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881.
Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. 总被引:81,自引:33,他引:48 下载免费PDF全文
A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines). 相似文献
882.
M T de los Frailes F Sánchez-Franco M J Lorenzo G Fernandez Vazquez L Cacicedo 《Regulatory peptides》1990,27(1):97-105
There is increasing evidence that persistent depolarization plays a critical role not only in excitation-secretion coupling, but also in the mechanisms linking excitation of neuronal cells to long-term adaptative changes in biosynthesis of neuropeptides. Somatostatin (SRIF) release and synthesis are affected by numerous agents, such as high concentrations of potassium that cause depolarization of cellular membrane. In the present work, we tried to determine whether prolonged exposure to veratridine (VTD) regulates SRIF synthesis. We found that exposure to VTD (100 microM) resulted in the stimulation of total (cell content + media) immunoreactive SRIF (IR-SRIF). This effect was calcium- and sodium-dependent, since it was prevented when verapamil (VPM) 20 microM or tetrodotoxin (TTX) 1 microM were added simultaneously with VTD. Cerebral cortical cells were exposed to high potassium concentrations, and the nature of the IR-SRIF was characterized by high-pressure liquid chromatography (HPLC) or gel filtration. It was evident that chronic exposure to high potassium concentrations modified the elution profile of medium IR-SRIF on HPLC and gel filtration, causing an increase in somatostatin-28 (S-28) and a decrease in somatostatin-14 (S-14). The results indicate that chronic exposure to VTD or high potassium concentration increases immunoreactive somatostatin and augments synthesis of its high-molecular-weight forms. This suggests that chronic membrane depolarization activating sodium and calcium channels initiates the entry of calcium ions, which triggers somatostatin release and causes a depletion of its intracellular stores. The stimulation of somatostatin secretion could be coupled to synthesis of the peptide. 相似文献
883.
Victor de Lorenzo Marta Herrero J. B. Neilands 《Molecular & general genetics : MGG》1988,213(2-3):487-490
Summary Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system. 相似文献
884.
Laura Lucarini Lucia Magnelli Nicola Schiavone Alfonso Crisci Alessio Innocenti Luca Puccetti 《Journal of enzyme inhibition and medicinal chemistry》2018,33(1):234-240
Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy. 相似文献
885.
Marwa G. El-Gazzar Nessma H. Nafie Alessio Nocentini Mostafa M. Ghorab Helmi I. Heiba 《Journal of enzyme inhibition and medicinal chemistry》2018,33(1):1565-1574
We report the synthesis and characterisation of a novel series of triazole benzenesulfonamide derivatives, which incorporate the general pharmacophore associated with carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The synthesised compounds were tested in vitro against four human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I, hCA II, hCA IV and hCA IX. The obtained results showed that the tumour-associated hCA IX was the most sensitive to inhibition with the synthesised derivatives, with the triazolo-pyridine benzenesulfonamides 14, 16 and 17 being the most effective inhibitors. Some selected compounds were chosen for a single dose anti-proliferative activity testing against a panel of 57 human tumour cell lines and show some anti-proliferative activity ex vivo. 相似文献
886.
In vitro models of multiple system atrophy from primary cells to induced pluripotent stem cells 下载免费PDF全文
Elena Abati Alessio Di Fonzo Stefania Corti 《Journal of cellular and molecular medicine》2018,22(5):2536-2546
Multiple system atrophy (MSA) is a rare neurodegenerative disease with a fatal outcome. Nowadays, only symptomatic treatment is available for MSA patients. The hallmarks of the disease are glial cytoplasmic inclusions (GCIs), proteinaceous aggregates mainly composed of alpha‐synuclein, which accumulate in oligodendrocytes. However, despite the extensive research efforts, little is known about the pathogenesis of MSA. Early myelin dysfunction and alpha‐synuclein deposition are thought to play a major role, but the origin of the aggregates and the causes of misfolding are obscure. One of the reasons for this is the lack of a reliable model of the disease. Recently, the development of induced pluripotent stem cell (iPSC) technology opened up the possibility of elucidating disease mechanisms in neurodegenerative diseases including MSA. Patient specific iPSC can be differentiated in glia and neurons, the cells involved in MSA, providing a useful human disease model. Here, we firstly review the progress made in MSA modelling with primary cell cultures. Subsequently, we focus on the first iPSC‐based model of MSA, which showed that alpha‐synuclein is expressed in oligodendrocyte progenitors, whereas its production decreases in mature oligodendrocytes. We then highlight the opportunities offered by iPSC in studying disease mechanisms and providing innovative models for testing therapeutic strategies, and we discuss the challenges connected with this technique. 相似文献
887.
