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1.
N Biotechnol ; 50: 44-51, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30668986

RESUMO

The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L-1 on day 15) and MnP (17,027.4 ± 87.2 IU L-1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L-1) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L-1). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain.


Assuntos
Lacase/biossíntese , Lignina/metabolismo , Azeite de Oliva/metabolismo , Peroxidases/biossíntese , Trametes/metabolismo , Eliminação de Resíduos Líquidos , Águas Residuárias/química , Peso Molecular , Azeite de Oliva/química , Trametes/enzimologia
2.
FEMS Microbiol Lett ; 364(13)2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28655193

RESUMO

White-rot fungi are the main natural producers of lignin-modifying enzymes, i.e. laccases and peroxidases, whose secretion and activity allows the depolymerization of lignin and the release of polysaccharides contained in lignocellulose. These enzymes are able to oxidize, in addition to lignin, a wide spectrum of natural and synthetic substrates, making their industrial and biotechnological application appealing. However, the complex regulation of the synthesis of lignin-modifying enzymes, as well as the heterogeneous physiology of fungi in response to nutrients, makes the use of white-rot fungi as production platforms challenging. Finally, yet importantly, analytical methods are not fully standardized, making evaluations and comparisons ambiguous. Consequently, robust and cost-effective fermentative processes for the production of lignin-modifying enzymes by fungi have not yet been fully established, limiting their industrial exploitation. In this review, we describe the importance of both the media composition and the fermentative conditions for leveraging the fungal potential in terms of production titer and enzymatic biodiversity of lignin-modifying enzymes.


Assuntos
Meios de Cultura/farmacologia , Fermentação , Fungos/enzimologia , Lacase/biossíntese , Lignina/metabolismo , Peroxidases/biossíntese , Biotecnologia , Fungos/metabolismo , Lignina/isolamento & purificação , Oxirredução
3.
J Biosci Bioeng ; 124(1): 91-98, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28319022

RESUMO

Amauroderma rugosum is a wild mushroom species widely distributed in tropics and is classified under the class of Basidiomycetes. Basidiomycetes are well-known for their abilities of producing lignocellulolytic enzymes such as lignin peroxidase (LiP), laccase (Lac) and manganese peroxidase (MnP). Different factors such as nutrient sources, incubation period and agitation affect the production of lignocellulolytic enzymes. The A. rugosum produced LiP in the medium supplemented with potato dextrose broth (PDB), 0.5% yeast and 1.0% saw dust at 26.70±3.31 U/mL. However, the LiP activity was increased to 106.32±5.32 U/mL when supplemented with 150 µm of copper (CuSO4). The aqueous two-phase system (ATPS) is a simple, rapid and low cost method for primary extraction and recovery of LiP. A total of 25 systems made from five different molecular weights of polyethylene glycol (PEG)/dipotassium hydrogen phosphate (K2HPO4) were tested. PEG 600 produced the highest top phase purification factor (PFT) of 1.33±0.62 with yield of 72.18±8.50%. The optimization of the ATPS parameters, such as volume ratio VR, pH and crude enzyme loading are the factors controlling the phase partition. Our results showed that significant improvement (PFT of 6.26±2.87 with yield of 87.31±3.14%) of LiP recovery can be achieved by optimized the parameters.


Assuntos
Fracionamento Químico/métodos , Fermentação , Peroxidases/isolamento & purificação , Polietilenoglicóis/química , Polyporales/metabolismo , Água/química , Imersão , Peso Molecular , Peroxidases/biossíntese , Peroxidases/química , Cloreto de Sódio/química
4.
Appl Biochem Biotechnol ; 181(3): 948-960, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27696141

RESUMO

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm3), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm3). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45-70 °C with the maximal activity at pH = 4.5.


Assuntos
Proteínas Fúngicas/biossíntese , Hidroxibenzoatos/metabolismo , Lacase/biossíntese , Peroxidases/biossíntese , Silagem/microbiologia , Trametes/crescimento & desenvolvimento , Zea mays , Animais , Bovinos
5.
Biochemistry ; 56(1): 143-148, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27957837

RESUMO

Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as N-terminal tags to the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., and Bren, K. L. (2010) Protein Sci. 19, 1830-1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides.


