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81.
Conrado RJ Wu GC Boock JT Xu H Chen SY Lebar T Turnšek J Tomšič N Avbelj M Gaber R Koprivnjak T Mori J Glavnik V Vovk I Benčina M Hodnik V Anderluh G Dueber JE Jerala R DeLisa MP 《Nucleic acids research》2012,40(4):1879-1889
Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications. 相似文献
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Busby GB Brisighelli F Sánchez-Diz P Ramos-Luis E Martinez-Cadenas C Thomas MG Bradley DG Gusmão L Winney B Bodmer W Vennemann M Coia V Scarnicci F Tofanelli S Vona G Ploski R Vecchiotti C Zemunik T Rudan I Karachanak S Toncheva D Anagnostou P Ferri G Rapone C Hervig T Moen T Wilson JF Capelli C 《Proceedings. Biological sciences / The Royal Society》2012,279(1730):884-892
Recently, the debate on the origins of the major European Y chromosome haplogroup R1b1b2-M269 has reignited, and opinion has moved away from Palaeolithic origins to the notion of a younger Neolithic spread of these chromosomes from the Near East. Here, we address this debate by investigating frequency patterns and diversity in the largest collection of R1b1b2-M269 chromosomes yet assembled. Our analysis reveals no geographical trends in diversity, in contradiction to expectation under the Neolithic hypothesis, and suggests an alternative explanation for the apparent cline in diversity recently described. We further investigate the young, STR-based time to the most recent common ancestor estimates proposed so far for R-M269-related lineages and find evidence for an appreciable effect of microsatellite choice on age estimates. As a consequence, the existing data and tools are insufficient to make credible estimates for the age of this haplogroup, and conclusions about the timing of its origin and dispersal should be viewed with a large degree of caution. 相似文献
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Manuela da Silva Solcà Leila Andrade Bastos Carlos Eduardo Sampaio Guedes Marcelo Bordoni Lairton Souza Borja Daniela Farias Larangeira Pétala Gardênia da Silva Estrela Tuy Leila Denise Alves Ferreira Amorim Eliane Gomes Nascimento Geraldo Gileno de Sá Oliveira Washington Luis Conrado dos-Santos Deborah Bittencourt Mothé Fraga Patrícia Sampaio Tavares Veras 《PloS one》2014,9(7)
Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4–100) in splenic aspirate; 79.2% (CI 68–90.3) in lymph nodes; 77.3% (CI 64.5–90.1) in skin; 75% (CI 63.1–86.9) in blood; 50% (CI 30–70) in bone marrow; 37.5% (CI 24.2–50.8) in left-eye; and 29.2% (CI 16.7–41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7–79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5–96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing. 相似文献
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María Ruth Pazos Rosa María Tolón Cristina Benito Conrado Fernández Rodríguez Juan José Gorgojo Manuel Nevado Mariano Alvarez Francisco Arias Francisca Almodóvar María Teresa Pérez Fernández José Luis Lledó Sara González José Javier Fernández-Ruiz Julián Romero 《The journal of histochemistry and cytochemistry》2008,56(5):511-516
Experimental data suggest that the endogenous cannabinoid system is involved in gastric function in different animal species. In most of them, CB(1) receptors have been localized on vagal terminals innervating the external wall of the stomach. We aimed at studying the putative presence and distribution of these receptors in the human gastric mucosa. To this end, we first performed Western blotting, RT-PCR, in situ hybridization, and immunohistochemical analysis of CB(1) protein distribution in biopsy samples of healthy individuals. To determine the precise cell populations expressing CB(1) receptors, we performed double immunofluorescence plus confocal microscopy analysis of the same samples. Our results show that CB(1) receptors are present in the gastric epithelium of the mucosa. Specifically, they are expressed by a subpopulation of mucosal cells, the acid-secreting parietal cells, as shown by double immunohistochemical staining and by their differential abundance in subregions of the gastric mucosa. These results reinforce the notion of a prominent role for the endocannabinoid system in the gastric function in humans and postulate the use of cannabinoid CB(1) receptors in parietal cells as new therapeutic targets for the regulation of gastric acid production. 相似文献
87.
Bernd Masepohl Sieglinde Angermüller Silke Hennecke Philipp Hübner Conrado Moreno-Vivian Werner Klipp 《Molecular genetics and genomics : MGG》1993,238(3):369-382
DNA sequence analysis of a 3494-bp HindIII-Bc1I fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifUI and NifUII share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneurnoniae. In contrast to nifA andnifB which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifU ISVW operon, which is preceded by a putative σ54-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifU I and nifW mutants as well as a nifU I/nifUII, double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV?NifW?), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase. 相似文献
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