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1.
Int J Nanomedicine ; 16: 3059-3071, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953555

RESUMO

Purpose: This study aimed to explain the influence of zein nanosphere (ZN NS) formulation on the pharmacotherapeutic profile of PTS in MCF7 cells. Methods: Liquid-liquid phase separation was used to formulate PTS-ZN NSs. The formulations developed were evaluated for particle-size analysis, encapsulation efficiency, and in vitro diffusion. Also, assays of cytotoxicity, uptake, cell-cycle progression, annexin V, apoptotic gene mRNA expression and biochemical assays were carried out. Results: The PTS-ZN NS formulation selected showed 104.5±6.2 nm, 33.4±1.8 mV, 95.1%±3.6%, and 89.1%±2.65% average particle size, zeta-potential, encapsulation efficiency and in vitro diffusion, respectively. With MCF7 cells, IC50 was reduced approximately 15-fold, with increased cellular uptake, accumulation in the G2/M phase, increased percentage of cells in the pre-G1 phase, amelioration of early and late apoptosis, raised mRNA expression of CASP3 and CASP7, lower expression of cyclin-CDK1, and enhanced oxidant potential through decreased glutathione reductase (GR) activity, and enhanced reactive oxygen-species generation and lipid-peroxidation products. Conclusion: PTS-ZN NSs indicated enhanced antiproliferative, proapoptotic, and oxidant potential toward MCF7 cells compared to free PTS. Ameliorated results of nanosized carriers, cellular uptake, and sustained diffusion may contribute to these outcomes.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Nanocompostos/química , Estilbenos/química , Estilbenos/farmacologia , Zeína/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Humanos , Células MCF-7 , Oxidantes/química , Oxidantes/farmacologia , Oxirredução , Tamanho da Partícula
2.
Molecules ; 26(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926119

RESUMO

Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive ß-93-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E0 of the ß-93-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E0 of -121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione-glutathione disulfide and of glutathionyl-hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.


Assuntos
Cisteína/química , Hemoglobinas/química , Oxidantes/química , Oxirredução , Compostos de Sulfidrila/química , Fenômenos Químicos , Eritrócitos/química , Eritrócitos/metabolismo , Glutationa/química , Glutationa/metabolismo , Dissulfeto de Glutationa/química , Hemoglobinas/metabolismo , Humanos , Peróxido de Hidrogênio/química , Cinética , Modelos Biológicos , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo
3.
J Vis Exp ; (170)2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33900289

RESUMO

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.


Assuntos
Drosophila melanogaster , Ovário/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Feminino , Peróxido de Hidrogênio/farmacologia , Microscopia , Mitocôndrias/efeitos dos fármacos , Ovário/citologia , Oxidantes/farmacologia
4.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924276

RESUMO

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape "Tirocytes". The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.


Assuntos
Eritrócitos/metabolismo , Microscopia de Fluorescência , Imagem Molecular , Oxidantes/metabolismo , Estresse Oxidativo , Antioxidantes/farmacologia , Descoberta de Drogas , Eritrócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Resveratrol/farmacologia , Fluxo de Trabalho
5.
Int J Mol Sci ; 22(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921206

RESUMO

Oxidation is an important degradation pathway of protein drugs. The susceptibility to oxidation is a common concern for therapeutic proteins as it may impact product efficacy and patient safety. In this work, we used 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) as an oxidative stress reagent to evaluate the oxidation of therapeutic antibodies. In addition to the oxidation of methionine (Met) and tryptophan (Trp) residues, we also observed an increase of protein aggregation. Size-exclusion chromatography and multi-angle light scattering showed that the soluble aggregates induced by AAPH consist of dimer, tetramer, and higher-order aggregate species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that inter-molecular disulfide bonds contributed to the protein aggregation. Furthermore, intrinsic fluorescence spectra suggested that dimerization of tyrosine (Tyr) residues could account for the non-reducible cross-links. An excipient screening study demonstrated that Trp, pyridoxine, or Tyr could effectively reduce protein aggregation due to oxidative stress. This work provides valuable insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins.


