budzichowski P Tsai G M Rosen 《Analytical biochemistry》2001,298(1):50-56
Nitric oxide synthase (NOS) generates nitric oxide (NO*) by the oxidation of l-arginine. Spin trapping in combination with electron paramagnetic resonance (EPR) spectroscopy using ferro-chelates is considered one of the best methods to detect NO* in real time and at its site of generation. The spin trapping of NO* from isolated NOS I oxidation of L-arginine by ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) and ferro-N-methyl-d-glucamide dithiocarbamate (Fe(MGD)2) in different buffers was investigated. We detected NO-Fe(DTCS)2, a nitrosyl complex, resulting from the reaction of NO* and Fe(DTCS)2, in phosphate buffer. However, Hepes and Tris buffers did not allow formation of NO-Fe(DTCS)2. Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular oxygen. Fe(DTCS)2 and Fe(MGD)2 were found to inhibit, to a small degree, NOS I activity with a greater effect observed with Fe(MGD)2. In contrast, Fe(MGD)2 was more efficient at spin trapping NO* from the lipopolysaccharide-activated macrophage cell line RAW264.7 than was Fe(DTCS)2. Data suggested that Fe(DTCS)2 and Fe(MGD)2 are efficient at spin trapping NO* but their maximal efficiency may be affected by experimental conditions. 相似文献
7.
The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules. Seven transfers were made between the initial broth culture and the final broth culture. A total of 40 single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing effectiveness of the population of one strain did not change on culturing, whereas there was considerable variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different nodules formed by the same Bradyrhizobium strain often differed in their effectiveness. 相似文献
8.
The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.). These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin (ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1' and S2' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117 degrees, and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 A, strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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10.
Christine L. Truitt Elizabeth A. Weaver William G. Haldenwang 《Molecular & general genetics : MGG》1988,212(1):166-171
Summary The E-37 gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc
- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc
- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis. 相似文献