Angelino Carta Brunello Pierini Francesco Roma-Marzio Gianni Bedini Lorenzo Peruzzi 《Plant biosystems》2018,152(4):831-839
Biodiversity studies traditionally use species as their analysis unit. However, observed biodiversity is not necessarily an indicator of significant biodiversity and therefore, should not be used alone, particularly when the results will be employed for conservation planning. Here, we examine pteridophyte diversity in Tuscany using 4668 geo-referenced pteridophyte records stored in Wikiplantbase #Toscana and calculating standard richness measures and phylogenetic indices; in addition, we assessed the environmental predictors of each diversity metric. Finally, we identified which areas hosting significantly high diversity are not adequately covered by protected areas or intersected by human activities. Although considerable diversity is distributed along Tuscany within areas traditionally recognised as biodiversity hotspots, important areas of diversity are identified only after the application of randomisation tests and through the phylogenetic metrics. Topographic ruggedness resulted the single most important variable predicting high value of all indices; temperature seasonality and temperature continentality resulted the secondary explanatory variables with a significant negative effect on most diversity indices. 相似文献
888.
The cereal pathogen Fusarium pseudograminearum produces a new class of active cytokinins during infection 下载免费PDF全文
Jens Laurids Sørensen Aurelie H. Benfield Rasmus Dam Wollenberg Klaus Westphal Reinhard Wimmer Mikkel Rank Nielsen Kristian Fog Nielsen Jason Carere Lorenzo Covarelli Giovanni Beccari Jonathan Powell Takafumi Yamashino Herbert Kogler Teis Esben Sondergaard Donald Max Gardiner 《Molecular Plant Pathology》2018,19(5):1140-1154
889.
Assessing uncertainties in crop and pasture ensemble model simulations of productivity and N2O emissions 下载免费PDF全文
Fiona Ehrhardt Jean‐François Soussana Gianni Bellocchi Peter Grace Russel McAuliffe Sylvie Recous Renáta Sándor Pete Smith Val Snow Massimiliano de Antoni Migliorati Bruno Basso Arti Bhatia Lorenzo Brilli Jordi Doltra Christopher D. Dorich Luca Doro Nuala Fitton Sandro J. Giacomini Brian Grant Matthew T. Harrison Stephanie K. Jones Miko U. F. Kirschbaum Katja Klumpp Patricia Laville Joël Léonard Mark Liebig Mark Lieffering Raphaël Martin Raia S. Massad Elizabeth Meier Lutz Merbold Andrew D. Moore Vasileios Myrgiotis Paul Newton Elizabeth Pattey Susanne Rolinski Joanna Sharp Ward N. Smith Lianhai Wu Qing Zhang 《Global Change Biology》2018,24(2):e603-e616
Simulation models are extensively used to predict agricultural productivity and greenhouse gas emissions. However, the uncertainties of (reduced) model ensemble simulations have not been assessed systematically for variables affecting food security and climate change mitigation, within multi‐species agricultural contexts. We report an international model comparison and benchmarking exercise, showing the potential of multi‐model ensembles to predict productivity and nitrous oxide (N2O) emissions for wheat, maize, rice and temperate grasslands. Using a multi‐stage modelling protocol, from blind simulations (stage 1) to partial (stages 2–4) and full calibration (stage 5), 24 process‐based biogeochemical models were assessed individually or as an ensemble against long‐term experimental data from four temperate grassland and five arable crop rotation sites spanning four continents. Comparisons were performed by reference to the experimental uncertainties of observed yields and N2O emissions. Results showed that across sites and crop/grassland types, 23%–40% of the uncalibrated individual models were within two standard deviations (SD) of observed yields, while 42 (rice) to 96% (grasslands) of the models were within 1 SD of observed N2O emissions. At stage 1, ensembles formed by the three lowest prediction model errors predicted both yields and N2O emissions within experimental uncertainties for 44% and 33% of the crop and grassland growth cycles, respectively. Partial model calibration (stages 2–4) markedly reduced prediction errors of the full model ensemble E‐median for crop grain yields (from 36% at stage 1 down to 4% on average) and grassland productivity (from 44% to 27%) and to a lesser and more variable extent for N2O emissions. Yield‐scaled N2O emissions (N2O emissions divided by crop yields) were ranked accurately by three‐model ensembles across crop species and field sites. The potential of using process‐based model ensembles to predict jointly productivity and N2O emissions at field scale is discussed. 相似文献
890.
Elena Andreucci Silvia Pietrobono Silvia Peppicelli Jessica Ruzzolini Francesca Bianchini Alessio Biagioni Barbara Stecca Lido Calorini 《Cell communication and signaling : CCS》2018,16(1):87