Assuntos
Anticorpos Monoclonais/química , Estresse Oxidativo/genética , Proteínas/química , Proteólise/efeitos dos fármacos , Amidinas , Anticorpos Monoclonais/genética , Dimerização , Radicais Livres/química , Radicais Livres/metabolismo , Humanos , Oxidantes/química , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Agregados Proteicos/genética , Proteínas/genética , Proteínas/uso terapêutico , Triptofano/química , Triptofano/genética
6.
J Biomed Nanotechnol ; 17(2): 279-290, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33785098

RESUMO

Retinopathy is an eye disease caused by the death of retinal cells in the macular area and the surrounding choroid. As the retinal rod cell dysfunction and death lead to the loss of night vision, the disease will lead to visual dysfunction and blindness as the disease progresses. Because of the irreversible nature of cell death, gene therapy has become a research hotspot in the field of retinopathy. But the technology is still in animal studies or clinical trials, and more research is needed to prove its feasibility. In this study, oxidative damage cell model was established and divided into a control group, H2O2 group, SS31 +NEC1 group, SS31 +H2O2 group, and SS31 +NEC1 +H2O2 group, for different interventions. The cell survival rate of the H2O2 group was significantly increased compared with those of the SS31 + H2O2 group, SS31 +NEC1 +H2O2 group, and NEC1 +H2O2 group. Nec1 combined treatment significantly reduced reactive oxygen species (ROS) production compared with that in the H2O2 group. The level of MDA in the SS31 group, Nec-1 group and combined treatment of SS31 +NEC1 group decreased significantly compared with the H2O2 group. The proportion of cells with decreased mitochondrial membrane potential in the H2O2 group significantly increased, and the rate of positivity for propidium iodide (PI) of 661W cells in the H2O2 group and the control group significantly increased. Nine hours after H2O2 treatment of 661W cells, the RIP3 expression level began to increase, and peaked at 24 h. The level of RIP3 in the H2O2 group was significantly increased, while this level was downregulated in the SS31 and NEC1 treatment groups. Therefore, this study suggests that SS31 has a partial protective effect on 661W cells by inhibiting necrosis, which has certain guiding significance for the treatment of retinal diseases.


Assuntos
Mitocôndrias , Oxidantes , Animais , Apoptose , Peróxido de Hidrogênio/toxicidade , Necroptose , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Oxidantes/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo , Peptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células Fotorreceptoras Retinianas Cones
7.
ACS Appl Mater Interfaces ; 13(2): 2204-2217, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33399455

RESUMO

In this article, we demonstrate that specifically engineered oxide nanoparticles (NPs) have the potential to act as theranostic materials that are able to generate or prevent oxidative stress through their oxi-redox activity in various types of malignant and nonmalignant cells. The oxi-redox activity is related to the type and presence of surface defects, which is modified with appropriate synthesis conditions. In the present work, we used MDA-MB-231 and MCF-7 human breast cancer cells and nonmalignant MCF-10A human breast cells to demonstrate how controlled oxidative stress mediated by specifically nanoengineered indium tin oxide (ITO) NPs can selectively induce cell death in the cancer cells while reducing the oxidative stress in the normal cells and supporting their proliferation. The ITO NPs are also promising nanotheranostic materials for cancer therapy and contrast agents because of their multimodal imaging capabilities. We demonstrate that the synthesized ITO NPs can selectively increase the generation of reactive oxygen species (ROS) in both breast tumor cell lines, resulting in activation of apoptosis, and can also greatly suppress the cellular proliferation in both types of tumor cells. In contrast, the ITO NPs exhibit ROS scavenging-like behavior, significantly decreasing the ROS levels in MCF-10A cells exposed to the additional ROS, hydrogen peroxide (H2O2), so that they protect the proliferation of nonmalignant MCF-10A cells from ROS damage. In addition, fluorescent microscopy images revealed that the ITO NPs emit strong fluorescence that could be used to reveal their location. Moreover, computed tomography imaging demonstrated that the ITO NPs exhibited a comparable capability toward anatomical contrast enhancement. These results suggest that the synthesized ITO NPs have the potential to be a novel selective therapeutic agent with a multimodal imaging property for anticancer treatment.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Nanopartículas , Estresse Oxidativo/efeitos dos fármacos , Compostos de Estanho/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Nanopartículas/química , Oxidantes/química , Oxidantes/farmacologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Nanomedicina Teranóstica , Compostos de Estanho/química , Tomografia Computadorizada por Raios X
8.
Nat Commun ; 11(1): 3881, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753572

