58 results on '"Carlos Vera"'
Search Results
2. Enzymatic production of prebiotic oligosaccharides
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Cecilia Guerrero, Carlos Vera, and Andrés Illanes
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0301 basic medicine ,chemistry.chemical_classification ,030109 nutrition & dietetics ,Chemistry ,Prebiotic ,medicine.medical_treatment ,04 agricultural and veterinary sciences ,040401 food science ,Applied Microbiology and Biotechnology ,Chemical synthesis ,03 medical and health sciences ,0404 agricultural biotechnology ,Enzyme ,Biochemistry ,Functional food ,Biocatalysis ,medicine ,Food Science - Abstract
Prebiotics are health promoting functional food ingredients. Main prebiotics are non-digestible oligosaccharides (NDOs), which are extracted from natural sources or enzymatically synthesized. The presence of equally reactive hydroxyl groups in carbohydrates makes the chemical synthesis of NDOS quite complex, so their enzymatic production is the preferred choice at large scale. The impressive advances in enzyme biocatalysis has allowed the development of robust biocatalysts which are compatible with their use under the rather harsh conditions usually required for the reactions of synthesis. In fact, nowadays several NDOs are being produced enzymatically at industrial level. In this article, the use of glycoside-hydrolases and glycosyltransferases for commercial NDOs production is reviewed.
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- 2021
3. Comparison of batch and repeated batch operation of lactulose synthesis with cross-linked aggregates of Bacillus circulans β-galactosidase
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Andrés Illanes, Claudia Ubilla, Carla Aburto, Carlos Vera, Nicolás Ramírez, Sebastián Suárez, and Cecilia Guerrero
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0106 biological sciences ,0303 health sciences ,Chromatography ,Bioengineering ,Fructose ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Propanol ,03 medical and health sciences ,chemistry.chemical_compound ,Lactulose ,chemistry ,Biocatalysis ,010608 biotechnology ,Bacillus circulans ,medicine ,Thermal stability ,Specific activity ,Lactose ,030304 developmental biology ,medicine.drug - Abstract
Synthesis of lactulose with crosslinked aggregates of Bacillus circulans β-galactosidase (CLAGs) has been compared in batch and repeated-batch operation for the first time. The effect of the type of the precipitating agent and its concentration, the crosslinker concentration and the time of crosslinking were evaluated for their effect on the parameters: immobilization yield, specific activity and thermal stability of the biocatalysts. The type and concentration of the precipitating agent were the variables that produced a significant variation in the immobilization parameters of the biocatalyst. CLAGs were obtained with a specific activity of 7790 IUH⋅g−1 at an immobilization yield of 46.2 % using 50 % v/v of propanol as precipitating agent, 5.5 gglutarldehyde gprotein−1 for crosslinking and 1 h of crosslinking time. This biocatalyst was more stable than the free enzyme with a stabilization factor of 11.3 h at 50 °C. Highest yield of lactulose synthesis with CLAGs was 0.42 g g−1 for a fructose/lactose molar ratio of 8. Repeated-batch operation allowed a significant increase in lactulose production per unit mass of biocatalyst and in cumulative productivity with respect to batch operation, yielding an efficiency of biocatalyst use of 2.43 kglactulose gbiocatalyst protein−1.
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- 2020
4. Improvements in the production of Aspergillus oryzae β-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose
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Andrés Illanes, Sebastián Suárez, Carla Aburto, Carlos Vera, and Cecilia Guerrero
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Aspergillus oryzae ,Chemistry Techniques, Synthetic ,02 engineering and technology ,Biochemistry ,Propanol ,03 medical and health sciences ,chemistry.chemical_compound ,Lactulose ,Structural Biology ,Enzyme Stability ,medicine ,Lactose ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Ethanol ,biology ,Temperature ,Fructose ,General Medicine ,Enzymes, Immobilized ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Galactosidases ,chemistry ,Yield (chemistry) ,Biocatalysis ,Methanol ,0210 nano-technology ,Nuclear chemistry ,medicine.drug - Abstract
Aspergillus oryzae β-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IUH g−1 were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50 °C, pH 4.5, 50% w/w total sugars, 200 IUH g−1 of enzyme and fructose/lactose molar ratio of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g g−1 and 0.24 g g−1 for fructose to lactose molar ratios of 4 mol mol−1 and 8 mol mol−1 while selectivities were 3.3 mol mol−1 and 6.6 mol mol−1 respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation.
- Published
- 2020
5. Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer
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Jessica D. Panes, Marcell Gatica, Eduardo Peña, Eveling Inostroza, Ilona I. Concha, Juan Carlos Vera, Mafalda Maldonado, Michelle Maurin, Kirsty Sotomayor, Pamela Mendoza, Tiare Silva-Grecchi, Claire Labrousse, Carolina Delgado, Francisco A. Gutierrez-Castro, Alejandro M. Reyes, Mauricio Ostria González, Gustavo Moraga-Cid, Francisco J. Roa, Carola Muñoz-Montesino, Carlos F. Aylwin, Karen Sweet, Coralia I. Rivas, and Jaime Madariaga
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0301 basic medicine ,Vitamin ,Breast Neoplasms ,Ascorbic Acid ,Mitochondrial Membrane Transport Proteins ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Breast cancer ,Physiology (medical) ,medicine ,Humans ,skin and connective tissue diseases ,Sodium-Coupled Vitamin C Transporters ,Vitamin C ,Sodium ,Glucose transporter ,Cancer ,Bystander Effect ,Ascorbic acid ,medicine.disease ,Mitochondria ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,chemistry ,Cancer cell ,MCF-7 Cells ,Cancer research ,Female ,Dehydroascorbic acid ,Reactive Oxygen Species ,Oxidation-Reduction ,030217 neurology & neurosurgery - Abstract
The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines. We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.
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- 2019
6. β-Galactosidase from Exiguobacterium acetylicum: Cloning, expression, purification and characterization
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Carlos Castillo, Mauricio Arenas-Salinas, Cecilia Guerrero, Carla Aburto, Claudio C. Vásquez, Fabián A. Cornejo, Andrés Illanes, Carlos Vera, and Felipe A. Arenas
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0106 biological sciences ,Hot Temperature ,Environmental Engineering ,Gene Expression ,Bioengineering ,010501 environmental sciences ,medicine.disease_cause ,01 natural sciences ,Substrate Specificity ,Lactulose ,Hydrolysis ,chemistry.chemical_compound ,Exiguobacterium acetylicum ,010608 biotechnology ,Escherichia coli ,medicine ,Cloning, Molecular ,Lactose ,Bacillaceae ,Waste Management and Disposal ,0105 earth and related environmental sciences ,Cloning ,Enzyme Gene ,chemistry.chemical_classification ,Renewable Energy, Sustainability and the Environment ,Chemistry ,General Medicine ,beta-Galactosidase ,Kinetics ,Enzyme ,Biochemistry ,Biocatalysis ,medicine.drug - Abstract
The main goal of this work was to evaluate the performance of β-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-β-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-β-d-glucopyranoside, butyl- and pentyl-β-d-galactosides. This unique and novel substrate specificity converts the E. acetylicum MF03 β-galactosidase into an ideal catalyst for the formulation of an enzymatic kit for lactulose quantification in thermally processed milk. This is because costly steps to eliminate glucose (resulting from hydrolysis of lactose when a customary β-galactosidase is used) can be avoided.
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- 2019
7. Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae β-galactosidase
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Cecilia Guerrero, Carlos Vera, Claudia Ubilla, Matías Gómez, Carla Aburto, Felipe Valdivia, Nicolás Ramírez, and Andrés Illanes
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0106 biological sciences ,Environmental Engineering ,Continuous operation ,Aspergillus oryzae ,Oligosaccharides ,Lactose ,Bioengineering ,Fructose ,010501 environmental sciences ,01 natural sciences ,chemistry.chemical_compound ,Lactulose ,010608 biotechnology ,medicine ,Waste Management and Disposal ,0105 earth and related environmental sciences ,Packed bed ,Chromatography ,biology ,Renewable Energy, Sustainability and the Environment ,Sepharose ,Glyoxylates ,General Medicine ,Enzymes, Immobilized ,beta-Galactosidase ,biology.organism_classification ,chemistry ,Yield (chemistry) ,Selectivity ,medicine.drug - Abstract
Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae β-galactosidase is reported for the first time. Alternative strategies to conventional batch synthesis have been scarcely explored for lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing material mass ratio (MB/MIM) were studied on reactor performance. Increase in any of these variables produced an increase in lactulose yield (YLu) being higher than obtained in batch synthesis at comparable conditions. Maximum YLu of 0.6 g·g−1 was obtained at 50 °C, pH 4.5, 50% w/w total sugars, 15 mL·min−1, fructose/lactose molar ratio of 12 and MB/MIM of 1/8 g·g−1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16 g·g−1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was 28%. Reactor operation with recycle had no significant effect on yield, producing only some decrease in productivity.
