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1.
Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway 总被引:1,自引:0,他引:1
RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies. 相似文献
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Michael S. Engel Diying Huang Abdulaziz S. Alqarni Chenyang Cai Mabel Alvarado Laura C.V. Breitkreuz Dany Azar 《Comptes Rendus Palevol》2017,16(1):5-11
A remarkably specialized parasitoid wasp of the family Scelionidae (Platygastroidea) is described and figured from mid-Cretaceous (Cenomanian) amber of the Hukawng Valley in northern Myanmar. Geoscelio mckellari Engel and Huang, gen. et sp. nov., is unique for its combination of a compact body, 12 antennal flagellomeres, a 1-2-2 tibial spur formula, a distinct malar sulcus, deeply impressed notauli, complete reduction of the wings, and basal crenulae on the metasomal terga and sterna, among many other features, and is placed within a separate tribe, Geoscelionini Engel and Huang, trib. nov. This is the first flightless species of Platygastroidea known from the Mesozoic, and its affinities with other Mesozoic and extant lineages are discussed. 相似文献
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Rebecca L. Hunter Michael Scott Webb Thomas M. Iliffe Jaime R. Alvarado Bremer 《Journal of Biogeography》2008,35(1):65-75
Aim To infer phylogenetic relationships among five species of the cave‐adapted shrimp genus Typhlatya in order to test competing hypotheses of dispersal and colonization of the disjunct cave localities occupied by these five species. Location Typhlatya species are found in caves and anchialine ponds across the northern margin of the Caribbean Sea, along the Mediterranean and Adriatic coasts and on oceanic islands in the Atlantic and eastern Pacific oceans. This study focuses on five species, one from Bermuda, one from the Caicos Islands and three from the Yucatan Peninsula of Mexico. Methods Partial sequences (c. 1400 bp) from the mitochondrial cytochrome b, 16S rDNA and COI genes were obtained from representative samples of the five species. Phylogenetic inference was carried out with maximum parsimony and maximum likelihood analyses. Parsimony networks were constructed for the Bermudian species Typhlatya iliffei and one Yucatan species Typhlatya mitchelli, to determine the degree of connectivity among populations inhabiting different cave systems. Results All three land masses were recovered as monophyletic. The two insular marine species from Bermuda and the Caicos Islands formed a clade, while the three continental freshwater species from the Yucatan Peninsula formed another. Within both Bermuda and the Yucatan, shared haplotypes were found in different cave systems, suggesting recent or ongoing gene flow among populations in both locales. Main conclusions The two insular marine Typhlatya species originated from an ancestral marine population, possibly already cave‐adapted, that is suggested to have colonized the Caicos Islands and subsequently dispersed to Bermuda via the Gulf Stream. Divergence estimates suggest that colonization occurred before the formation of present‐day anchialine cave habitat, which did not form on either island until the late Pliocene to early Pleistocene. Divergence estimates also indicate that the Yucatan freshwater species split before the formation of freshwater cave habitat in the Yucatan. These species could have inhabited crevicular marine habitats before the late Pliocene/early Pleistocene in the Yucatan or elsewhere in the Caribbean, and subsequently migrated to freshwater caves once they formed. 相似文献
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Savannah S. Cooley;Naiara Pinto;Milagros Becerra;Jorge Washington Vela Alvarado;Jocelyn C. Fahlen;Ovidio Rivera;G. Andrew Fricker;Augusto Rafael De Los Ríos Dantas;Naikoa Aguilar-Amuchastegui;Yunuen Reygadas;Julie Gan;Ruth DeFries;Duncan N. L. Menge; 《Ecology and evolution》2024,14(8):e70116
Improving our ability to monitor fragmented tropical ecosystems is a critical step in supporting the stewardship of these complex landscapes. We investigated the structural characteristics of vegetation classes in Ucayali, Peru, employing a co-production approach. The vegetation classes included three agricultural classes (mature oil palm, monocrop cacao, and agroforestry cacao plantations) and three forest regeneration classes (mature lowland forest, secondary lowland forest, and young lowland vegetation regrowth). We combined local knowledge with spaceborne lidar from NASA's Global Ecosystem Dynamics Investigation mission to classify vegetation and characterize the horizontal and vertical structure of each vegetation class. Mature lowland forest had consistently higher mean canopy height and lower canopy height variance than secondary lowland forest (μ = 29.40 m, sd = 6.89 m vs. μ = 20.82 m, sd = 9.15 m, respectively). The lower variance of mature forest could be attributed to the range of forest development ages in the secondary forest patches. However, secondary forests exhibited a similar vertical profile to mature forests, with each cumulative energy percentile increasing at similar rates. We also observed similar mean and standard deviations in relative height ratios (RH50/RH95) for mature forest, secondary forest, and oil palm even when removing the negative values from the relative height ratios and interpolating from above-ground returns only (mean RH50/RH95 of 0.58, 0.54, and 0.53 for mature forest, secondary forest, and oil palm, respectively) (p < .0001). This pattern differed from our original expectations based on local knowledge and existing tropical forest succession studies, pointing to opportunities for future work. Our findings suggest that lidar-based relative height metrics can complement local information and other remote sensing approaches that rely on optical imagery, which are limited by extensive cloud cover in the tropics. We show that characterizing ecosystem structure with a co-production approach can support addressing both the technical and social challenges of monitoring and managing fragmented tropical landscapes. 相似文献
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Alvarado Claudia Ramírez Jose Martín Herrera-López Enrique J. Cortez-Valladolid Diego Ramírez Gerardo 《Biological trace element research》2020,193(1):226-233
Biological Trace Element Research - Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis.... 相似文献
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Tanya Majumder Min Liu Vicky Chen Marife Martinez Danielle Alvarado Karl V. Clemons David A. Stevens 《Mycopathologia》2014,178(3-4):189-195
Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 × 107; 2S, 1.2 × 108; 3S, 2.4 × 108) began 28 (×3), 35 (×4), or 42 (×6) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S×4, or 2S reduced brain CFU; liver or spleen CFU was reduced by S×4 or 2S. In a more lethal second experiment, 2S×6, 2S, or 3S×4 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S×6, 2S, or 3S×4 most efficacious. Dose size appears more important than the number of doses: Regimens >S were superior, and 2S and 2S×6 were equivalent. 2S and 3S were equivalent, suggesting doses >2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine. 相似文献
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