Publications (6)11.49 Total impact
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Dataset: Mercuri fluorescent flowers PlantSci2002 copia
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Article: Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide.
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ABSTRACT: Ethidium bromide (EtBr) is the most widely used fluorescent dye in nucleic acid gel electrophoresis since decades. However it has been essentially forgotten in DNA quantification by spectrofluorimetry. While investigating sensitivity and dynamic range of available fluorochromes, we found that EtBr permits much more sensitive fluorimetric measurements than previously thought. We report here a revised, accurate, and easy-to-use protocol for EtBr-based DNA quantification in solution, which usefully complements the widely used indirect quantification on agarose gels.BioTechniques 09/2007; 43(2):173-4, 176. · 2.67 Impact Factor -
Article: Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells.
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ABSTRACT: Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum-free medium (containing 5mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K(+) (5mM) concentrations, the potential anti-apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10 microg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K(+). Since there is evidence that low K(+)-induced apoptosis in CGNs is associated with a drop in intracellular Ca(2+) concentration ([Ca(2+)](i)), activation of the cell death effector proteases caspase-3 and caspase-9, and of the transcription factor nuclear factor kappa B (NF-kappaB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in [Ca(2+)](i) induced by low K(+), whereas it completely blocked caspase-3, caspase-9, and NF-kappaB activation. It is concluded that CAPE could exert its anti-apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.International Journal of Developmental Neuroscience 12/2003; 21(7):379-89. · 2.42 Impact Factor -
Article: Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore.
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ABSTRACT: The coral red fluorescent protein (DsRed) absorbs and emits light at much higher wavelengths than the structurally homologous green fluorescent protein, raising questions about the properties of its chromophore. We have analyzed the relationship between the aggregation state and fluorescence of native, 6-histidine-tagged, or maltose-binding protein-fused DsRed. In all cases, newly synthesized DsRed molecules were largely monomeric and devoid of covalently closed chromophores. Maturation in vitro induces the expression of red fluorescent chromophores but only in oligomeric forms of the protein, whereas monomers are essentially devoid of fluorescence. NaOH-denatured samples demonstrated a generalized breakdown of the DsRed oligomers to monomers, which refolded after neutralization into weakly green fluorescent and still monomeric species. Red fluorescent chromophores were regenerated only upon oligomerization. These findings demonstrate that 'red' chromophores form and are functional only as oligomers, and suggest that the smallest red fluorescent functional unit is a dimer. A comparison of alkali-, acid- and guanidinium-denatured DsRed indicates that stabilization of the DsRed chromophore by concerted steps of folding and oligomerization may play a critical role in its maturation process.FEBS Letters 09/2002; 525(1-3):13-9. · 3.54 Impact Factor -
Article: Green Fluorescent Protein variants fold differentially in prokaryotic and eukaryotic cells
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ABSTRACT: Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding efficiency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3–4 times better than the wtGFP or S65T in E. coli, and 10–20-fold or more than 95-fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding efficiency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or fluorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that specific GFP variants follow different folding trajectories in mammalian versus bacterial cells. The specificity of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo. J. Cell. Biochem. Suppl. 36: 117–128, 2001. © 2001 Wiley-Liss, Inc.Journal of Cellular Biochemistry 03/2001; 81(S36):117 - 128. · 2.87 Impact Factor -
Article: Green fluorescent flowers
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ABSTRACT: The Green Fluorescent Protein (GFP) is efficiently expressed in plant cells and its use as a flower fluorescent dye is appealing. However, the generation of fluorescent flowers has remained as yet elusive. In this work we demonstrate the generation of green-fluorescent flowers. Non-fluorescent GFP-transgenic flowers were analysed by Western blot and spectrofluorimetry, and were demonstrated to efficiently express GFP. This indicated that the lack of GFP fluorescence could be due to opacity to the exciting light. Thus, flowers that are largely transparent to UV, Eustoma grandiflorum (Lisianthus), or that present a transparent petal cuticle, Osteospermum ecklonis, were selected for gfp transformation. Strikingly, the transformed Eustoma flowers fluoresced brightly upon illumination with UV light. GFP-transgenic Osteospermum flowers also appeared distinctly green upon UV illumination. GFP was expressed in comparable amounts by the fluorescent Osteospermum and by the non-fluorescent Limonium petals, confirming that an efficient excitation of GFP fluorescence is a limiting factor in its detection. These results demonstrate the feasibility of using GFP as a fluorescent dye for flower petals. Chromophores excited at longer, more deeply penetrating wavelengths, e.g. DsRed, may extend the use this technology also to flowers opaque to UV light.Plant Science.
Top Journals
Institutions
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2001–2012
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Istituto di Ricerche Farmacologiche Mario Negri
- Laboratory of Experimental Neurology
Milano, Lombardy, Italy
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2007
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Università degli Studi G. d'Annunzio Chieti e Pescara
Chieti, Abruzzo, Italy
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