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81.
The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden. 相似文献
82.
Elain Gutierrez‐Carbonell Giuseppe Lattanzio Alfonso Albacete Juan José Rios Julia Kehr Anunciación Abadía Michael A. Grusak Javier Abadía Ana Flor López‐Millán 《Proteomics》2015,15(22):3835-3853
The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF‐SDS‐PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty‐three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe‐deficient plants may have an impaired capacity to heal sieve elements upon injury. 相似文献
83.
Hélène Cheap Sophie Bernad Valérie Derrien László Gerencsér Julia Tandori Pedro de Oliveira Deborah K. Hanson Péter Maróti Pierre Sebban 《BBA》2009,1787(12):1505-1515
Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely QA and QB. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P+QA? state (P is the primary electron donor) and temperature dependence of the rate of P+QA? charge recombination that are in good agreement show that in the two RC variants, both QA? and QB? are destabilized by about the same free energy amount: respectively ~ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of QA? and QB?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein. 相似文献
84.
85.
Sara Kanje Raminta Venskutonytė Julia Scheffel Johan Nilvebrant Karin Lindkvist-Petersson Sophia Hober 《Journal of molecular biology》2018,430(18):3427-3438
Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification. 相似文献
86.
Testa G Schaft J van der Hoeven F Glaser S Anastassiadis K Zhang Y Hermann T Stremmel W Stewart AF 《Genesis (New York, N.Y. : 2000)》2004,38(3):151-158
Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications. 相似文献
87.
Background
The ability to predict antibody binding sites (aka antigenic determinants or B-cell epitopes) for a given protein is a precursor to new vaccine design and diagnostics. Among the various methods of B-cell epitope identification X-ray crystallography is one of the most reliable methods. Using these experimental data computational methods exist for B-cell epitope prediction. As the number of structures of antibody-protein complexes grows, further interest in prediction methods using 3D structure is anticipated. This work aims to establish a benchmark for 3D structure-based epitope prediction methods. 相似文献88.
Podocarpus falcatus is an indigenous evergreen conifer species of tropical mountain forests in southeastern Ethiopia, showing potential tree ages of around 500 years. To study the influence of seasonal climate on the growth pattern of P. falcatus, we combined high-resolution electronic dendrometer measurements with wood anatomical investigations of microcores from the outermost stem parts collected in monthly intervals. At any time of the year sufficient rain events are able to cause cambial activity in P. falcatus. This permanent growing readiness leads to irregular wood formation with the formation of intra-annual density fluctuations and missing rings. Wood anatomical studies of microcores collected around the circumference of a mature P. falcatus revealed locally different activity status of the cambium on different lobes of the stem. Tree-ring width measurements of stem disks resulted in tentative tree ages that were confirmed by radiocarbon dating of selected wood samples. Although our efforts to cross-date ring-width series from several stem disks were not successful, further sampling in areas with different rainfall regimes, additional radiocarbon dating and measurements of stable isotopes hopefully would enable the establishment of a multi-century-long tree-ring series for climate reconstruction. 相似文献
89.
Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations 下载免费PDF全文
Julia Jabs Franziska M Zickgraf Jeongbin Park Steve Wagner Xiaoqi Jiang Katharina Jechow Kortine Kleinheinz Umut H Toprak Marc A Schneider Michael Meister Saskia Spaich Marc Sütterlin Matthias Schlesner Andreas Trumpp Martin Sprick Roland Eils Christian Conrad 《Molecular systems biology》2017,13(11)
Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests. 相似文献
90.
Root Hairs as a Model System for Studying Plant Cell Growth 总被引:5,自引:0,他引:5
Root hairs are tip-growing projections that form on specializedepidermal cells. Physiological studies are identifying key transportersrequired for hair growth, and drug studies have been instructivein defining the role of the cytoskeleton in cell morphogenesis.Genetic analysis is identifying proteins involved in cell growthand the phenotypes of the mutants are instructive in definingthe precise function of these proteins in cellular morphogenesis.Recent progress in our understandings of cell growth using thearabidopsis root hair as a model system is reviewed. Copyright2001 Annals of Botany Company Arabidopsis, root hair, trichoblast, actin, microtubules, cell wall, genetics, calcium, potassium, phosphorus 相似文献