Browsing by Author "Bravo, Denisse"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Biomimetic, Mild Chemical Synthesis of CdTe-GSH Quantum Dots with Improved Biocompatibility
    (PUBLIC LIBRARY SCIENCE, 2012) Perez Donoso, Jose M.; Monras, Juan P.; Bravo, Denisse; Aguirre, Adam; Quest, Andrew F.; Osorio Roman, Igor O.; Aroca, Ricardo F.; Chasteen, Thomas G.; Vasquez, Claudio C.
    Multiple applications of nanotechnology, especially those involving highly fluorescent nanoparticles (NPs) or quantum dots (QDs) have stimulated the research to develop simple, rapid and environmentally friendly protocols for synthesizing NPs exhibiting novel properties and increased biocompatibility. In this study, a simple protocol for the chemical synthesis of glutathione (GSH)-capped CdTe QDs (CdTe-GSH) resembling conditions found in biological systems is described. Using only CdCl2, K2TeO3 and GSH, highly fluorescent QDs were obtained under pH, temperature, buffer and oxygen conditions that allow microorganisms growth. These CdTe-GSH NPs displayed similar size, chemical composition, absorbance and fluorescence spectra and quantum yields as QDs synthesized using more complicated and expensive methods. CdTe QDs were not freely incorporated into eukaryotic cells thus favoring their biocompatibility and potential applications in biomedicine. In addition, NPs entry was facilitated by lipofectamine, resulting in intracellular fluorescence and a slight increase in cell death by necrosis. Toxicity of the as prepared CdTe QDs was lower than that observed with QDs produced by other chemical methods, probably as consequence of decreased levels of Cd+2 and higher amounts of GSH. We present here the simplest, fast and economical method for CdTe QDs synthesis described to date. Also, this biomimetic protocol favors NPs biocompatibility and helps to establish the basis for the development of new, "greener" methods to synthesize cadmium-containing QDs.
  • No Thumbnail Available
    Item
    Capsular-defective Porphyromonas gingivalis mutant strains induce less alveolar bone resorption than W50 wild-type strain due to a decreased Th1/Th17 immune response and less osteoclast activity
    (2019) Monasterio, Gustavo; Fernandez, Baltasar; Castillo, Francisca; Rojas, Carolina; Cafferata, Emilio A.; Rojas, Leticia; Alvarez, Carla; Fernandez, Alejandra; Hernandez, Marcela; Bravo, Denisse; Vernal, Rolando
    Background Encapsulation of Porphyromonas gingivalis has been demonstrated as responsible of several host immunological changes, which have been associated with the pathogenesis of periodontitis. Using a murine model of periodontitis and two isogenic non-capsulated mutants of P. gingivalis, this study aimed to analyze whether P. gingivalis encapsulation induces more severe alveolar bone resorption, and whether this bone loss is associated with a T-helper (Th)1 and Th17-pattern of immune response. Methods Experimental periodontal infections were generated by oral inoculation with the encapsulated W50 wild-type strain or isogenic non-encapsulated Delta PG0116-PG0120 (GPA) and Delta PG0109-PG0118 (GPC) mutants of P. gingivalis. Periodontal infections induced with the encapsulated HG184 or non-encapsulated ATCC 33277 strains of P. gingivalis were used as controls. Alveolar bone resorption was analyzed using microcomputed tomography and scanning electron microscopy. The expression levels of Th1, Th2, Th17, or T regulatory-associated cytokines and RANKL, as well as the periodontal bacterial load, were quantified by quantitative polymerase chain reaction. The detection of Th1 and Th17 lymphocytes was analyzed by flow cytometry. Results In the periodontal lesions, both capsular-defective knockout mutant strains of P. gingivalis induced less alveolar bone resorption than the encapsulated W50 wild-type strain. This decreased bone loss was associated with a dismissed RANKL expression, decreased Th1- and Th17-type of cytokine expression, reduced Th1 and Th17 lymphocyte detection, and low osteoclast finding. Conclusion These data demonstrate that encapsulation of P. gingivalis plays a key role in the alveolar bone resorption induced during periodontitis, and this bone loss is associated with a Th1- and Th17-pattern of immune response triggered in the periodontal lesions.
  • No Thumbnail Available
    Item
    Cysteine-Mediated Green Synthesis of Copper Sulphide Nanoparticles: Biocompatibility Studies and Characterization as Counter Electrodes
    (2022) Saona, Luis A.; Campo-Giraldo, Jessica L.; Anziani-Ostuni, Giovanna; Ordenes-Aenishanslins, Nicolas; Venegas, Felipe A.; Giordana, Maria F.; Diaz, Carlos; Isaacs, Mauricio; Bravo, Denisse; Perez-Donoso, Jose M.