Assuntos
Heme/metabolismo , Peptídeos/metabolismo , Peroxidases/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Azurina/química , Azurina/genética , Azurina/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Heme/química , Heme/genética , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Peptídeos/genética , Peroxidases/química , Peroxidases/genética , Conformação Proteica , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
J Gen Appl Microbiol ; 62(6): 303-312, 2017 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-27885193

RESUMO

The biodegradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, fluorene, and pyrene, by a newly isolated thermotolerant white rot fungal strain RYNF13 from Thailand, was investigated. The strain RYNF13 was identified as Trametes polyzona, based on an analysis of its internal transcribed spacer sequence. The strain RYNF13 was superior to most white rot fungi. The fungus showed excellent removal of PAHs at a high concentration of 100 mg·L-1. Complete degradation of phenanthrene in a mineral salt glucose medium culture was observed within 18 days of incubation at 30°C, whereas 90% of fluorene and 52% of pyrene were degraded under the same conditions. At a high temperature of 42°C, the strain RYNF13 was still able to grow, and degraded approximately 68% of phenanthrene, whereas 48% of fluorene and 30% of pyrene were degraded within 32 days. Thus, the strain RYNF13 is a potential fungus for PAH bioremediation, especially in a tropical environment where the temperature can be higher than 40°C. The strain RYNF13 secreted three different ligninolytic enzymes, manganese peroxidase, laccase, and lignin peroxidase, during PAH biodegradation at 30°C. When the incubation temperature was increased from 30°C to 37°C and 42°C, only two ligninolytic enzymes, manganese peroxidase and laccase, were detectable during the biodegradation. Manganese peroxidase was the major enzyme produced by the fungus. In the culture containing phenanthrene, manganese peroxidase showed the highest enzymatic activity at 179 U·mL-1. T. polyzona RYNF13 was determined as a potential thermotolerant white rot fungus, and suitable for application in the treatment of PAH-containing contaminants.


Assuntos
Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Trametes/metabolismo , Biodegradação Ambiental , Carcinógenos/metabolismo , Carcinógenos/farmacologia , Meios de Cultura/química , DNA Espaçador Ribossômico , Fluorenos/metabolismo , Fluorenos/farmacologia , Glucose/farmacologia , Lacase/biossíntese , Peroxidases/biossíntese , Fenantrenos/metabolismo , Fenantrenos/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Pirenos/metabolismo , Pirenos/farmacologia , Temperatura Ambiente , Tailândia , Trametes/genética , Trametes/crescimento & desenvolvimento , Trametes/isolamento & purificação
7.
Molecules ; 21(11)2016 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-27869681

RESUMO

Ligninolytic enzymes, such as laccase, lignin peroxidase and manganese peroxidase, are biotechnologically-important enzymes. The ability of five white-rot fungal strains Daedaleopsis confragosa, Fomes fomentarius, Trametes gibbosa, Trametes suaveolens and Trametes versicolor to produce these enzymes has been studied. Three different copper(II) complexes have been prepared ((Him)[Cu(im)4(H2O)2](btc)·3H2O, where im = imidazole, H3btc = 1,3,5-benzenetricarboxylic acid, [Cu3(pmdien)3(btc)](ClO4)3·6H2O) and [Cu3(mdpta)3(btc)](ClO4)3·4H2O, where pmdien = N,N,N',N'',N''-pentamethyl-diethylenetriamine and mdpta = N,N-bis-(3-aminopropyl)methyl- amine), and their potential application for laccase and peroxidases induction have been tested. The enzyme-inducing activities of the complexes were compared with that of copper sulfate, and it has been found that all of the complexes are suitable for the induction of laccase and peroxidase activities in white-rot fungi; however, the newly-synthesized complex M1 showed the greatest potential for the induction. With respect to the different copper inducers, this parameter seems to be important for enzyme activity, which depends also on the fungal strains.


Assuntos
Complexos de Coordenação/farmacologia , Proteínas Fúngicas/genética , Lacase/genética , Peroxidases/genética , Trametes/enzimologia , Complexos de Coordenação/química , Cobre/química , Indução Enzimática , Proteínas Fúngicas/biossíntese , Regulação Enzimológica da Expressão Gênica , Ligações de Hidrogênio , Lacase/biossíntese , Peroxidases/biossíntese
8.
Bioengineered ; 7(3): 145-54, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27295524

RESUMO

Lignin is 1 of the 3 major components of lignocellulose. Its polymeric structure includes aromatic subunits that can be converted into high-value-added products, but this potential cannot yet been fully exploited because lignin is highly recalcitrant to degradation. Different approaches for the depolymerization of lignin have been tested, including pyrolysis, chemical oxidation, and hydrolysis under supercritical conditions. An additional strategy is the use of lignin-degrading enzymes, which imitates the natural degradation process. A versatile set of enzymes for lignin degradation has been identified, and research has focused on the production of recombinant enzymes in sufficient amounts to characterize their structure and reaction mechanisms. Enzymes have been analyzed individually and in combinations using artificial substrates, lignin model compounds, lignin and lignocellulose. Here we consider progress in the production of recombinant lignin-degrading peroxidases, the advantages and disadvantages of different expression hosts, and obstacles that must be overcome before such enzymes can be characterized and used for the industrial processing of lignin.


