International Journal of Molecular Sciences, Feb 24, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The International Journal of Biochemistry & Cell Biology, Sep 1, 1998
In the present study we investigated the influence of several nonsteroidal anti-inflammatory drug... more In the present study we investigated the influence of several nonsteroidal anti-inflammatory drugs on calcium efflux in isolated rat renal cortex mitochondria in order to assess their potential to disrupt cell calcium homeostasis, as well as aspects of the mechanisms associated with oxidation of mitochondrial pyridine nucleotides (NAD(P)H) and with inhibition of the process by cyclosporin A (CsA). Calcium efflux
Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked... more Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Because breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from A. fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. Association of AfCel6A and AfAA9_B inhibits the activity of AfCel6A, an outcome that needs to be further investigated. However, addition of AfCel6A or AfAA9_B boosts the enzymatic saccharific...
Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act ... more Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. Objective: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. Methods: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. Results: LPMO was highly ...
Journal of bioenergetics and biomembranes, Jan 11, 2017
Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Asp... more Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD(+)), generating a large mitochondrial membrane potential. The NAD(+) carrier utilized the electrochemical proton gradient to drive NAD(+) entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.
Atherosclerosis has been described as an inflammatory disease in which polymorphonuclear leukocyt... more Atherosclerosis has been described as an inflammatory disease in which polymorphonuclear leukocytes (PMNLs) seem to be involved. These cells may induce atherosclerotic lesions by releasing reactive oxygen species (ROS) and a sort of pro-inflammatory mediators. In this study, the PMNL oxidative metabolic status of Golden Syrian hamsters fed a normal diet (ND), or a high-fat diet (10% coconut oil plus 0.2% cholesterol) supplemented (R-HCD) or not (HCD) with 0.1% (w/w) rutin was evaluated after 120 days of treatment. PMNL oxidative metabolism was assessed by whole blood luminol-enhanced chemiluminescence and 2',7'-dichlorofluorescein diacetate-dependent flow cytometry. The results obtained by both methods were similar and showed no significant changes in ROS generation by PMNLs in blood samples from HCD or R-HCD animals when compared to ND. Furthermore it was shown that rutin supplementation did not significantly affect plasma lipid and lipoprotein levels in the hypercholestero...
International Journal of Molecular Sciences, Dec 29, 2020
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
International Journal of Molecular Sciences, May 7, 2019
In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic bio... more In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters K m and V max were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 • C for 72 h. Low Cu 2+ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast ® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.
Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (⌬⌿) of tachyzoi... more Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (⌬⌿) of tachyzoites of the apicomplexan parasite Toxoplasma gondii were assayed in situ using very low concentrations of digitonin to render their plasma membrane permeable to succinate, ADP, safranin O, and other small molecules. The rate of basal respiration was slightly increased by digitonin when the cells were incubated in medium containing succinate. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide, and N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) was oxidized by antimycin A-poisoned mitochondria. The addition of ADP after TMPD/ascorbate also resulted in phosphorylating respiration. The antitoxoplasmosis drug atovaquone, at a very low concentration (0.03 M), totally inhibited respiration and disrupted the mitochondrial membrane potential. Atovaquone was shown to inhibit the respiratory chain of T. gondii and mammalian mitochondria between cytochrome b and c 1 as occurs with antimycin A 1. Phosphorylation of ADP could not be obtained in permeabilized tachyzoites in the presence of either pyruvate, 3-oxo-glutarate, glutamate, isocitrate, dihydroorotate, ␣-glycerophosphate, or endogenous substrates. Although ADP phosphorylation was detected in the presence of malate, this activity was rotenone-insensitive and was probably due to the conversion of malate into succinate through a fumarate reductase activity that was detected in mitochondrial extracts. Together these results provide the first direct biochemical evidence that the respiratory chain and oxidative phosphorylation are functional in apicomplexan parasites, although the terminal respiratory pathway is different from that in the mammalian host. * This work was supported by National Institutes of Health Grants AI-37805 and AI-39185 (to S. N. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ Biotechnology Career Fellow, Rockefeller Foundation and supported by grants from the Fundaçao de Amparo a Pesquisa do Estado de Sã o Paulo (FAPESP, Brazil) and Programa de Apoio a Desenvolvimento Científico e Tecnológico (PADCT, Brazil). ʈ Graduate fellow of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).