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- 2019
8. Phytolith analyses from Khil and Kaf Taht el-Ghar (Western Maghreb): Plant use trajectories in a long-term perspective
- Author
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Lancelotti, Carla, primary, Martínez Sánchez, Rafael M., additional, Carlos Vera Rodríguez, Juan, additional, Pérez-Jordà, Guillem, additional, Peña-Chocarro, Leonor, additional, Biagetti, Stefano, additional, and Madella, Marco, additional
- Published
- 2021
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9. Effect of particle size and enzyme load on the simultaneous reactions of lactose hydrolysis and transgalactosylation with glyoxyl-agarose immobilized β-galactosidase from Aspergillus oryzae
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Carlos Vera, Andrés Illanes, Sebastián Suárez, and Cecilia Guerrero
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0106 biological sciences ,chemistry.chemical_classification ,Chromatography ,biology ,010405 organic chemistry ,Bioengineering ,biology.organism_classification ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,0104 chemical sciences ,Reaction rate ,chemistry.chemical_compound ,Hydrolysis ,Enzyme ,chemistry ,Aspergillus oryzae ,Biocatalysis ,010608 biotechnology ,Particle ,Particle size ,Lactose - Abstract
The simultaneous reactions of lactose hydrolysis and transgalactosylation in the production of galacto-oligosaccharides (GOS) were evaluated with immobilized Aspergillus oryzae β-galactosidase in glyoxyl-agarose of different particle sizes (fine and macro) and enzyme loads (1, 10 and 30 mgprotein/gsupport) to produce biocatalysts subjected to different magnitudes of internal diffusional restrictions. The ratio of initial reaction rates depended on the initial concentration of lactose: at values higher than 800 mM, the ratio of hydrolysis to total reaction rate (rhydr/rtotal), which reflects the hydrolytic potential, had values of 16 and 30%, and the ratio of transgalactosylation to total reaction rates (rtransgal/rtotal), which reflects the transgalactosylation potential, had values of 84 and 70% with the biocatalysts of smaller size and enzyme load, and bigger size and enzyme load, respectively. Results obtained highlight the fact that the biocatalyst should be optimized with regard to its intended use; β-galactosidase biocatalysts that have been optimized for lactose hydrolysis can be quite inadequate for performing GOS synthesis.
- Published
- 2018
10. Effect of the type of immobilization of β-galactosidase on the yield and selectivity of synthesis of transgalactosylated oligosaccharides
- Author
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Andrés Illanes, Carla Aburto, Cecilia Guerrero, Sebastián Suárez, and Carlos Vera
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0106 biological sciences ,0301 basic medicine ,Chromatography ,biology ,Substrate (chemistry) ,Bioengineering ,biology.organism_classification ,01 natural sciences ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrolysis ,030104 developmental biology ,chemistry ,Aspergillus oryzae ,Covalent bond ,010608 biotechnology ,Yield (chemistry) ,Agarose ,Lactose ,Selectivity ,Agronomy and Crop Science ,Food Science ,Biotechnology - Abstract
Aspergillus oryzae β-galactosidase was immobilized by crosslinking and aggregation (CLA), and by covalent attachment to monofunctional glyoxyl agarose (GA) and to the heterofunctional supports: amino-glyoxyl agarose (Am-GA), carboxy-glyoxyl agarose (Cx-GA) and chelate-glyoxyl agarose (Che-GA) with the purpose of determining the effect of immobilization on the performance of the biocatalysts in terms of yield, productivity, kinectic parameters and product distribution in the synthesis of transgalactosylated oligosaccharides (lactulose, galacto-oligosaccharides and fructosyl-galacto-oligosaccharides). In all transgalactosylation reactions, yield was barely affected by the type of immobilization; however, a strong effect on productivity was observed, being the highest with the enzyme immobilized in CLA-βG and Am-GA and the lowest with the enzyme immobilized as Cx-GA and Che-GA. The type of immobilization had a strong influence on the apparent kinetic parameters of the biocatalysts, affecting product composition during the synthesis of transgalactosylated oligosaccharides. Diffusional restrictions of the biocatalysts increased with the degree of crosslinking, particle size and enzyme load; however their effect on the synthesis of the transgalactosylated products was low since reactions are conducted at very high substrate concentrations. Results suggest that, because of the operation conditions, immobilization has a more critical impact on the hydrolysis of lactose than in its transgalactosylation where the use of very high substrate concentrations is mandatory.
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- 2018
11. Selective bioconversion with yeast for the purification of raw lactulose and transgalactosylated oligosaccharides
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Carlos Vera, Cecilia Guerrero, and Andrés Illanes
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0106 biological sciences ,chemistry.chemical_classification ,Chromatography ,biology ,Chemistry ,Bioconversion ,Saccharomyces cerevisiae ,04 agricultural and veterinary sciences ,Carbohydrate ,biology.organism_classification ,040401 food science ,01 natural sciences ,Applied Microbiology and Biotechnology ,Yeast ,Lactulose ,0404 agricultural biotechnology ,Aspergillus oryzae ,Kluyveromyces marxianus ,010608 biotechnology ,medicine ,Monosaccharide ,Food Science ,medicine.drug - Abstract
Raw transgalactosylated oligosaccharides (raw TOS) obtained by enzymatic synthesis with Aspergillus oryzae β-galactosidase were purified by selective bioconversion with Saccharomyces cerevisiae ATCC4126 and Kluyveromyces marxianus NRLLY-1109. Monosaccharides were completely removed by S. cerevisiae and K. marxianus from raw TOS without any nutrient supplementation. The effect of the biomass to carbohydrate mass ratio on the purity and specific productivity of removal of sugars was assessed with S. cerevisiae, where both effects were significant. Purity obtained with a collection strain of S. cerevisiae and bakers’ yeast was compared, the latter being higher after 24 h of bioconversion. A product free of monosaccharides was obtained with a purity (0.48; 28% lactulose and 20% oligosaccharides), similar to that of commercial galacto-oligosaccharide preparations. The possibility of yeast recovery and reuse in sequential batch operation was assessed: two batches could be performed before a significant reduction in monosaccharide removal was seen.
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- 2018
12. Fed-batch operation for the synthesis of lactulose with β-galactosidase of Aspergillus oryzae
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Carlos Vera, Cecilia Guerrero, and Andrés Illanes
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0106 biological sciences ,0301 basic medicine ,Environmental Engineering ,Aspergillus oryzae ,Oligosaccharides ,Lactose ,Bioengineering ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrolysis ,Lactulose ,010608 biotechnology ,medicine ,Waste Management and Disposal ,Chromatography ,biology ,Renewable Energy, Sustainability and the Environment ,Chemistry ,Substrate (chemistry) ,Fructose ,General Medicine ,beta-Galactosidase ,biology.organism_classification ,030104 developmental biology ,Biochemistry ,Yield (chemistry) ,Selectivity ,medicine.drug - Abstract
Fed-batch synthesis of lactulose from lactose and fructose with Aspergillus oryzae β-galactosidase was evaluated, obtaining a concentration of 40.4 g·L−1, which is 20% higher than obtained in batch, while the concentration of transgalactosylated oligosaccharides (TOS) was reduced by 98%. Therefore, selectivity of lactulose synthesis can be significantly higher by operating in fed-batch mode. The enzyme-limiting substrate mass ratio (E/S) is a critical variable in fed-batch operation. Higher values favor lactose hydrolysis over transgalactosylation, being 400 IU/g the limit for proper lactulose synthesis in fed-batch operation. Selectivity of lactulose synthesis increased with E/S being quite high at 800 IUH·g−1 or higher. However, this increase was obtained at the expense of lactulose yield. Lactulose synthesis in fed-batch operation was a better option than conventional batch synthesis, since higher product concentration and selectivity of lactulose over TOS synthesis were obtained.
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- 2017
13. Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β- d -galactoside
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Cecilia Guerrero, Andrés Illanes, Carlos Vera, and Lorena Wilson
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0106 biological sciences ,0301 basic medicine ,biology ,Water activity ,Leaving group ,Substrate (chemistry) ,Bioengineering ,biology.organism_classification ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Galactoside ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Aspergillus oryzae ,chemistry ,010608 biotechnology ,Yield (chemistry) ,Acetone ,Organic chemistry ,Lactose ,Nuclear chemistry - Abstract
Reaction conditions were optimized and the donor substrate selected for maximizing the reaction yield and productivity of the enzymatic synthesis of hexyl-β-galactoside with β-galactosidase from Aspergillus oryzae . We independently studied the effect of water content, type of cosolvent, temperature, donor substrate concentration, and leaving group of the donor substrate on the yield and productivity of hexyl-β-galactoside synthesis. Reaction yield was maximum in the medium with 70% water content and acetone as cosolvent, corresponding to a water activity of 0.94. Temperature and donor substrate concentration had very little effect on the yield. The leaving group of the donor substrate was the most relevant variable. Lactose, lactulose, o-nitrophenyl-β- d -galactopyranoside, and propyl- and butyl-β- d -galactoside (the last two are enzymatically synthesized from lactose) were evaluated as donor substrates. Use of propyl- and butyl-β-galactoside as donor substrates allowed us to increase the product yield by 683% and 716% [vs. lactose (0.06 mol/mol)]. Because propyl- and butyl-β-galactosides can be synthesized at low cost from lactose, using them in a two-step process could be much better alternative than a one-step process with lactose; leading to a theoretical global molar yield of 0.41 and 0.37 mol/mol from propyl- and butyl-β-galactosides, respectively.
