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991.
992.
Immobilization and affinity purification of recombinant proteins using histidine peptide fusions 总被引:4,自引:0,他引:4
C Ljungquist A Breitholtz H Brink-Nilsson T Moks M Uhlén B Nilsson 《European journal of biochemistry》1989,186(3):563-569
A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics. 相似文献
993.
Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor 总被引:1,自引:0,他引:1
The use of an amperometric graphite-Teflon composite tyrosinase biosensor for the rapid monitoring of alkaline phosphatase (ALP), with no need of an incubation step and using phenyl phosphate as the substrate, is reported. Phenol generated by the action of ALP is monitored at the tyrosinase composite electrode through the electrochemical reduction of the o-quinone produced to catechol, which produces a cycle between the tyrosinase substrate and the electroactive product, giving rise to the amplification of the biosensor response and to the sensitive detection of ALP. The current was measured at -0.10 V 5 min after the addition of ALP. As a compromise between high ALP activity and high sensitivity for the detection of phenol, a pH of 8.5 was chosen. The substrate concentration was also optimized. A linear calibration plot was obtained for ALP between 2.0 x 10(-13) and 2.5 x 10(-11), with a detection limit of 6.7 x 10(-14) M. Different types of milk were analyzed with good results, using an extremely simple and rapid procedure. 相似文献
994.
995.
Plant cyclotides: an unusual class of defense compounds 总被引:2,自引:0,他引:2
Plant cyclotides are unusual peptides with low molecular masses and a three-dimensional structure characterized by the presence of a cyclic fold. Synthetic peptides can adopt this circular conformation, but it is not a common feature for most members of other peptide groups. Cyclotides present a wide range of functions, such as the ability to induce stronger contractions during childbirth and anti-tumor activity. Additionally, some cyclotides present anti-viral, insecticidal or proteinase inhibitory activity. In this paper, we describe the structural and functional characteristics of plant cyclotides, their most conserved features and the development of these peptides for human health and biotechnological applications. 相似文献
996.
Sudano MJ Paschoal DM Rascado Tda S Magalhães LC Crocomo LF de Lima-Neto JF Landim-Alvarenga Fda C 《Theriogenology》2011,75(7):1211-1220
The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification. 相似文献
997.
E. Cortés J.A. Goolsby P.J. Moran M.A. Marcos-García 《Biocontrol Science and Technology》2011,21(5):535-545
The effect of feeding by the armored scale, Rhizaspidiotus donacis (Leonardi, 1920) (Hemiptera: Diaspididae) on the growth of the plant Arundo donax L. (Poaceae) was evaluated under field conditions in its native range. The study was designed to evaluate the impact of R. donacis, a candidate agent for biological control of A. donax which is invasive in arid riparian ecosystems of the Southwestern USA and Mexico. The study was carried out at five A. donax sites in the Province of Alicante, Spain, differing in altitude and climate. At each site, 30 infested lateral shoots were selected and 15 were randomly treated monthly with imidacloprid insecticide. Shoot lengths were measured monthly over a 1-year period in a comparative growth analysis. Shoots infested with R. donacis had an over 2-fold reduced growth rate as compared to treated shoots. Growth of shoots varied by site, and the effect of R. donacis on growth was most pronounced in the late spring, when mature females produced first instar scale crawlers. The impact of R. donacis on A. donax growth under field conditions in the native range, combined with its narrow host specificity, indicate that R. donacis is a promising candidate for biological control of A. donax in North America and other areas invaded by this weed. 相似文献
998.
Differential effect of pertussis toxin on the affinity state for agonists of renal alpha 1- and alpha 2- adrenoceptors 总被引:4,自引:0,他引:4
J L Boyer A García C Posadas J A García-Sáinz 《The Journal of biological chemistry》1984,259(13):8076-8079
The effect of pertussis toxin treatment on the guanine nucleotide-induced modulation of the affinity of renal alpha 1- and alpha 2-adrenergic receptors was investigated. Pretreatment of rats with pertussis toxin did not induce any change in the number of or affinity for antagonists of alpha 1- or alpha 2-receptors studied using [3H]prazosin and [3H]yohimbine, respectively. Guanyl-5'-yl imidodiphosphate induced an "up-shift" in the number of alpha 2-adrenergic receptors; this up-shift was not observed for alpha 1-adrenergic receptors. Pertussis toxin treatment decreased the affinity of epinephrine for the [3H]yohimbine-binding sites and reduced the ability of guanine nucleotides to modulate alpha 2-adrenoceptor agonist affinity. The regulation by guanine nucleotides of alpha 1-adrenoceptor affinity for agonists was not altered. These results suggest that the modulation of alpha 1- and alpha 2-adrenoceptors by guanine nucleotides is probably exerted through different molecular entities. 相似文献
999.
1000.
Luis Giménez-Benavides Raúl García-Camacho José María Iriondo Adrián Escudero 《Evolutionary ecology》2011,25(4):777-794
Under climate warming, plants will undergo novel selective pressures to adjust reproductive timing. Adjustment between reproductive
phenology and environment is expected to be higher in arctic and alpine habitats because the growing season is considerably
short. As early- and late-flowering species reproduce under very different environmental conditions, selective pressures on
flowering phenology and potential effects of climate change are likely to differ between them. However, there is no agreement
on the magnitude of the benefits and costs of early- vs. late-flowering species under a global warming scenario. In spite
of its relevance, phenotypic selection on flowering phenology has rarely been explored in alpine plants and never in Mediterranean
high mountain species, where selective pressures are very different due to the summer drought imposed over the short growth
season. We hypothesized that late-flowering plants in Mediterranean mountains should present stronger selective pressures
towards early onset of reproduction than early-flowering species, because less water is available in the soil as growing season
progresses. We performed selection analyses on flowering onset and duration in two high mountain species of contrasting phenology.
Since phenotypic selection can be highly context-dependent, we studied several populations of each species for 2 years, covering
their local altitudinal ranges and their different microhabitats. Surrogates of biotic selective agents, like fruitset for
pollinators and flower and fruit loss for flower and seed predators, were included in the analysis. Differences between the
early- and the late-flowering species were less than expected. A consistent negative correlational selection of flowering
onset and duration was found affecting plant fitness, i.e., plants that bloomed earlier flowered for longer periods improving
plant fitness. Nevertheless, the late-flowering species may experience higher risks under climate warming because in extremely
warm and dry years the earlier season does not bring about a longer flowering duration due to summer drought. 相似文献