International Journal of Molecular Sciences, Feb 24, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The International Journal of Biochemistry & Cell Biology, Sep 1, 1998
In the present study we investigated the influence of several nonsteroidal anti-inflammatory drug... more In the present study we investigated the influence of several nonsteroidal anti-inflammatory drugs on calcium efflux in isolated rat renal cortex mitochondria in order to assess their potential to disrupt cell calcium homeostasis, as well as aspects of the mechanisms associated with oxidation of mitochondrial pyridine nucleotides (NAD(P)H) and with inhibition of the process by cyclosporin A (CsA). Calcium efflux
Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked... more Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Because breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from A. fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. Association of AfCel6A and AfAA9_B inhibits the activity of AfCel6A, an outcome that needs to be further investigated. However, addition of AfCel6A or AfAA9_B boosts the enzymatic saccharific...
Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act ... more Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. Objective: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. Methods: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. Results: LPMO was highly ...
Journal of bioenergetics and biomembranes, Jan 11, 2017
Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Asp... more Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD(+)), generating a large mitochondrial membrane potential. The NAD(+) carrier utilized the electrochemical proton gradient to drive NAD(+) entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.
Atherosclerosis has been described as an inflammatory disease in which polymorphonuclear leukocyt... more Atherosclerosis has been described as an inflammatory disease in which polymorphonuclear leukocytes (PMNLs) seem to be involved. These cells may induce atherosclerotic lesions by releasing reactive oxygen species (ROS) and a sort of pro-inflammatory mediators. In this study, the PMNL oxidative metabolic status of Golden Syrian hamsters fed a normal diet (ND), or a high-fat diet (10% coconut oil plus 0.2% cholesterol) supplemented (R-HCD) or not (HCD) with 0.1% (w/w) rutin was evaluated after 120 days of treatment. PMNL oxidative metabolism was assessed by whole blood luminol-enhanced chemiluminescence and 2',7'-dichlorofluorescein diacetate-dependent flow cytometry. The results obtained by both methods were similar and showed no significant changes in ROS generation by PMNLs in blood samples from HCD or R-HCD animals when compared to ND. Furthermore it was shown that rutin supplementation did not significantly affect plasma lipid and lipoprotein levels in the hypercholestero...
International Journal of Molecular Sciences, Dec 29, 2020
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
International Journal of Molecular Sciences, May 7, 2019
In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic bio... more In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters K m and V max were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 • C for 72 h. Low Cu 2+ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast ® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.
Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (⌬⌿) of tachyzoi... more Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (⌬⌿) of tachyzoites of the apicomplexan parasite Toxoplasma gondii were assayed in situ using very low concentrations of digitonin to render their plasma membrane permeable to succinate, ADP, safranin O, and other small molecules. The rate of basal respiration was slightly increased by digitonin when the cells were incubated in medium containing succinate. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide, and N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) was oxidized by antimycin A-poisoned mitochondria. The addition of ADP after TMPD/ascorbate also resulted in phosphorylating respiration. The antitoxoplasmosis drug atovaquone, at a very low concentration (0.03 M), totally inhibited respiration and disrupted the mitochondrial membrane potential. Atovaquone was shown to inhibit the respiratory chain of T. gondii and mammalian mitochondria between cytochrome b and c 1 as occurs with antimycin A 1. Phosphorylation of ADP could not be obtained in permeabilized tachyzoites in the presence of either pyruvate, 3-oxo-glutarate, glutamate, isocitrate, dihydroorotate, ␣-glycerophosphate, or endogenous substrates. Although ADP phosphorylation was detected in the presence of malate, this activity was rotenone-insensitive and was probably due to the conversion of malate into succinate through a fumarate reductase activity that was detected in mitochondrial extracts. Together these results provide the first direct biochemical evidence that the respiratory chain and oxidative phosphorylation are functional in apicomplexan parasites, although the terminal respiratory pathway is different from that in the mammalian host. * This work was supported by National Institutes of Health Grants AI-37805 and AI-39185 (to S. N. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ Biotechnology Career Fellow, Rockefeller Foundation and supported by grants from the Fundaçao de Amparo a Pesquisa do Estado de Sã o Paulo (FAPESP, Brazil) and Programa de Apoio a Desenvolvimento Científico e Tecnológico (PADCT, Brazil). ʈ Graduate fellow of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).
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