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- 2017
14. Immobilization of Aspergillus oryzae β-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose
- Author
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Carlos Vera, Andrés Illanes, Nestor Serna, and Cecilia Guerrero
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0106 biological sciences ,0301 basic medicine ,Glycosylation ,Environmental Engineering ,Aspergillus oryzae ,Bioengineering ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Lactulose ,010608 biotechnology ,Enzyme Stability ,medicine ,Particle Size ,Waste Management and Disposal ,chemistry.chemical_classification ,Chromatography ,biology ,Renewable Energy, Sustainability and the Environment ,Sepharose ,Temperature ,Glyoxylates ,General Medicine ,Enzymes, Immobilized ,beta-Galactosidase ,biology.organism_classification ,030104 developmental biology ,Enzyme ,chemistry ,Biocatalysis ,Yield (chemistry) ,Agarose ,Specific activity ,Selectivity ,Biotechnology ,medicine.drug - Abstract
Aspergillus oryzae β-galactosidase was immobilized in monofunctional glyoxyl-agarose and heterofunctional supports (amino-glyoxyl, carboxy-glyoxyl and chelate-glyoxyl agarose), for obtaining highly active and stable catalysts for lactulose synthesis. Specific activities of the amino-glyoxyl agarose, carboxy-glyoxyl agarose and chelate-glyoxyl agarose derivatives were 3676, 430 and 454 IU/g biocatalyst with half-life values at 50 °C of 247, 100 and 100 h respectively. Specific activities of 3490, 2559 and 1060 IU/g were obtained for fine, standard and macro agarose respectively. High immobilization yield (39.4%) and specific activity of 7700 IU/g was obtained with amino-glyoxyl-agarose as support. The highest yields of lactulose synthesis were obtained with monofunctional glyoxyl-agarose. Selectivity of lactulose synthesis was influenced by the support functionalization: glyoxyl-agarose and amino-glyoxyl-agarose derivatives retained the selectivity of the free enzyme, while selectivity with the carboxy-glyoxyl-agarose and chelate-glyoxyl-agarose derivatives was reduced, favoring the synthesis of transgalactosylated oligosaccharides over lactulose.
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- 2017
15. Synthesis of butyl-β- d -galactoside with commercial β-galactosidases
- Author
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Carlos Vera, Cecilia Guerrero, Lorena Wilson, and Andrés Illanes
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0106 biological sciences ,0301 basic medicine ,Kluyveromyces lactis ,biology ,Immobilized enzyme ,Chemistry ,General Chemical Engineering ,Electrospray ionization ,Substrate (chemistry) ,biology.organism_classification ,01 natural sciences ,Biochemistry ,Galactoside ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,010608 biotechnology ,Yield (chemistry) ,Bacillus circulans ,Organic chemistry ,Lactose ,Food Science ,Biotechnology ,Nuclear chemistry - Abstract
Three commercial β-galactosidase preparations from Aspergillus oryzae, Bacillus circulans and Kluyveromyces lactis were evaluated in the synthesis of butyl-β-galactoside. The enzyme from A. oryzae performed the best, producing the highest product yield and having the highest operational stability, being selected for further studies. Then, lactose and o -NPG were evaluated as substrates, and temperature and 1-butanol concentration were optimized using response surface methodology. Higher yields (ranging between 0.7 and 0.92 mol/mol) were obtained with o -NPG rather than lactose. However, under optimized conditions, a yield of 0.58 mol/mol was obtained with lactose as substrate, which is interesting because of being much cheaper than o -NPG. Three immobilization strategies were evaluated, the catalyst immobilized in glyoxyl-agarose being selected for producing a yield from lactose of 0.76 mol/mol. Catalyst reuse was evaluated in the synthesis of butyl-β-galactoside in consecutive batch mode during ten cycles of 2 h. Immobilization allowed an increase in the efficiency of catalyst use with respect to the soluble enzyme, the amount of product per unit mass of enzyme protein, being higher from the fifth batch on. Butyl-β-galactoside was easily purified by extraction with acetone and characterized by liquid electrospray ionization mass spectrometry.
- Published
- 2017
16. Synthesis of propyl-β-d-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae
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Lorena Wilson, Cecilia Guerrero, Andrés Illanes, and Carlos Vera
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0106 biological sciences ,Chromatography ,Immobilized enzyme ,biology ,010405 organic chemistry ,Extraction (chemistry) ,Bioengineering ,biology.organism_classification ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Galactoside ,0104 chemical sciences ,Catalysis ,chemistry.chemical_compound ,chemistry ,Aspergillus oryzae ,010608 biotechnology ,Yield (chemistry) ,Acetone ,Response surface methodology - Abstract
Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.
- Published
- 2017
17. Optimizing the location of ambulances in Tijuana, Mexico
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Leonardo Trujillo, Juan Carlos Dibene, Oliver Schtze, Carlos Vera, Yazmin Maldonado, and Mauricio C. de Oliveira
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Emergency Medical Services ,Optimization problem ,Operations research ,Computer science ,Ambulances ,0211 other engineering and technologies ,Health Informatics ,02 engineering and technology ,Set (abstract data type) ,Time of day ,0502 economics and business ,Emergency medical services ,Humans ,Mexico ,Integer programming ,Simulation ,050210 logistics & transportation ,021103 operations research ,business.industry ,05 social sciences ,Models, Theoretical ,Red Cross ,Computer Science Applications ,Work (electrical) ,Geographic Information Systems ,business ,Medical Informatics - Abstract
In this work we report on modeling the demand for Emergency Medical Services (EMS) in Tijuana, Baja California, Mexico, followed by the optimization of the location of the ambulances for the Red Cross of Tijuana (RCT), which is by far the largest provider of EMS services in the region. We used data from more than 10,000 emergency calls surveyed during the year 2013 to model and classify the demand for EMS in different scenarios that provide different perspectives on the demand throughout the city, considering such factors as the time of day, work and off-days. A modification of the Double Standard Model (DSM) is proposed and solved to determine a common robust solution to the ambulance location problem that simultaneously satisfies all specified constraints in all demand scenarios selecting from a set of almost 1000 possible base locations. The resulting optimization problems are solved using integer linear programming and the solutions are compared with the locations currently used by the Red Cross. Results show that demand coverage and response times can be substantially improved by relocating the current bases without the need for additional resources.
- Published
- 2017
18. Modification of chitosan to deliver grapes proanthocyanidins: Physicochemical and biological evaluation
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Marlene Roeckel, Valeria Muñoz, Tomás Kappes, Katherina Fernández, and Juan Carlos Vera
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0301 basic medicine ,Chromatography ,02 engineering and technology ,Permeation ,021001 nanoscience & nanotechnology ,Chitosan ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Proanthocyanidin ,chemistry ,Caco-2 ,Monolayer ,sense organs ,Phenols ,0210 nano-technology ,Cytotoxicity ,IC50 ,Food Science - Abstract
Chitosan (CS) and lauryl succinyl chitosan (LSC) particles were synthesized by ionic gelation to encapsulate high molecular weight proanthocyanidins (PAs) extracted from grape seed in an attempt to improve the cellular transport and delivery of the PAs. The extracts and particles were subjected to simulated gastrointestinal assays, cytotoxicity studies and cell permeation studies. The LSC and CS physicochemical properties were also evaluated and compared. The LSC particle sizes were 3640 ± 33 nm and the CS particles were 458 ± 11 nm; both were loaded with seed extract. The LSC released a lower PAs amount than the CS, protecting the encapsulated extract from the stomach pH. The cytotoxicity studies on HEK-293 cells showed that the half maxima inhibitory concentration (IC50) of the raw extract was 0.006 mg/mL, and with the stabilization (on LSC or CS) this value increased to 1.7 mg/mL. The encapsulation of the extracts decreased their toxicity, allowing higher concentrations to be used. The transport studies through Caco-2 monolayer cells indicated the effective release of PAs from the particles. The phenols released by the LSC particles were significantly higher than that released by the native CS during the Caco- 2 cell permeation study.
- Published
- 2016
19. Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides in batch operation
- Author
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Andrés Illanes, Carla Aburto, Lorena Wilson, Cecilia Guerrero, and Carlos Vera
- Subjects
0106 biological sciences ,0301 basic medicine ,chemistry.chemical_classification ,Chromatography ,biology ,Chemistry ,Substrate (chemistry) ,biology.organism_classification ,01 natural sciences ,Yeast ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Kluyveromyces marxianus ,Aspergillus oryzae ,Biochemistry ,010608 biotechnology ,Yield (chemistry) ,Bioreactor ,Monosaccharide ,Lactose ,Food Science - Abstract
The production of prebiotic galacto-oligosaccharides (GOS) considers the enzymatic synthesis by lactose transgalactosylation and the purification from the reacted mixture (raw GOS). Simultaneous synthesis and purification of GOS (SSP) was conducted in a one-pot operation using β-galactosidase from Aspergillus oryzae and Saccharomyces cerevisiae or Kluyveromyces marxianus cells, allowing the selective removal of unwanted carbohydrates (monosaccharides and lactose). The reaction was carried out in a stirred reactor at 40 °C, pH 4.5 and 200 rpm, using lactose without additional supplements, evaluating the effect of yeast genus ( S . cerevisiae or K . marxianus ), enzyme-substrate ratio (20–400 IU H /g lactose ), initial lactose concentration (20–50% w/w) and substrate mass-biomass ratio (0.05–0.4). The best GOS yield was obtained with S . cerevisiae at 50 IU H /g lactose and 0.25 g cell biomass /g carbohydrates . These conditions were used for scaling-up into a bioreactor (1000 g of 40% w/w lactose) operated under temperature and pH control at an aeration rate of 5 vvm, obtaining a GOS yield of 40% at 24 h of reaction, which is significantly higher than obtained in the conventional system of GOS synthesis. SSP may represent a technological advantage in terms of productivity and yield of GOS production.