    A one-pot green method for aqueous synthesis of fluorescent copper sulphide nanoparticles (NPs) was developed. The reaction was carried out in borax-citrate buffer at physiological pH, 37 degrees C, aerobic conditions and using Cu (II) and the biological thiol cysteine. NPs exhibit green fluorescence with a peak at 520 nm when excited at 410 nm and an absorbance peak at 410 nm. A size between 8-12 nm was determined by dynamic light scattering and transmission electron microscopy. An interplanar atomic distance of (3.5 +/- 0.1) A and a hexagonal chalcocite crystalline structure (beta Ch) of Cu2S NPs were also determined (HR-TEM). Furthermore, FTIR analyses revealed a Cu-S bond and the presence of organic molecules on NPs. Regarding toxicity, fluorescent Cu2S NPs display high biocompatibility when tested in cell lines and bacterial strains. Electrocatalytic activity of Cu2S NPs as counter electrodes was evaluated, and the best value of charge transfer resistance (R-ct) was obtained with FTO/Cu2S (four layers). Consequently, the performance of biomimetic Cu2S NPs as counter electrodes in photovoltaic devices constructed using different sensitizers (ruthenium dye or CdTe NPs) and electrolytes (S2-/S-n(2-) or I-/I3-) was successfully checked. Altogether, novel characteristics of copper sulfide NPs such as green, simple, and inexpensive production, spectroscopic properties, high biocompatibility, and particularly their electrochemical performance, validate its use in different biotechnological applications.
  • Thumbnail Image
    Item
    Enhanced Glutathione Content Allows the In Vivo Synthesis of Fluorescent CdTe Nanoparticles by Escherichia coli
    (PUBLIC LIBRARY SCIENCE, 2012) Monras, Juan P.; Diaz, Victor; Bravo, Denisse; Montes, Rebecca A.; Chasteen, Thomas G.; Osorio Roman, Igor O.; Vasquez, Claudio C.; Perez Donoso, Jose M.
    The vast application of fluorescent semiconductor nanoparticles (NPs) or quantum dots (QDs) has prompted the development of new, cheap and safer methods that allow generating QDs with improved biocompatibility. In this context, green or biological QDs production represents a still unexplored area. This work reports the intracellular CdTe QDs biosynthesis in bacteria. Escherichia coli overexpressing the gshA gene, involved in glutathione (GSH) biosynthesis, was used to produce CdTe QDs. Cells exhibited higher reduced thiols, GSH and Cd/Te contents that allow generating fluorescent intracellular NP-like structures when exposed to CdCl2 and K2TeO3. Fluorescence microscopy revealed that QDs-producing cells accumulate defined structures of various colors, suggesting the production of differently-sized NPs. Purified fluorescent NPs exhibited structural and spectroscopic properties characteristic of CdTe QDs, as size and absorption/emission spectra. Elemental analysis confirmed that biosynthesized QDs were formed by Cd and Te with Cd/Te ratios expected for CdTe QDs. Finally, fluorescent properties of QDs-producing cells, such as color and intensity, were improved by temperature control and the use of reducing buffers.
  • Thumbnail Image
    Item
    Helicobacter pylori-Induced Loss of the Inhibitor-of-Apoptosis Protein Survivin Is Linked to Gastritis and Death of Human Gastric Cells
    (OXFORD UNIV PRESS INC, 2010) Valenzuela, Manuel; Perez Perez, Guillermo; Corvalan, Alejandro H.; Carrasco, Gonzalo; Urra, Hery; Bravo, Denisse; Toledo, Hector; Quest, Andrew F. G.
    Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.
  • No Thumbnail Available
    Item
    Steady State Kinetics for Enzymes with Multiple Binding Sites Upstream of the Catalytic Site
    (2023) Osorio, Manuel I.; Petrache, Mircea; Salinas, Dino G.; Valenzuela-Ibaceta, Felipe; Gonzalez-Nilo, Fernando; Tiznado, William; Perez-Donoso, Jose M.; Bravo, Denisse; Yanez, Osvaldo
    The Michaelis-Menten mechanism, which describes the binding of a substrate to an enzyme, is a simplification of the process on a molecular scale. A more detailed model should include the binding of the substrate to precatalytic binding sites (PCBSs) prior to the transition to the catalytic site. Our work shows that the incorporation of PCBSs, in steady-state conditions, generates a Michaelis-Menten-type expression, in which the kinetic parameters KM and Vmax adopt more complex expressions than in the model without PCBSs. The equations governing reaction kinetics can be seen as generalized symmetries, relative to time translation actions over the state space of the underlying chemical system. The study of their structure and defining parameters can be interpreted as looking for invariants associated with these time evolution actions. The expression of KM decreases as the number of PCBSs increases, while Vmax reaches a minimum when the first PCBSs are incorporated into the model. To evaluate the trend of the dynamic behavior of the system, numerical simulations were performed based on schemes with different numbers of PCBSs and six conditions of kinetic constants. From these simulations, with equal kinetic constants for the formation of the Substrate/PCBS complex, it is observed that KM and Vmax are lower than those obtained with the Michaelis-Menten model. For the model with PCBSs, the Vmax reaches a minimum at one PCBS and that value is maintained for all of the systems evaluated. Since KM decreases with the number of PCBSs, the catalytic efficiency increases for enzymes fitting this model. All of these observations are consistent with the general equation obtained. This study allows us to explain, on the basis of the PCBS to KM and Vmax ratios, the effect on enzyme parameters due to mutations far from the catalytic site, at sites involved in the first enzyme/substrate interaction. In addition, it incorporates a new mechanism of enzyme activity regulation that could be fundamental to search for new activity-modulating sites or for the design of mutants with modified enzyme parameters.