RESUMO

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.


Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais/instrumentação , Cálcio/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Imagem Óptica/instrumentação , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Propanolaminas/farmacologia
9.
Nat Struct Mol Biol ; 27(8): 726-734, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32601441

RESUMO

The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated virions in ligand-free, antibody-bound and CD4-bound forms at subnanometer resolution. Tomographic reconstructions document molecular features consistent with high-resolution structures of engineered soluble and detergent-solubilized Env trimers. One of three conformational states previously predicted by smFRET was not observed by cryo-ET, potentially owing to AT-2 inactivation. We did observe Env trimers to open in situ in response to CD4 binding, with an outward movement of gp120-variable loops and an extension of a critical gp41 helix. Overall features of Env trimer embedded in AT-2-treated virions appear well-represented by current engineered trimers.


Assuntos
2,2'-Dipiridil/análogos & derivados , Dissulfetos/farmacologia , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/efeitos dos fármacos , Vírion/efeitos dos fármacos , 2,2'-Dipiridil/farmacologia , Linhagem Celular , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Proteína gp120 do Envelope de HIV/ultraestrutura , Proteína gp41 do Envelope de HIV/ultraestrutura , Infecções por HIV/virologia , HIV-1/química , Humanos , Modelos Moleculares , Oxidantes/farmacologia , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Solubilidade , Vírion/química
11.
Toxicol Lett ; 332: 27-35, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32585298

RESUMO

Reactive oxygen species (ROS) within the cell are rapidly detoxified by antioxidants such as glutathione. Depletion of glutathione will therefore increase levels of intracellular ROS, which can lead to oxidative DNA damage and the induction of apoptosis. The working hypothesis was that Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) would be more sensitive in response to GSH depletion due to their deficiency in the removal of the oxidative DNA modification, 8-oxo-7,8-dihydroguanine (8-oxoG). Following GSH depletion, an increase in intracellular ROS and a subsequent induction of apoptosis was measured in mOgg1-/- MEFs; as expected. Unexpectedly, an elevated basal level of ROS was identified in mOgg1-/- MEFs compared to wild type MEFs; which we suggest is partly due to the differential expression of key anti-oxidant genes. The elevated basal ROS levels in mOgg1-/- MEFs were not accompanied by a deficiency in ATP production or a large increase in 8-oxoG levels. Although 8-oxoG levels did increase following GSH depletion in mOgg1-/- MEFs; this increase was significantly lower than observed following treatment with a non-toxic dose of hydrogen peroxide. Reconstitution of Ogg1 into mOgg1-/- MEFs resulted in an increased viability following glutathione depletion, however this rescue did not differ between a repair-proficient and a repair-impaired variant of Ogg1. The data indicates that induction of apoptosis in response to oxidative stress in mOgg1-/- MEFs is independent of DNA damage and OGG1-initiated DNA repair.


Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA , DNA Glicosilases/genética , Fibroblastos/efeitos dos fármacos , Glutationa/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Butionina Sulfoximina/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
12.
Arch Microbiol ; 202(7): 1873-1880, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32448965

RESUMO

The aim is to evaluate the prooxidant and antimicrobial effects of Fe3O4 and TiO2 nanoparticles and thalicarpine by luminescent and standard microbiological assays. Their effect on the kinetics of free-radical oxidation reactions (at pH 7.4 and pH 8.5) is studied in the following model systems, using activated chemiluminescence: chemical, with Fenton's reagent (H2O2-FeSO4)-for the generation of hydroxyl radicals (.OH); chemical, with oxidant hydrogen peroxide (H2O2); chemical (NAD.H-PhMS), for the generation of superoxide radicals (O2.-). Fe3O4 nanoparticles exhibit highly pronounced antioxidant properties; TiO2 nanoparticles exhibit mild to moderate prooxidant properties at neutral and alkaline conditions. Those properties are tested by the chemiluminescent method for the first time. Thalicarpine and its combination with TiO2 nanoparticles exhibit pronounced antioxidant activities at pH 8.5 which are lost and transformed into well-presented prooxidant effects at pH 7.4. That is a result-supported proof on the observed typical properties of thalicarpine and TiO2, namely antibacterial, organic-preserving and anti-pathogenic activities. The antimicrobial effect is tested on Gram-positive and Gram-negative bacteria: two strains of Escherichia coli, Bacillus cereus 1095 and Staphylococcus aureus. All bacteria are destroyed after the application of TiO2, but not Fe3O4 nanoparticles, showing their antibacterial effect. Thalicarpine, in combination with TiO2, showed even synergetic antibacterial effect.


Assuntos
Aporfinas/farmacologia , Bactérias/efeitos dos fármacos , Nanopartículas , Titânio/farmacologia , Anti-Infecciosos/farmacologia , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Ferro/química , Oxidantes/farmacologia , Oxirredução , Espécies Reativas de Oxigênio
13.
Microvasc Res ; 131: 104012, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32428522

RESUMO

Recent evidences have shown that reactive oxygen species (ROS) are involved in regulating angiogenesis and preventing tissue injury. However, the precise molecular mechanisms behind ROS-induced angiogenesis are still unknown. The aim of the present study was to investigate the effects of ROS-induced angiogenesis in rat brain microvessel endothelial cells (rBMECs) and identify involving the signal pathways. For initial experiments, the rBMECs were incubated with different concentrations of hydrogen peroxide (H2O2). For the second experiments, the rBMECs were respectively treated with ROS scavenger dimethylthiourea (DMTU), NADPH oxidase (Nox) inhibitor apocynin, small interfering RNAs-mediated knock down Nox2 or Nox4, or pretreated with c-Jun N-terminal kinase (JNK) inhibitor SP600125. The cell proliferation, migration, tube formation, and the expressions of several important neuroangiogenic factors including vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), matrix metalloproteinase (MMP) -9 and phos-JNK were measured. Low level of H2O2 significantly promoted endothelial cell (EC) proliferation, migration and tube formation and upregulated levels of VEGF, BDNF, MMP-9 and phos-JNK. DMTU and apocynin significantly inhibited endothelial angiogenesis and downregulated these protein levels. As expected, knockdown of Nox2 or Nox4 expression blocked endothelial angiogenesis and downregulated the expressions of pro-neuroangiogenic factors. Furthermore, H2O2-induced endothelial angiogenesis and high expressions of pro-neuroangiogenic factors were decreased by SP600125. In conclusion, Nox-derived ROS were required for endothelial angiogenesis. Low level of ROS may activate JNK signaling pathway and upregulate pro-neuroangiogenic factors, ultimately mediating endothelial angiogenesis.