- Published
- 2016
20. Assessment of the fouling mechanisms of an ultrafiltration membrane bioreactor during synthesis of galacto-oligosaccharides: Effect of the operational variables
- Author
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Andrés Illanes, Cecilia Guerrero, Andrés Córdova, Carlos Vera, and Carolina Astudillo
- Subjects
Fouling ,Chemistry ,Mechanical Engineering ,General Chemical Engineering ,Membrane fouling ,Ultrafiltration ,Analytical chemistry ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Membrane bioreactor ,01 natural sciences ,0104 chemical sciences ,Viscosity ,Membrane ,Chemical engineering ,Bioreactor ,General Materials Science ,0210 nano-technology ,Flux (metabolism) ,Water Science and Technology - Abstract
The flux decay of an ultrafiltration membrane bioreactor for the synthesis of galacto-oligosaccharides was modeled, by varying the processing conditions: temperature (40–60 °C), transmembrane pressure (2.5–4 bar) and cross-flow velocity (3.5–7 m/s) according to a 2 k design. Fouling mechanisms varied according to the operational condition, showing that the predominant mechanism were the intermediate fouling, which was associated to the higher flux decay due to the partial adsorption of the enzyme on the membrane, and the cake fouling mechanism, which was associated to those runs where the flux declined by around 20% because of the high concentration of substrate used (40% w/w). Strong statistical analysis allowed validation or rejection of initial adjustments given by a simple R-square statistical test, showing that misinterpretation of the fouling mechanism can be done under particular conditions, but also revealed that fouling equations have some limitations when drastic flux decay occurs or when this remains virtually unchanged. The experimental design also showed that temperature was the variable having the main positive effect on flux stability by decreasing the solution viscosity; however, the interaction between cross-flow velocity and transmembrane pressure had the main effect on flux decay.
- Published
- 2016
21. The introduction of South-Western Asian domesticated plants in North-Western Africa: An archaeobotanical contribution from Neolithic Morocco
- Author
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Jacob Morales, Leonor Peña-Chocarro, Jörg Linstädter, Guillem Pérez Jordà, Rafael María Martínez Sánchez, Youssef Bokbot, Juan Carlos Vera Rodríguez, European Research Council, Pérez Jordá, Guillem [0000-0003-1459-0219], Peña-Chocarro, Leonor [0000-0002-7807-8778], Vera Rodríguez, Juan Carlos [0000-0002-5989-2694], Morales Mateos, Jacob [0000-0002-6781-2121], Pérez Jordá, Guillem, Peña-Chocarro, Leonor, Vera Rodríguez, Juan Carlos, and Morales Mateos, Jacob
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Radiocarbon dating ,Mediterranean climate ,010506 paleontology ,Crops ,Early farming ,01 natural sciences ,law.invention ,Peninsula ,law ,0601 history and archaeology ,Neolithic ,Domestication ,0105 earth and related environmental sciences ,Earth-Surface Processes ,geography ,geography.geographical_feature_category ,Cultivated plant taxonomy ,Macro-botanical remains ,060102 archaeology ,business.industry ,06 humanities and the arts ,15. Life on land ,North Africa ,Archaeology ,Agriculture ,Hordeum vulgare ,Pottery ,business - Abstract
This paper focuses on the new macro-botanical evidence of South-Western Asian cultivated plants from northern Moroccan Neolithic sites. Due to the reduced presence of plant remains from previous excavations in the region, archaeological evidence of agriculture is rare and the arrival of domesticated plants and the role of farming in the Early Neolithic of North Africa are still poorly understood. Here we present results of the analysis carried out in three sites recently excavated: Kaf Taht el-Ghar, Khil, and Ifri Oudadane. Charred seeds of domesticated cereals (Triticum dicoccum, Triticum monococcum/dicoccum, Triticum durum, Triticum aestivum/durum, Hordeum vulgare, Hordeum vulgare var. nudum) and pulses (Lens culinaris, Pisum sativum, Vicia faba) have been recorded in all sites analyzed. Radiocarbon dating of crop seeds indicates that farming, along animal herding and pottery, was most probably introduced in the region at the interval between 5500 and 5000 cal. BC. Absence of evidence for Neolithic farming in other regions of North-Western Africa suggests that the first crops arrived into Morocco through a maritime route, more likely from the Central or Northern Mediterranean shores. Similarities in both radiocarbon dates and crop assemblages from early Neolithic sites in Northern Morocco and the south of the Iberian Peninsula point to an almost simultaneous East to West maritime spread of agriculture along both shores of the Western Mediterranean., Research has been carried out within the framework of the AGRIWESTMED project ERC-AdG 230561 coordinated by Leonor Peña-Chocarro. J.M. is part of the Research Group IT-622-13/UFI 11/09 of the University of the Basque Country and his work is funded by ERC-CoG 614960. GPJ work has been carried out within the postdoctoral contract FPDI-2013-16034 funded by the Ministerio de Economía y Competitividad, Spain. J.L. would like to express his thanks to Abdessalam Mikdad from INSAP (Institut National des Sciences de l'Archéologie et du Patrimoine) in Rabat, Morocco, and to Josef Eiwanger, DAI (Deutsches Archäologisches Institut), Bonn, Germany for long-term, amicable cooperative work and the providing of site data. J.L. also thanks the German Research Foundation (DFG) for financing his fieldwork in the framework of the CRC 806 ‘Our way to Europe’.
- Published
- 2016
22. The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects
- Author
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Leslie A. Leinwand, Marieke J. Bloemink, Michael A. Geeves, Jonathan Walklate, and Carlos Vera
- Subjects
recombinant protein expression ,0301 basic medicine ,myosin subfragment 1 ,transient kinetics ,ATPase ,Embryonic Germ Cells ,medicine.disease_cause ,human myosin ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,0302 clinical medicine ,enzyme kinetics ,Myosin ,Molecular motor ,medicine ,Humans ,Protein Isoforms ,skeletal muscle ,Molecular Biology ,Cells, Cultured ,Adenosine Triphosphatases ,Genetics ,Mutation ,biology ,Craniofacial Dysostosis ,Hydrolysis ,Myosin Subfragments ,Skeletal muscle ,Molecular Bases of Disease ,Cell Biology ,Cell biology ,molecular motor ,Cytoskeletal Proteins ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,muscle disease ,biology.protein ,MYH7 ,medicine.symptom ,Adenosine triphosphate ,030217 neurology & neurosurgery ,Muscle Contraction ,Muscle contraction - Abstract
The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.
- Published
- 2016
23. Activity of diphenyl ether benzyl amines against Human African Trypanosomiasis
- Author
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Rosalie C. Warner, Katie Higgins, Monica Cal, Bret Belter, Samuel Anderson, Tasloach Wol, Derek A. Leas, Victoria Mashinson, Grant Darner, Carlos Vera-Esquivel, Alexander I. Wallick, James P. Hagen, Paul H. Davis, Marcel Kaiser, and Ananya Mitra
- Subjects
Trypanosoma brucei rhodesiense ,Benzylamines ,Trypanosoma brucei ,Pharmacology ,01 natural sciences ,Biochemistry ,Cell Line ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Discovery ,medicine ,Humans ,African trypanosomiasis ,Available drugs ,Molecular Biology ,biology ,010405 organic chemistry ,Phenyl Ethers ,Organic Chemistry ,Diphenyl ether ,Human cell ,biology.organism_classification ,medicine.disease ,Trypanocidal Agents ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Trypanosomiasis, African ,chemistry - Abstract
Insect-borne parasite Trypanosoma brucei plagues humans and other animals, eliciting the disease Human African trypanosomiasis, also known as African sleeping sickness. This disease poses the biggest threat to the people in Sub-Saharan Africa. Given the high toxicity and difficulties with administration of currently available drugs, a novel treatment is needed. Building on known Human African trypanosomiasis structure–activity relationship (SAR), we now describe a number of functionally simple diphenyl ether analogs which give low micromolar activity (IC50 = 0.16–0.96 μM) against T. b. rhodesiense. The best compound shows favorable selectivity against the L6 cell line (SI = 750) and even greater selectivity (SI = 1200) against four human cell lines. The data herein provides direction for the ongoing optimization of antitrypanosomal diphenyl ethers.
