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Boje Müller Gundula A. Noll Antonia M. Ernst Boris Rüping Sira Groscurth Richard M. Twyman Lawrence M. Kawchuk Dirk Prüfer 《Applied microbiology and biotechnology》2010,88(3):689-698
Forisomes are mechanoproteins that undergo ATP-independent contraction–expansion cycles triggered by divalent cations, pH
changes, and electrical stimuli. Although native forisomes from Medicago truncatula comprise a number of subunits encoded by separate genes, here we show that at least two of those subunits (MtSEO1 and MtSEO4)
can assemble into homomeric forisome bodies that are functionally similar to their native, multimeric counterparts. We expressed
these subunits in plants and yeast, resulting in the purification of large quantities of artificial forisomes with unique
characteristics depending on the expression platform. These artificial forisomes were able to contract and expand in vitro
like native forisomes and could respond to electrical stimulation when immobilized between interdigital transducer electrodes.
These results indicate that recombinant artificial forisomes with specific characteristics can be prepared in large amounts
and used as components of microscale and nanoscale devices. 相似文献
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Noll GA Müller B Ernst AM Rüping B Groscurth S Twyman RM Kawchuk LM Prüfer D 《Bioengineered bugs》2011,2(2):111-114
Forisomes are protein bodies found exclusively in the phloem of the Fabaceae (legumes). In response to wounding, the influx of Ca ( 2+) induces a conformational change from a condensed to a dispersed state which plugs the sieve tubes and prevents the loss of photoassimilates. This reversible, ATP-independent reaction can be replicated with purified forisomes in vitro by adding divalent cations or electrically inducing changes in pH, making forisomes ideal components of technical devices. Although native forisomes comprise several subunits, we recently showed that functional homomeric forisomes with distinct properties can be expressed in plants and yeast, providing an abundant supply of forisomes with tailored properties. Forisome subunits MtSEO-F1 and MtSEO-F4 can each assemble into homomeric artificial forisomes, which indicates functional redundancy. However, we provide further evidence that both proteins are subunits of the native heteromeric forisome body in planta. We also show that the properties of artificial forisomes can be modified by immobilization, which is a prerequisite for their incorporation into technical devices. 相似文献
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Echevarría-Zomeño S Pérez-de-Luque A Jorrín J Maldonado AM 《Journal of experimental botany》2006,57(15):4189-4200
Sunflower broomrape (Orobanche cumana Wallr.) is a root holoparasitic angiosperm considered as one of the major constraints for sunflower production in Mediterranean areas. Breeding for resistance is regarded as the most effective, feasible, and environmentally friendly solution to control this parasite. However, the existing sources of genetic resistance are defeated by the continuous emergence of new more virulent races of the parasite. In this work, the interaction between sunflower and O. cumana has been analysed in order to gain insights into the mechanisms involved in resistance. Two sunflower genotypes were selected showing different behaviour against the new race F of O. cumana, HE-39998 (susceptible) and HE-39999 (resistant), and both compatible and incompatible interactions were compared. Pot and Petri dish bioassays revealed that only HE-39998 plants were severely affected, supporting a high number of successfully established broomrapes to mature flowering, whereas in HE-39999 root tubercles were never observed, resistance being associated with browning symptoms of both parasite and host tissues. Histological aspects of the resistance were further investigated. Suberization and protein cross-linking at the cell wall were seen in the resistant sunflower cells in contact with the parasite, preventing parasite penetration and connection to the host vascular system. In addition, fluorescence and confocal laser microscopy (CLM) observations revealed accumulation of phenolic compounds during the incompatible reaction, which is in agreement with these metabolites playing a defensive role during H. annuus-O. cumana interaction. 相似文献
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Eila Torvinen Teija Meklin Pirjo Torkko Sini Suomalainen Marjut Reiman Marja-Leena Katila Lars Paulin Aino Nevalainen 《Applied microbiology》2006,72(10):6822-6824
In contrast to the growth of fungi, the growth of mycobacteria in moisture-damaged building materials has rarely been studied. Environmental mycobacteria were isolated from 23% of samples of moisture-damaged materials (n = 88). The occurrence of mycobacteria increased with increasing concentrations of fungi. Mycobacteria may contribute to indoor exposure and associated adverse health effects. 相似文献
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Sira Echevarría-Zomeño Nieves Abril Julia Ruiz-Laguna Jesús Jorrín-Novo Ana M. Maldonado-Alconada 《Acta Physiologiae Plantarum》2012,34(2):793-805
Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular
biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations
contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol
adapted to the specific plant sample and the pursued research objectives. We have evaluated several protocols to establish
the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising
yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silica-membrane purification,
as the most convenient for Q. ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation
process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples
in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efficiency
than other more labour and time-consuming protocols. 相似文献
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Maldonado AM Echevarría-Zomeño S Jean-Baptiste S Hernández M Jorrín-Novo JV 《Journal of Proteomics》2008,71(4):461-472
This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued. 相似文献
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Luigi F Agnati Kjell Fuxe Maria Torvinen Susanna Genedani Rafael Franco Stan Watson Gastone G Nussdorfer Giuseppina Leo Diego Guidolin 《The journal of histochemistry and cytochemistry》2005,53(8):941-953
An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein-coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor-receptor interaction, and their distribution patterns in dual-color confocal laser microscopy. 相似文献
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Estrogenic regulation of skeletal muscle proteome: a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy 下载免费PDF全文
Eija K. Laakkonen Rabah Soliymani Sira Karvinen Jaakko Kaprio Urho M. Kujala Marc Baumann Sarianna Sipilä Vuokko Kovanen Maciej Lalowski 《Aging cell》2017,16(6):1276-1287
Female middle age is characterized by a decline in skeletal muscle mass and performance, predisposing women to sarcopenia, functional limitations, and metabolic dysfunction as they age. Menopausal loss of ovarian function leading to low circulating level of 17β‐estradiol has been suggested as a contributing factor to aging‐related muscle deterioration. However, the underlying molecular mechanisms remain largely unknown and thus far androgens have been considered as a major anabolic hormone for skeletal muscle. We utilized muscle samples from 24 pre‐ and postmenopausal women to establish proteome‐wide profiles, associated with the difference in age (30–34 years old vs. 54–62 years old), menopausal status (premenopausal vs. postmenopausal), and use of hormone replacement therapy (HRT; user vs. nonuser). None of the premenopausal women used hormonal medication while the postmenopausal women were monozygotic (MZ) cotwin pairs of whom the other sister was current HRT user or the other had never used HRT. Label‐free proteomic analyses resulted in the quantification of 797 muscle proteins of which 145 proteins were for the first time associated with female aging using proteomics. Furthermore, we identified 17β‐estradiol as a potential upstream regulator of the observed differences in muscle energy pathways. These findings pinpoint the underlying molecular mechanisms of the metabolic dysfunction accruing upon menopause, thus having implications for understanding the complex functional interactions between female reproductive hormones and health. 相似文献