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排序方式: 共有135条查询结果,搜索用时 15 毫秒
1.
Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli 总被引:4,自引:0,他引:4
E Ciccarelli M Massaer J P Guillaume A Herzog R Loriau A Cravador P Jacobs A Bollen 《Biochemical and biophysical research communications》1989,161(2):865-872
DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product. 相似文献
2.
l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism?
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Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H+ efflux from the cells by 111% and the uptake of l-[U-14C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO2 evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and 14CO2 evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas 14CO2 evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-14C]glutamate were higher in the light while rates of 14CO2 evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO2 and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H+/l-glutamate uptake process. 相似文献
3.
Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands. 总被引:5,自引:2,他引:3
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A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli. 相似文献
4.
5.
S. Martinoia M. Meloni M. T. Parodi M. Tedesco C. Ciccarelli M. Grattarola 《Cytotechnology》1993,11(Z1):S86-S88
A silicon microsensor (ISFET — Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density. 相似文献
6.
7.
Dario Beruto Margherita Beruto Carlo Ciccarelli Pierre Debergh 《Physiologia plantarum》1995,94(1):151-157
A new method for evaluating the matric potential of gelled media has been developed. The method allows the derivation of the matric potential as a limit of a series of measurements of water potential values from gelled media prepared without added components, from agar powders progressively cleaned of mineral impurities. Three commercial agar brands were tested, and for these the matric potential was found to contribute only between 1 and 2% of their total water potential. Thermodynamic features relating matric and osmotic potentials are described. New hypotheses for understanding the water flux mechanism from gel to tissue cultured explants are discussed. Movement of water along polymeric chains is postulated to be a facilitated step in comparison with bulk movement. 相似文献
8.
Genetic variation at the Major Histocompatibility Complex locus DQ beta was
analyzed in 233 beluga whales (Delphinapterus leucas) from seven
populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi
Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island,
and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the
High Arctic beluga population. Variation was assessed by amplification of
the exon coding for the peptide binding region via the polymerase chain
reaction, followed by either cloning and DNA sequencing or single-stranded
conformation polymorphism analysis. Five alleles were found across the
beluga populations and one in the narwhal. Pairwise comparisons of these
alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per
site leading to eight amino acid differences, five of which were
nonconservative substitutions, centered around positions previously shown
to be important for peptide binding. Although the amount of allelic
variation is low when compared with terrestrial mammals, the nature of the
substitutions in the peptide binding sites indicates an important role for
the DQ beta locus in the cellular immune response of beluga whales.
Comparisons of allele frequencies among populations show the High Arctic
population to be different (P < or = .005) from the other beluga
populations surveyed. In these other populations an allele, Dele-DQ
beta*0101-2, was found in 98% of the animals, while in the High Arctic it
was found in only 52% of the animals. Two other alleles were found at high
frequencies in the High Arctic population, one being very similar to the
single allele found in narwhal.
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9.
John J. Malinowski Bruce L. Grasberger Gary Trakshel Edward E. Huston Tracey M. Banks Patricia G. Brake Richard B. Ciccarelli Barry N. Jones James A. Koehn Diane Kratz Nicole Lundberg Panayiotis E. Stevis Carla T. Helaszek Mark A. Ator Angela M. Small Wood Travis Stams Byron Rubin Richard S. Alexander 《Protein science : a publication of the Protein Society》1995,4(10):2149-2155
10.
Cultural and physiological characteristics of Clostridium botulinum type G and the susceptibility of certain animals to its toxin. 总被引:9,自引:5,他引:4
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A S Ciccarelli D N Whaley L M McCroskey D F Gimenez V R Dowell Jr C L Hatheway 《Applied microbiology》1977,34(6):843-848
Strain 89 of Clostridium botulinum type G, isolated by Gimenez and Ciccarelli in 1969, was characterized culturally, biochemically, and toxigenically. It was motile, hemolytic asaccharolytic, weakly proteolytic, lipase and lecithinase negative, and it produced acetic, isobutyric, butyric, and isovaleric acids in peptone-yeast extract-glucose broth. No spores were seen in smears from solid or liquid media. Very low levels of toxin were produced in regular broth cultures, but dialysis cultures yielded 30,000 mouse 50% mean lethal doses (LD50 per kg, orally and subcutaneously, respectively; and for guinea pigs, 10,000 to 20,000 and 100 mouse LD50 per kig, intragastrically and intraperitoneally, respectively. 相似文献