- Published
- 2020
24. Comparative analysis of relocation strategies for ambulances in the city of Tijuana, Mexico
- Author
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Guadalupe Álvarez-Hernández, Leonardo Trujillo, Yazmin Maldonado, and Carlos Vera
- Subjects
0301 basic medicine ,Emergency Medical Services ,Operations research ,business.industry ,Computer science ,Ambulances ,Health Informatics ,Travel cost ,Red Cross ,Computer Science Applications ,Travel time ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Work (electrical) ,Emergency medical services ,Relocation ,business ,Mexico ,Limited resources ,030217 neurology & neurosurgery - Abstract
This paper analyzes the problem of locating ambulances for the Red Cross of Tijuana, Baja California, Mexico, considering demand changes over time, a problem that usually requires relocating ambulances at specific moments in time. This problem is usually solved using optimization models, seeking to maximize coverage and reduce the response time using an appropriate relocation strategy, while also minimizing the cost of relocating ambulances across the city. The goal of this work is to determine the benefits and costs, advantages and disadvantages, of using different strategies to solve this problem for the Red Cross of Tijuana. Therefore, different solution strategies are evaluated, all of them are based on the Double Standard Model (DSM) for ambulance locations. The first approach is to apply a robust version of the DSM, Robust DSM, that finds the best trade-off solutions across all possible time periods, or scenarios, throughout the day. The second approach is to apply the DSM to each scenario independently, and then perform relocations based on the different configurations of the ambulances in different scenarios. The final approach is to use an explicit relocation model, the multi-period DSM. The approaches are evaluated based on the percentage of double coverage, total number of relocations, relocation travel time, relocation travel distance and the financial cost of performing relocations. More than 13,000 calls for Emergency Medical Services (EMS) were analyzed, received by the Red Cross from August 2016 to April 2017, such that the results obtained are based on a comprehensive characterization of this real-world case study. Results show that while the relocation approaches do provide better overall coverage, for the Red Cross of Tijuana, an EMS provider with limited resources and funding, the increased coverage may not justify the additional costs.
- Published
- 2020
25. Conventional and non-conventional applications of β-galactosidases
- Author
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Carlos Vera, Cecilia Guerrero, Andrés Illanes, Carla Aburto, and Andrés Córdova
- Subjects
0106 biological sciences ,0303 health sciences ,Biophysics ,Lactose ,Dairy industry ,beta-Galactosidase ,01 natural sciences ,Biochemistry ,Catalysis ,Analytical Chemistry ,Industrial enzymes ,03 medical and health sciences ,chemistry.chemical_compound ,Fresh air ,chemistry ,010608 biotechnology ,Paradigm shift ,Sustainability ,Organic synthesis ,Business ,Asset (economics) ,Biochemical engineering ,Molecular Biology ,Commodity (Marxism) ,030304 developmental biology - Abstract
β-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of β-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide. However, a fresh air has come into the β-galactosidase business as non-conventional applications arose in recent decades based on its transgalactosylation activity. This capacity is certainly a major asset for a commodity enzyme that can be used now as a catalyst for the upgrading of readily available and cheap lactose into high added-value glycosides in processes of organic synthesis in tune with green chemistry principles within the framework of sustainability. This is a reflection of a paradigm shift, where enzymes are now being considered as apt catalysts for the synthesis of valuable organic compounds. This article reviews the main applications of β-galactosidase, going from its conventional use related to its hydrolytic activity to the ongoing non-conventional applications in the synthesis of high added-value oligosaccharides based on its transgalactosylation activity.
- Published
- 2020
26. Improvement in the yield and selectivity of lactulose synthesis with Bacillus circulans β-galactosidase
- Author
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Carlos Vera, Cecilia Guerrero, Andrés Illanes, and Carla Aburto
- Subjects
0106 biological sciences ,chemistry.chemical_classification ,Fructose ,04 agricultural and veterinary sciences ,040401 food science ,01 natural sciences ,Lactulose ,chemistry.chemical_compound ,0404 agricultural biotechnology ,Enzyme ,chemistry ,010608 biotechnology ,Yield (chemistry) ,Bacillus circulans ,medicine ,Lactose ,Selectivity ,Sugar ,Food Science ,medicine.drug ,Nuclear chemistry - Abstract
A commercial preparation of β-galactosidase from Bacillus circulans was evaluated as catalyst in the synthesis of lactulose considering temperature, pH, initial total sugar concentration, enzyme to initial lactose mass ratio and fructose to lactose molar ratio (F/L) as variables. The results obtained in terms of selectivity (SLu) yield (YLu) and productivity (πLu) of lactulose synthesis showed that only the initial total sugar concentration and lactose to fructose molar ratio were significant, the latter strongly affecting SLu. Best results were obtained at F/L of 20, being YLu = 0.54, πLu = 0.043 g h−1·mg enz−1, and SLu = 30.9. Under these conditions lactulose synthesis was favored over transgalactosylated oligosaccharides (TOS), due to the higher chance of transferring the galactosyl group into fructose at high F/L. Yields of lactulose and TOS depended on the enzyme origin, while their maximum concentrations were obtained at different lactose conversions (at 0.7 for lactulose and at 0.45 for TOS) and depended on the kinetics of reactions. Yields attained with Bacillus circulans β-galactosidase are higher than obtained with enzymes from other origin at comparable conditions, so this system is a preferred option for lactulose production.
- Published
- 2020
27. Photodynamic therapy (PDT) for malignant brain tumors – Where do we stand?
- Author
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Jennifer Connelly, Peter S. LaViolette, Harry T. Whelan, Brendan J. Quirk, Andrew B. Foy, Garth Brandal, Steven Donlon, Sachin Jogal, Thomas S. Mang, Sean M. Lew, Albert W. Girotti, and Juan Carlos Vera
- Subjects
medicine.medical_specialty ,Porphyrins ,Standard of care ,Research groups ,medicine.medical_treatment ,Biophysics ,Infratentorial Neoplasms ,Photodynamic therapy ,Dermatology ,Nitric Oxide ,Fluorescence ,Resection ,medicine ,Humans ,Hematoporphyrin Derivative ,Pharmacology (medical) ,Clinical Trials as Topic ,Photosensitizing Agents ,Cell Death ,Brain Neoplasms ,business.industry ,Talaporfin ,Aminolevulinic Acid ,Malignant brain tumors ,PDT light penetration ,Effective depth ,Surgery ,Clinical Practice ,Clinical trial ,Mesoporphyrins ,Photochemotherapy ,Surgery, Computer-Assisted ,Oncology ,Radiology ,business ,Signal Transduction ,medicine.drug - Abstract
Summary Introduction What is the current status of photodynamic therapy (PDT) with regard to treating malignant brain tumors? Despite several decades of effort, PDT has yet to achieve standard of care. Purpose The questions we wish to answer are: where are we clinically with PDT, why is it not standard of care, and what is being done in clinical trials to get us there. Method Rather than a meta-analysis or comprehensive review, our review focuses on who the major research groups are, what their approaches to the problem are, and how their results compare to standard of care. Secondary questions include what the effective depth of light penetration is, and how deep can we expect to kill tumor cells. Current results A measurable degree of necrosis is seen to a depth of about 5 mm. Cavitary PDT with hematoporphyrin derivative (HpD) results are encouraging, but need an adequate Phase III trial. Talaporfin with cavitary light application appears promising, although only a small case series has been reported. Foscan for fluorescence guided resection (FGR) plus intraoperative cavitary PDT results were improved over controls, but are poor compared to other groups. 5-Aminolevulinic acid-FGR plus postop cavitary HpD PDT show improvement over controls, but the comparison to standard of care is still poor. Conclusion Continued research in PDT will determine whether the advances shown will mitigate morbidity and mortality, but certainly the potential for this modality to revolutionize the treatment of brain tumors remains. The various uses for PDT in clinical practice should be pursued.
- Published
- 2015
28. Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation
- Author
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Carlos Vera, Cecilia Guerrero, Fernando Acevedo, and Andrés Illanes
- Subjects
medicine.medical_treatment ,Oligosaccharides ,Bioengineering ,Fructose ,Applied Microbiology and Biotechnology ,Bacteria, Anaerobic ,Feces ,Lactulose ,chemistry.chemical_compound ,Gastrointestinal Agents ,medicine ,Food science ,Lactose ,Prebiotic ,Galactose ,General Medicine ,Carbohydrate ,beta-Galactosidase ,Product distribution ,Prebiotics ,chemistry ,Batch Cell Culture Techniques ,Yield (chemistry) ,Fermentation ,Biotechnology ,medicine.drug - Abstract
Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with β-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum. Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for the synthesis of lactulose from fructose and lactose with Aspergillus oryzae β-galactosidase. Synthesis was optimized using response surface methodology, considering temperature, initial concentrations of acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of 4). Under optimal conditions (50°C, 50%w/w total initial concentrations of sugars and fructose/lactose molar ratio of 6.44), lactulose yield of 0.26g of lactulose produced per g of initial lactose was obtained at the optimal product distribution.