Assuntos
Córtex Cerebral/irrigação sanguínea , Células Endoteliais/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Microvasos/enzimologia , NADP/metabolismo , Neovascularização Fisiológica , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Microvasos/efeitos dos fármacos , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Oxidantes/farmacologia , Fosforilação , Ratos , Transdução de Sinais
14.
J Photochem Photobiol B ; 205: 111851, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32172134

RESUMO

Molecular clocks are known to mediate cellular responses during oxidative stress. This important interplay is less understood in fish, particularly at mucosal surfaces. Here we report the coordinated modulation of the molecular clocks and antioxidant defence following chemically induced oxidative stress in the gill mucosa of Atlantic salmon (Salmo salar). A short-term gill explant (GE) culture was used as a model in a series of experiments aiming to demonstrate how photoperiod during culture, levels of environmental reactive oxygen species (ROS), time of oxidative stress induction, and the daily light-dark cycle affect the expression of molecular clocks and antioxidant genes in the gills. Photoperiod (either 12 light:12 dark cycle, LD or 0 light:24 dark cycle, DD) during explant culture affected the transcription of two clock genes, circadian locomotor output cycles kaput (clk) and period 1 (per1), as well as one antioxidant gene, glutathione peroxidase (gpx). When the GEs were exposed to two ROS-generating oxidants (i.e., peracetic acid, PAA and hydrogen peroxide, H2O2), photoperiod condition was demonstrated to have a significant impact on the transcription of the core genes. PAA significantly downregulated the expression of reverb alpha (reverbα) under LD, while per1 and per2 expression were significantly upregulated under DD. Nevertheless, there was no distinct pattern in the oxidant-induced expression of clock genes. On the other hand, photoperiod was shown to influence the antioxidant defence under increased ROS level, where significant transcriptional upregulation was a hallmark response under LD. Interestingly, no changes were identified under DD. Induction of oxidative stress either at ZT2 (2 h after lights on) or at ZT14 (2 h after lights off) revealed striking differences that highlighted the temporal sensitivity of the oxidative defence repertoire. Per1 was significantly modulated following time-dependent induction of oxidative stress among the clock genes. Inducing oxidative stress at ZT2 resulted in a significant upregulation of antioxidant genes; but when the same stimuli were given at ZT14, all antioxidant genes exhibited downregulation. It was further revealed that neither of the genes demonstrated daily rhythmicity in their expression in the GE cultures. Collectively, the study revealed the coordinated expression of the core elements in the molecular clock and antioxidant systems in the gill mucosa following oxidative stress. Furthermore, the results reveal that the time of day plays a crucial influence on how defences are mobilised during oxidative stress, adding new insights into the rhythms of oxidative stress response in mucosal tissues in fish.


Assuntos
Relógios Circadianos/genética , Brânquias/metabolismo , Membrana Mucosa/metabolismo , Estresse Oxidativo , Fotoperíodo , Animais , Regulação da Expressão Gênica , Brânquias/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Membrana Mucosa/efeitos dos fármacos , Oxidantes/farmacologia , Ácido Peracético/farmacologia , Salmo salar/metabolismo
15.
Org Biomol Chem ; 18(11): 2076-2084, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32108208

RESUMO

An Auxiliary Activity Family 5 (AA5) copper-radical alcohol oxidase (AlcOx) with promiscuous activity towards simple alkyl and aromatic alcohols was evaluated using real-time reaction progress monitoring. Reaction kinetics using variable time normalization analysis (VTNA) were determined from reaction progress curves. By this approach, a detailed view of the entire reaction time course under various conditions was obtained and used to identify parameters that will inform further process optimization development. Optimal activity was found impacted by several factors, including reaction pH, oxygen saturation, and the source of a co-oxidant, either HRP or a chemical alternative, potassium ferricyanide. Analysis of reaction progress curves demonstrated that reaction stalling occurred as a result of oxygen depletion and from a loss of enzyme activity over time. The cooperativity between AlcOx, horseradish peroxidase (HRP), and catalase that result in enhanced reactivity was explored, with reaction pH being identified as a key factor for optimal activity. The results show that a process with HRP is more robust than with potassium ferricyanide, but that both oxidants likely activate AlcOx by a similar mechanism. The phenomenon of product inhibition was investigated for representative reactants, revealing that reaction inhibition was more significant for butyraldehyde than for benzaldehyde. Our analysis suggests that this is linked to the greater proportion in which butyraldehyde exists in the hydrated form.