- Published
- 2015
29. Cis-regulatory elements involved in species-specific transcriptional regulation of the SVCT1 gene in rat and human hepatoma cells
- Author
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Jorge R. Toledo, Alejandro M. Reyes, Alejandra Muñoz, Elizabeth Escobar, Sergio A. Onate, Marcelo Villagrán, Mafalda Maldonado, Carlos Soliz, Coralia I. Rivas, Juan Carlos Vera, Marcell Gatica, Carlos F. Aylwin, Karen Sweet, Paula Guzmán, and Oliberto Sánchez
- Subjects
Reporter gene ,Response element ,Promoter ,Regulatory Sequences, Nucleic Acid ,Biology ,Ascorbic acid ,Biochemistry ,Molecular biology ,Rats ,Species Specificity ,Transcription (biology) ,Cell Line, Tumor ,Physiology (medical) ,Transcriptional regulation ,Animals ,Humans ,Promoter Regions, Genetic ,Sodium-Coupled Vitamin C Transporters ,Transcription factor ,Gene - Abstract
Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute oxidative stress in cells from species that differ in their capacity to synthesize vitamin C, with a marked decrease in SVCT1 mRNA and protein levels in rat hepatoma cells that was not observed in human hepatoma cells. To better understand the regulatory aspects involved, we performed a structural and functional analysis of the proximal promoter of the SVCT1 rat gene. We cloned a 1476-bp segment containing the proximal promoter of the rat SVCT1 gene and generated deletion-derived truncated promoters of decreasing sizes and mutant promoters by modification of consensus binding sites for transcription factors by site-directed mutagenesis. We next analyzed their capacity to direct the transcription of a reporter gene after transfection into rat H4IIE and human HepG2 hepatoma cells, in experiments involving the coexpression of transcription factors whose consensus binding sequences are present in the SVCT1 promoter. This analysis revealed the presence of two critical cis-regulatory elements of the transcriptional activity of the rat SVCT1 gene promoter, sites containing consensus sequences for the binding of the transcription factors Bach1 and HNF4 that are not present in equivalent locations in the human SVCT1 gene promoter. Moreover, a consensus site for HNF1 that is crucial for the regulation of the human SVCT1 promoter is present in the SVCT1 rat promoter but has no effect on its transcriptional activity. These findings imply that regulation of vitamin C metabolism in the rat, a species with the capacity to synthesize large amounts of ascorbic acid, may differ from that of humans, a species that must obtain ascorbic acid from the diet through a transport mechanism that depends on proper SVCT1 expression.
- Published
- 2015
30. Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates
- Author
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Raúl Conejeros, Andrés Illanes, Carlos Vera, and Cecilia Guerrero
- Subjects
Glycosylation ,Aspergillus oryzae ,Carbohydrates ,Oligosaccharides ,Bacillus ,Lactose ,Bioengineering ,Fructose ,Applied Microbiology and Biotechnology ,Biochemistry ,Kluyveromyces ,chemistry.chemical_compound ,Hydrolysis ,Lactulose ,Species Specificity ,medicine ,Beta-galactosidase ,biology ,Galactose ,beta-Galactosidase ,Enzyme assay ,Kinetics ,Prebiotics ,chemistry ,biology.protein ,Bacillus circulans ,Biotechnology ,medicine.drug - Abstract
β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.
- Published
- 2015
31. Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2
- Author
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Mafalda Maldonado, Eduardo Peña, Coralia I. Rivas, Francisco J. Roa, Carola Muñoz-Montesino, Mauricio Ostria González, Alejandro M. Reyes, Eveling Inostroza, Juan Carlos Vera, Carolina Muñoz, Carlos Soliz, Iván González, and Kirsty Sotomayor
- Subjects
Free Radicals ,Ascorbic Acid ,Mitochondrion ,Biochemistry ,chemistry.chemical_compound ,Physiology (medical) ,Humans ,RNA, Small Interfering ,Sodium-Coupled Vitamin C Transporters ,biology ,Endoplasmic reticulum ,Glucose transporter ,Biological Transport ,Ascorbic acid ,Mitochondria ,Cell biology ,HEK293 Cells ,Gene Expression Regulation ,chemistry ,biology.protein ,GLUT1 ,Dehydroascorbic acid ,ATP–ADP translocase ,Oxidation-Reduction ,Intracellular - Abstract
Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport Km of 0.6mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.
- Published
- 2014
32. Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with β-galactosidases of different origin
- Author
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Cecilia Guerrero, Carlos Vera, and Andrés Illanes
- Subjects
Aspergillus oryzae ,Oligosaccharides ,Bacillus ,Analytical Chemistry ,Fungal Proteins ,Kluyveromyces ,Lactulose ,Bacterial Proteins ,medicine ,Response surface methodology ,Food science ,Sugar ,Kluyveromyces lactis ,biology ,Galactosidases ,Chemistry ,General Medicine ,beta-Galactosidase ,biology.organism_classification ,Kinetics ,Yield (chemistry) ,Biocatalysis ,Bacillus circulans ,Food Science ,medicine.drug - Abstract
Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial β-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g−1 fructosyl-galacto-oligosaccharides was obtained at 70 °C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h−1 mg−1 was obtained at 70 °C and 40% w/w lactulose concentration.
- Published
- 2013
33. Essential role of intracellular glutathione in controlling ascorbic acid transporter expression and function in rat hepatocytes and hepatoma cells
- Author
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Kirsty Sotomayor, David Escobar, Juan Carlos Vera, Marcelo Villagrán, Alejandro M. Reyes, Gloria Oñate, Juan G. Cárcamo, Coralia I. Rivas, Pamela Mendoza, Ilona I. Concha, Constanza Angulo, Felipe Zuñiga, Lorena Mardones, Mauricio Ostria González, Mafalda Maldonado, Valeria Barra, and Valeska Ormazabal
- Subjects
Carcinoma, Hepatocellular ,Ascorbic Acid ,Biology ,Biochemistry ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Physiology (medical) ,Extracellular ,Animals ,Humans ,Buthionine Sulfoximine ,Sodium-Coupled Vitamin C Transporters ,DNA Primers ,Base Sequence ,Vitamin C ,Liver Neoplasms ,Glucose transporter ,Glutathione ,Metabolism ,Ascorbic acid ,Subcellular localization ,Immunohistochemistry ,Rats ,chemistry ,Hepatocytes ,Dehydroascorbic acid - Abstract
Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We show here that disruption of glutathione metabolism with buthionine-( S,R )-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na + -coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.
- Published
- 2012
34. Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose
- Author
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Andrés Illanes, Cecilia Guerrero, Raúl Conejeros, and Carlos Vera
- Subjects
Aspergillus oryzae ,medicine.medical_treatment ,Oligosaccharides ,Lactose ,Bioengineering ,Applied Microbiology and Biotechnology ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Bioreactors ,medicine ,chemistry.chemical_classification ,Supersaturation ,biology ,Precipitation (chemistry) ,Prebiotic ,Temperature ,Galactose ,Substrate (chemistry) ,Hydrogen-Ion Concentration ,beta-Galactosidase ,biology.organism_classification ,Solutions ,Kinetics ,Prebiotics ,Enzyme ,chemistry ,Yield (chemistry) ,Biotechnology ,Nuclear chemistry - Abstract
The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae β-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55 °C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5 °C. Highest specific productivity of 0.38 g GOS h−1 mg enzyme−1 was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55 °C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 g GOS h−1 mg enzyme−1were obtained, respectively.
- Published
- 2012
35. ATPase Cycle Analysis Predicts that Mutations Linked to Dilated Cardiomyopathy in Human Beta Myosin Will Impair Force Generation
- Author
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Leslie A. Leinwand, Srbolujub M. Mijailovich, Carlos Vera, Michael A. Geeves, Marina Svicevic, and Zoltan Ujfalusi
- Subjects
Force generation ,Beta-Myosin ,biology ,Chemistry ,ATPase ,Biophysics ,biology.protein ,medicine ,Dilated cardiomyopathy ,medicine.disease ,Cell biology - Published
- 2018
36. A Hybrid Model for Erythrocyte Membrane: A Single Unit of Protein Network Coupled with Lipid Bilayer
- Author
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Carlos Vera, Paul Sche, Qiang Zhu, L. Amy Sung, and Robert J. Asaro
- Subjects
Lipid Bilayers ,Biophysics ,Biophysical Theory and Modeling ,macromolecular substances ,In Vitro Techniques ,Models, Biological ,Biophysical Phenomena ,Quantitative Biology::Cell Behavior ,Quantitative Biology::Subcellular Processes ,Cell membrane ,Orientations of Proteins in Membranes database ,medicine ,Humans ,Spectrin ,Lipid bilayer ,Cytoskeleton ,Physics::Biological Physics ,Chemistry ,Bilayer ,Erythrocyte Membrane ,Membrane Proteins ,Lipid bilayer mechanics ,Condensed Matter::Mesoscopic Systems and Quantum Hall Effect ,Actins ,Elasticity ,Condensed Matter::Soft Condensed Matter ,Crystallography ,medicine.anatomical_structure ,Brownian dynamics ,Thermodynamics ,Algorithms ,Mathematics ,Nanomechanics - Abstract
To investigate the nanomechanics of the erythrocyte membrane we developed a hybrid model that couples the actin-spectrin network to the lipid bilayer. This model features a Fourier space Brownian dynamics model of the bilayer, a Brownian dynamics model of the actin protofilament, and a modified wormlike-chain model of the spectrin (including a cable-dynamics model to predict the oscillation in tension). This model enables us to predict the nanomechanics of single or multiple units of the protein network, the lipid bilayer, and the effect of their interactions. The present work is focused on the attitude of the actin protofilament at the equilibrium states coupled with the elevations of the lipid bilayer through their primary linkage at the suspension complex in deformations. Two different actin-spectrin junctions are considered at the junctional complex. With a point-attachment junction, large pitch angles and bifurcation of yaw angles are predicted. Thermal fluctuations at bifurcation may lead to mode-switching, which may affect the network and the physiological performance of the membrane. In contrast, with a wrap-around junction, pitch angles remain small, and the occurrence of bifurcation is greatly reduced. These simulations suggest the importance of three-dimensional molecular junctions and the lipid bilayer/protein network coupling on cell membrane mechanics.
- Published
- 2007
- Full Text
- View/download PDF
37. Vitamin C Is an Essential Antioxidant That Enhances Survival of Oxidatively Stressed Human Vascular Endothelial Cells in the Presence of a Vast Molar Excess of Glutathione
- Author
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Catherine Guzmán, Sergio A. Onate, Elizabeth Escobar, Osmán Vásquez, Alejandro Godoy, María Rosa Bono, Coralia I. Rivas, Marcelo Villagrán, Viviana P. Montecinos, Lorena Mardones, Juan G. Cárcamo, Victoria Gallardo, Paula Sotomayor, Marcelo E. Bustamante, Carola Muñoz-Montesino, Brigitte van Zundert, Juan Carlos Vera, Valeria Barra, Paula Guzmán, and Kirsty Sotomayor
- Subjects
GPX1 ,Antioxidant ,Cell Survival ,medicine.medical_treatment ,Glutathione reductase ,Ascorbic Acid ,Biology ,medicine.disease_cause ,Biochemistry ,Antioxidants ,chemistry.chemical_compound ,medicine ,Humans ,Molecular Biology ,Respiratory Burst ,Dose-Response Relationship, Drug ,Vitamin C ,Endothelial Cells ,Hydrogen Peroxide ,Cell Biology ,Glutathione ,Oxidants ,Ascorbic acid ,Oxidative Stress ,chemistry ,Dehydroascorbic acid ,Oxidoreductases ,Oxidation-Reduction ,Oxidative stress - Abstract
Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.
- Published
- 2007
38. Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2
- Author
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Marcelo Villagrán, Juan G. Cárcamo, Alejandro M. Reyes, Sergio A. Onate, Juan Carlos Vera, Valeska Ormazabal, Gustavo Moraga-Cid, Viviana P. Montecinos, Alejandro Godoy, Valeria Barra, Catherine Guzmán, Paula Sotomayor, Coralia I. Rivas, Luis G. Aguayo, Osmán Vásquez, Felipe Zuñiga, and Lorena Mardones
- Subjects
chemistry.chemical_classification ,Organic anion transporter 1 ,biology ,Stereochemistry ,Sodium ,Sodium-Coupled Vitamin C Transporters ,chemistry.chemical_element ,Bicarbonate transporter protein ,Cooperativity ,Transporter ,Cell Biology ,Ascorbic acid ,Biochemistry ,Amino acid ,chemistry ,biology.protein ,Molecular Biology - Abstract
We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.
- Published
- 2007
39. Mapping the tropomyosin isoform 5 binding site on human erythrocyte tropomodulin: Further insights into E-Tmod/TM5 interaction
- Author
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Donald Hamelberg, Lanping Amy Sung, Carlos Vera, and Jianmin Lao
- Subjects
Models, Molecular ,Protein Conformation ,Molecular Sequence Data ,Biophysics ,Tropomyosin ,Plasma protein binding ,Biochemistry ,Protein structure ,Protein Interaction Mapping ,Humans ,Computer Simulation ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Actin ,Binding Sites ,biology ,Molecular biology ,Heptad repeat ,Amino Acid Substitution ,Models, Chemical ,biology.protein ,Tropomodulin ,Protein Binding - Abstract
Actin protofilaments in the erythrocyte membrane skeleton are uniformly approximately 37nm. This length may be in part attributed to a "molecular ruler" made of erythrocyte tropomodulin (E-Tmod) and tropomyosin (TM) isoforms 5 or 5b. We previously mapped the E-Tmod binding site to TM5 N-terminal heptad repeat residues "a" (I(7), I(14)), "d" (V(10)) and "f" (R(12)). We now map the TM5 binding site to E-Tmod residues at L(116), E(117) and/or E(118) by identifying among 35 deletion clones and a series of point mutations that no longer bind to human TM5 and rat TM5b. Upstream residues 71-104 contain an actin binding site. The N-terminal "KRK ring" may participate in balancing electrostatic force with hydrophobic interaction in dimerization of TM and its binding to E-Tmod.
- Published
- 2005
40. Up-regulation and Polarized Expression of the Sodium-Ascorbic Acid Transporter SVCT1 in Post-confluent Differentiated CaCo-2 Cells
- Author
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Francisco Nualart, Adolph Grünert, Juan Carlos Vera, Alexandra Schmid-Kotsas, Nancy P. Maulén, Sybille Kempe, Marcelo E. Bustamante, Esther A. Henrı́quez, Juan G. Cárcamo, and Max G. Bachem
- Subjects
Time Factors ,Monosaccharide Transport Proteins ,Protein Conformation ,Organic Anion Transporters, Sodium-Dependent ,Ascorbic Acid ,Deoxyglucose ,Biochemistry ,chemistry.chemical_compound ,Tumor Cells, Cultured ,Humans ,RNA, Messenger ,Sodium-Coupled Vitamin C Transporters ,Molecular Biology ,Cells, Cultured ,Hexoses ,Glucose Transporter Type 1 ,Dose-Response Relationship, Drug ,Symporters ,biology ,Vitamin C ,Reverse Transcriptase Polymerase Chain Reaction ,Glucose Transporter Type 5 ,Glucose transporter ,Biological Transport ,Cell Differentiation ,DNA ,Cell Biology ,Apical membrane ,Ascorbic acid ,Dehydroascorbic Acid ,Up-Regulation ,Kinetics ,Protein Transport ,chemistry ,biology.protein ,Dehydroascorbic acid ,GLUT1 ,GLUT3 - Abstract
Human cells acquire vitamin C using two different transporter systems, the sodium-ascorbic acid co-transporters with specificity for ascorbic acid, and the facilitative glucose transporters with specificity for dehydroascorbic acid. There is no information on the mechanism of vitamin C transport across the intestinal barrier, a step that determines the bioavailability of vitamin C in humans. We used the colon carcinoma cell line CaCo-2 as an in vitro model for vitamin C transport in enterocyte-like cells. The results of transport kinetics, sodium dependence, inhibition studies, and reverse transcriptase-PCR analysis indicated that CaCo-2 cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the dehydroascorbic acid transporters GLUT1 and GLUT3, and a third dehydroascorbic acid transporter with properties expected for GLUT2. Analysis by real time quantitative PCR revealed that the post-confluent differentiation of CaCo-2 cells was accompanied by a marked increase (4-fold) in the steady-state level of SVCT1 mRNA, without changes in SVCT2 mRNA levels. Functional studies revealed that the differentiated cells expressed only one functional ascorbic acid transporter having properties expected for SVCT1, and transported ascorbic acid with a V(max) that was increased at least 2-fold compared with pre-confluent cells. Moreover, post-confluent Caco-2 cells growing as monolayers in permeable filter inserts showed selective sorting of SVCT1 to the apical membrane compartment, without functional evidence for the expression of SVCT2. The identification of SVCT1 as the transporter that allows vectorial uptake of ascorbic acid in differentiated CaCo-2 cells has a direct impact on our understanding of the mechanism for vitamin C transport across the intestinal barrier.
- Published
- 2003
41. Modelling the Discrete Part of Hybrid Dynamical Systems
- Author
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Martin Carlos Vera Estrada, Hasane Alla, and Jean-Marie Flaus
- Subjects
Discrete system ,Controllability ,Discrete event system ,Supervisory control ,Dynamical systems theory ,ComputingMethodologies_SIMULATIONANDMODELING ,Control theory ,Event (relativity) ,Topology ,Dynamical system ,Discrete event dynamic system ,Mathematics - Abstract
This paper proposes a framework for modelling and controlling the discrete behavior of a hybrid dynamical system. The notion of discrete machine, and the notion of extended discrete event system (EDES) are defined. An EDES is a discrete event system that uses event and condition signals to model the discrete behavior of the (hybrid dynamical) system.
- Published
- 2000
42. Tropomodulin-Binding Site Mapped to Residues 7–14 at the N-Terminal Heptad Repeats of Tropomyosin Isoform 5
- Author
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Jim J.-C. Lin, Alka Sood, Leland J. Yee, Carlos Vera, Ke-Ming Gao, and L. Amy Sung
- Subjects
Models, Molecular ,Repetitive Sequences, Amino Acid ,Gene isoform ,Globular protein ,Molecular Sequence Data ,Biophysics ,Tropomyosin ,Binding, Competitive ,Biochemistry ,Escherichia coli ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Actin ,DNA Primers ,chemistry.chemical_classification ,Coiled coil ,Binding Sites ,Base Sequence ,biology ,Ligand binding assay ,Microfilament Proteins ,Antibodies, Monoclonal ,Recombinant Proteins ,Rats ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Carrier Proteins ,Tropomodulin ,Epitope Mapping - Abstract
Tropomodulin is a globular protein that caps the pointed end of actin filaments by complexing with the N-terminus of a tropomyosin (TM) molecule. TM consists of coiled coils except for the N-terminus, which may be globular. Here we report that human TM isoform 5 (hTM5) lacking the N-terminal 18 residues lost its binding activity toward tropomodulin. We further characterized the tropomodulin-binding site by creating a series of deletion and missense mutations within this region, followed by a solid-phase binding assay. I 7 , V 10 , and I 14 , hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropomodulin binding. R 12 , a positively charged residue at the f position, is also involved in recognition. In contrast, A2R and G3Y mutations, each creating a bulky N-terminus, did not alter the binding. In addition, rat TM5b, which differs from hTM5 in residues 4–6, exhibits a similar binding affinity. The tropomodulin-binding site, therefore, is mapped to residues 7–14 at the beginning of the long heptad repeats. Column chromatography revealed that hTM5 mutants remained capable of dimerization. Results also suggest tropomodulin has a groove-type, rather than a cavity-type, binding site for hTM5. We also mapped the epitope of monoclonal antibody LC1 to residues 4–10 of hTM5 and showed the competition between mAb LC1 and tropomodulin in hTM5 binding. Since the N-terminal residues need to overlap with the C-terminus of TM in their head-to-tail association, this investigation elucidates the mechanisms by which the tropomodulin–hTM5 complex is formed and functions in regulating the actin filaments.