Assuntos
Oxirredutases do Álcool/metabolismo , Biocatálise , Aldeídos , Catalase/farmacologia , Cobre , Peroxidase do Rábano Silvestre/farmacologia , Cinética , Oxidantes/farmacologia , Oxigênio/metabolismo , Oxigênio/farmacologia
16.
J Food Prot ; 83(5): 779-787, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31869255

RESUMO

ABSTRACT: Antimicrobial seed treatments recommended by Canadian guidance for sprouted vegetable production (2,000 ppm of hypochlorite for 15 to 20 min or 6 to 10% hydrogen peroxide for 10 min at room temperature) are not fully compliant with organic production principles. We investigated the effect of a sequential treatment consisting of a 10-min soak at 50°C in water followed by exposure to a 2.0% H2O2 plus 0.1% AcOH sanitizing solution against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica inoculated onto alfalfa and radish seed. The sequential treatment was as effective as the recommended treatments and could reduce populations of all three species by a minimum of 3 log CFU/g using a reduced (1:2) ratio of seed to sanitizing solution and low concentrations of sanitizers approved for use in organic food production. However, the efficacy of all the treatments examined in this work was considerably reduced by storage of the seed for 4 weeks at either 11 or 75% relative humidity prior to treatment and assessment. None of the treatments could eradicate the target pathogens from seed, irrespective of time elapsed since inoculation. The results of this work suggest that the effect of storage should be considered in the assessment of antimicrobial treatments for sprouting vegetable seed.


Assuntos
Desinfecção/métodos , Manipulação de Alimentos/métodos , Peróxido de Hidrogênio/farmacologia , Medicago sativa , Raphanus , Canadá , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Alimentos Orgânicos , Germinação , Medicago sativa/microbiologia , Oxidantes/farmacologia , Raphanus/microbiologia , Sementes
17.
Am J Physiol Cell Physiol ; 318(1): C137-C149, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31721616

RESUMO

Reactive oxygen species (ROS) are important signaling molecules mediating the exercise-induced adaptations in skeletal muscle. Acute exercise also drives the expression of genes involved in reesterification and glyceroneogenesis in white adipose tissue (WAT), but whether ROS play any role in this effect has not been explored. We speculated that exercise-induced ROS would regulate acute exercise-induced responses in WAT. To address this question, we utilized various models to alter redox signaling in WAT. We examined basal and exercise-induced gene expression in a genetically modified mouse model of reduced mitochondrial ROS emission [mitochondrial catalase overexpression (MCAT)]. Additionally, H2O2, various antioxidants, and the ß3-adrenergic receptor agonist CL316243 were used to assess gene expression in white adipose tissue culture. MCAT mice have reduced ROS emission from WAT, enlarged WAT depots and adipocytes, and greater pyruvate dehydrogenase kinase-4 (Pdk4) gene expression. In WAT culture, H2O2 reduced glyceroneogenic gene expression. In wild-type mice, acute exercise induced dramatic but transient increases in Pdk4 and phosphoenolpyruvate carboxykinase (Pck1) mRNA in both subcutaneous inguinal WAT and epididymal WAT depots, which was almost completely absent in MCAT mice. Furthermore, the induction of Pdk4 and Pck1 in WAT culture by CL316243 was markedly reduced in the presence of antioxidants N-acetyl-cysteine or vitamin E. Genetic and nutritional approaches that attenuate redox signaling prevent exercise- and ß-agonist-induced gene expression within WAT. Combined, these data suggest that ROS represent important mediators of gene expression within WAT.