- Published
- 2000
43. Efficient Transport and Accumulation of Vitamin C in HL-60 Cells Depleted of Glutathione
- Author
-
Juan Carlos Vera, Charles M. Farber, Victor H. Guaiquil, and David W. Golde
- Subjects
Vitamin ,chemistry.chemical_classification ,Molar concentration ,Vitamin C ,Antimetabolites ,Chemistry ,Maleates ,Temperature ,Biological Transport ,HL-60 Cells ,Ascorbic Acid ,Cell Biology ,Glutathione ,Ascorbic acid ,Dehydroascorbic Acid ,Biochemistry ,chemistry.chemical_compound ,Enzyme ,Humans ,Dehydroascorbic acid ,Buthionine Sulfoximine ,Molecular Biology ,Intracellular - Abstract
Human myeloid leukemia cells (HL-60) transport only the oxidized form of vitamin C (dehydroascorbic acid) and accumulate the vitamin in the reduced form, ascorbic acid. We performed a detailed study of the role of glutathione in the intracellular trapping/accumulation of ascorbic acid in HL-60 cells. Uptake studies using HL-60 cells depleted of glutathione by treatment with L-buthionine-(S,R) sulfoximine and diethyl maleate, revealed no changes in the cells' ability to transport dehydroascorbic acid and accumulate ascorbic acid. Similar transport and accumulation rates were obtained using HL-60 cells containing intracellular glutathione concentrations from 6 mM to 1 microM. HL-60 cells, containing as little as 5 microM glutathione, were able to accumulate up to 150 mM ascorbic acid intracellularly when incubated with dehydroascorbic acid. Glutathione was capable of reducing dehydroascorbic acid by a direct chemical reaction, but only when present in a greater than 10-fold stoichiometric excess over dehydroascorbic acid. The accumulation of ascorbic acid by HL-60 cells was strongly temperature-dependent and was very inefficient at 16 degrees C. On the other hand, the direct chemical reduction of dehydroascorbic acid by excess glutathione proceeded efficiently at temperatures of 16 degrees C. Our data indicate that glutathione-dependent reductases in HL-60 cells are not responsible for the ability of these cells to accumulate millimolar concentrations of ascorbic acid. These findings indicate that alternative enzymatic mechanisms are involved in the cellular reduction of dehydroascorbic acid.
- Published
- 1997
44. N-Glycosylation of the Human Granulocyte-Macrophage Colony-stimulating Factor Receptor α Subunit Is Essential for Ligand Binding and Signal Transduction
- Author
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Dawn Xiao-Hong Ding, David W. Golde, Juan Carlos Vera, and Mark L. Heaney
- Subjects
Glycosylation ,Macromolecular Substances ,Protein subunit ,Interleukin 5 receptor alpha subunit ,HL-60 Cells ,Biology ,Ligands ,Transfection ,Biochemistry ,Gamma-aminobutyric acid receptor subunit alpha-1 ,Cell Line ,Interleukin 10 receptor, alpha subunit ,chemistry.chemical_compound ,Chlorocebus aethiops ,Animals ,Humans ,Molecular Biology ,G alpha subunit ,COS cells ,Tunicamycin ,Cell Membrane ,Granulocyte-Macrophage Colony-Stimulating Factor ,Tyrosine phosphorylation ,Cell Biology ,Molecular biology ,Recombinant Proteins ,carbohydrates (lipids) ,Kinetics ,chemistry ,Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ,Signal Transduction - Abstract
The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein containing 11 potential N-glycosylation sites in the extracellular domain. We examined the role of N-glycosylation on alpha subunit membrane localization and function. Tunicamycin, an N-glycosylation inhibitor, markedly inhibited GM-CSF binding, GM-CSF-induced deoxyglucose uptake, and protein tyrosine phosphorylation in HL-60(eos) cells but did not affect cell surface expression of the alpha subunit as detected by an anti-alpha subunit monoclonal antibody. In COS cells expressing the alpha subunit and treated with tunicamycin, N-unglycosylated alpha subunit was expressed and transported to the cell surface but was not capable of binding GM-CSF. High affinity binding in COS cells expressing both alpha and beta subunits was also blocked by tunicamycin treatment. These studies indicate that N-linked oligosaccharides are essential for alpha subunit ligand binding and signaling by the human GM-CSF receptor.
- Published
- 1995
45. Resolution of the Facilitated Transport of Dehydroascorbic Acid from Its Intracellular Accumulation as Ascorbic Acid
- Author
-
Coralia I. Rivas, Rong Hua Zhang, Ilona I. Concha, Juan Carlos Vera, David W. Golde, and Fernando V. Velásquez
- Subjects
Intracellular Fluid ,Monosaccharide Transport Proteins ,Biological Transport, Active ,Ascorbic Acid ,Deoxyglucose ,Dehydroascorbic acid transport ,Binding, Competitive ,Models, Biological ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Glucoside ,Humans ,Molecular Biology ,Glucose Transporter Type 1 ,Sodium ,Sodium-Coupled Vitamin C Transporters ,Glucose transporter ,Methylglucosides ,Cell Biology ,Ascorbic acid ,Dehydroascorbic Acid ,Kinetics ,chemistry ,3-O-Methylglucose ,Dehydroascorbic acid ,Oxidation-Reduction ,Intracellular - Abstract
We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external Na+, whereas the intracellular accumulation of ascorbic acid was found to be a Na(+)-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na(+)-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation.
- Published
- 1995
46. Optimization of synthesis of propyl and butyl β-galactosides with commercial β-galactosidases
- Author
-
Andrés Illanes, Cecilia Guerrero, Carlos Vera, and Lorena Wilson
- Subjects
Galactosidases ,Galactosides ,Chemistry ,Stereochemistry ,Bioengineering ,General Medicine ,Molecular Biology ,Biotechnology - Published
- 2016
47. Effect of precipitants on the kinetic parameters of β-galactosidase from Aspergillus oryzae and Bacillus circulans immobilized by aggregation and crosslinking
- Author
-
Cecilia Guerrero, Carlos Vera, and Andrés Illanes
- Subjects
Biochemistry ,Aspergillus oryzae ,biology ,Chemistry ,Bacillus circulans ,Bioengineering ,General Medicine ,biology.organism_classification ,Molecular Biology ,Biotechnology - Published
- 2016
48. Cis-regulatory elements involved in species-specific transcriptional regulation of the SVCT1 gene in rat and human hepatoma cells
- Author
-
Muñoz, Alejandra, primary, Villagrán, Marcelo, additional, Guzmán, Paula, additional, Solíz, Carlos, additional, Gatica, Marcell, additional, Aylwin, Carlos, additional, Sweet, Karen, additional, Maldonado, Mafalda, additional, Escobar, Elizabeth, additional, Reyes, Alejandro M., additional, Toledo, Jorge R., additional, Sánchez, Oliberto, additional, Oñate, Sergio A., additional, Carlos Vera, Juan, additional, and Rivas, Coralia I., additional
- Published
- 2015
- Full Text
- View/download PDF
49. Functional Analysis of Freeman-Sheldon Syndrome Causing Mutations on Embryonic Myosin
- Author
-
Velazquez, Carlos Vera, primary, Walklate, Jonathan, additional, Deacon, Jonathan, additional, Geeves, Michael A., additional, and Leinwand, Leslie A., additional
- Published
- 2015
- Full Text
- View/download PDF
50. A method of obtaining a structural dynamic matrix using bond graph techniques
- Author
-
Francisco Buil and Carlos Vera
- Subjects
Mathematical optimization ,Mechanical Engineering ,Aerospace Engineering ,Degrees of freedom (mechanics) ,Finite element method ,Computer Science Applications ,Mechanical system ,Matrix (mathematics) ,Graph energy ,Control and Systems Engineering ,Signal Processing ,Algorithm ,Bond graph ,Eigenvalues and eigenvectors ,Civil and Structural Engineering ,Resolution (algebra) ,Mathematics - Abstract
A large number of methods have been developed in order to solve the eigenvalues and eigenvectors of mechanical systems, from a generic treatment through to finite elements. For accuracy to be guaranteed in the results, a through discrete-parameters procedure should be performed on the model which, when many degrees of freedom are involved, implies resolution of a high-order system at the expense of considerable computer time. An alternative method is proposed in this paper for calculating natural frequencies and modes based on building the dynamic matrix through Bond Graph techniques.
- Published
- 1991
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