Assuntos
Adipócitos/enzimologia , Tecido Adiposo Branco/enzimologia , Metabolismo Energético , Mitocôndrias/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Adipócitos/efeitos dos fármacos , Adipogenia , Tecido Adiposo Branco/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Antioxidantes , Catalase/genética , Catalase/metabolismo , Metabolismo Energético/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Oxidantes/farmacologia , Oxirredução , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Esforço Físico , Transdução de Sinais , Fatores de Tempo , Técnicas de Cultura de Tecidos
18.
Cancer Lett ; 471: 1-11, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31811907

RESUMO

Deregulated metabolism of oxygen with increased generation of reactive oxygen species (ROS) is characteristic for a majority of cancers. The elevated ROS levels are in part responsible for further progression of cancer, but when produced in large excess, they endanger the viability of the cancer cells. To protect themselves from ROS-mediated toxicity, many types of cancers enhance the intrinsic antioxidant defenses, which make them dependent on the efficacy of a given ROS-detoxifying system. This poses an attractive target for anticancer therapy by two main approaches: the use of ROS-generating agents (i.e., prooxidants) or by inhibition of a chosen antioxidant system. However, the clinical efficacy of either of these approaches used alone is modest at best. The solution may rely on combining these strategies into an advanced prooxidant therapy (APoT) in order to produce a synergistic and cancer-specific effect. Indeed, such strategies have proven efficient in preclinical models, e.g., in B cell malignancies and breast cancer. Following promising experimental reports on APoT, this approach needs to be further extensively tested in order to become a potential alternative or an enhancement for classical chemotherapy.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oxidantes/farmacologia , Animais , Antioxidantes/metabolismo , Humanos , Oxidantes/uso terapêutico , Oxirredução , Ensaios Clínicos Controlados Aleatórios como Assunto , Espécies Reativas de Oxigênio/metabolismo
19.
Appl Environ Microbiol ; 86(4)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31811037

RESUMO

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx 2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx 2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.


Assuntos
Cloro/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Loci Gênicos , Ilhas Genômicas , Oxidantes/farmacologia , Termotolerância/genética , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/genética
20.
Biochimie ; 170: 10-20, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31830513

RESUMO

Oxidative stress leads to intestinal epithelial cells damage, which induces tight junction injury and systemic endogenous stress syndrome. The evidence suggests that SIRT1/PGC-1α pathway is closely associated with oxidative damage. However, the mechanism in protecting intestinal epithelial cells against oxidative stress dependant on autopahgy/mitophagy remains to be elucidated. In the current study, we investigated the functional role of SIRT1/PGC-1α pathway on regulation of autopahgy/mitophagy and tight junction protein expression underlying the oxidative dysfunction in porcine intestinal epithelial cells (IPEC-1). Results demonstrated that H2O2 exposure caused high accumulation of ROS, with a decrease of mitochondrial membrane potential and an inhibition of the tight junction molecules in IPEC-1 cells. Also, COX IV mRNA expression and SIRT1/PGC-1α pathway were suppressed. Autophagy and PINK1/Parkin dependant-mitophagy were activated following H2O2 treatment. Further research indicated that activation of SIRT1/PGC-1α pathway caused by specific activator SRT 1720 resulted in elevating autophagy/mitophagy related markers and SIRT1 inhibitor EX 527 reversed these effects. Additionally, SIRT1 activation significantly suppressed the ROS generation, leading to increase mitochondrial membrane potential and COX IV expression. Most importantly, the expression of tight junction molecules contributing to maintain intestinal barrier integrity was significantly up-regulated. Collectively, these findings indicated that autophagy/mitophagy elevation caused by SIRT1/PGC-1α pathway activation might be a protective mechanism to increase tight junction integrity against oxidative stress-mediated ROS production in IPEC-1 cells.


Assuntos
Autofagia , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mitofagia , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 1/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxidantes/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Sirtuína 1/genética
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