Assuntos
Lignina/metabolismo , Engenharia Metabólica/métodos , Peroxidases/biossíntese , Phanerochaete/enzimologia , Pichia/enzimologia , Saccharomyces cerevisiae/enzimologia , Clonagem Molecular , Expressão Gênica , Humanos , Hidrólise , Cinética , Lignina/química , Oxirredução , Peroxidases/química , Peroxidases/genética , Phanerochaete/genética , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética
9.
Prikl Biokhim Mikrobiol ; 52(2): 210-6, 2016 Mar-Apr.
Artigo em Russo | MEDLINE | ID: mdl-27266250

RESUMO

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Sidel) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H2O2, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls a, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.


Assuntos
Peroxidação de Lipídeos/fisiologia , Methylobacteriaceae/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ervilhas/fisiologia , Aerobiose/fisiologia , Catalase/biossíntese , Paraquat/farmacologia , Ervilhas/efeitos dos fármacos , Ervilhas/microbiologia , Peroxidases/biossíntese , Fotossíntese/fisiologia , Superóxido Dismutase/biossíntese
10.
Mol Biol (Mosk) ; 50(2): 336-46, 2016 Mar-Apr.
Artigo em Russo | MEDLINE | ID: mdl-27239855

RESUMO

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1-6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10-63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1-6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1-6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.


Assuntos
Proteínas de Homeodomínio/genética , Estresse Oxidativo/genética , Peroxidases/genética , Peroxirredoxinas/genética , Proteínas de Xenopus/genética , Xenopus laevis/crescimento & desenvolvimento , Animais , Citoplasma/genética , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Estabilidade Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Peroxidases/biossíntese , Peroxidases/química , Peroxirredoxinas/biossíntese , Peroxirredoxinas/química , Espécies Reativas de Oxigênio/metabolismo , Xenopus laevis/genética
11.
Microvasc Res ; 103: 1-10, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26409120

RESUMO

H2O2 mediates autocrine and paracrine signaling in the vasculature and can propagate endothelial dysfunction. However, it is not clear how endothelial cells withstand H2O2 exposure and promote H2O2-induced vascular remodeling. To understand the innate ability of endothelial cells for sustaining excess H2O2 exposure, we investigated the genotypic and functional regulation of redox systems in primary HUVECs following an H2O2 treatment. Primary HUVECs were exposed to transient H2O2 exposure and consistent H2O2 exposure. Following H2O2 treatments for 24, 48 and 72 h, we measured O2(-) production, mitochondrial membrane polarization (MMP), and gene expressions of pro-oxidative enzymes, peroxidase enzymes, and cytoprotective intermediates. Our results showed that the 24 h H2O2 exposure significantly increased O2(-) levels, hyperpolarized MMP, and downregulated CAT, GPX1, TXNRD1, NFE2L2, ASK1, and ATF2 gene expression in HUVECs. At 72 h, HUVECs in both treatment conditions were shown to adapt to reduce O2(-) levels and normalize MMP. An upregulation of GPX1, TXNRD1, and HMOX1 gene expression and a recovery of NFE2L2 and PRDX1 gene expression to control levels were observed in both consistent and transient treatments at 48 and 72 h. The response of endothelial cells to excess levels of H2O2 involves a complex interaction amongst O2(-) levels, mitochondrial membrane polarization and anti- and pro-oxidant gene regulation. As a part of this response, HUVECs induce cytoprotective mechanisms including the expression of peroxidase and antioxidant enzymes along with the downregulation of pro-apoptotic genes. This adaptation assists HUVECs to withstand subsequent exposures to H2O2.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peroxidases/biossíntese , Adaptação Fisiológica , Células Cultivadas , Relação Dose-Resposta a Droga , Indução Enzimática , Glutationa Peroxidase/biossíntese , Heme Oxigenase-1/biossíntese , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Peroxidases/genética , Peroxirredoxinas/biossíntese , Superóxidos/metabolismo , Tiorredoxina Redutase 1/biossíntese , Fatores de Tempo
12.
Eur J Histochem ; 59(4): 2540, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26708178

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. The selenium is an essential trace mineral implicated as a key factor in the early stage of cancer and exerts its biological function through the selenoproteins. In the last years our group has been studying the involvement of some selenoproteins in HCC. However, no many data are reported in literature about the correlation between HCC and the glutathione peroxidases (GPXs), both selenium and non selenium-containing GPXs. In this paper we have evaluated the GPX4 and GPX7 expression in some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV)-related cirrhosis and HCC by immunohistochemistry and RT-qPCR analysis. Our results evidenced that i) GPX4 and GPX7 had a statistically significant over-expression in HCC tissues compared to cirrhotic counterparts used as non tumor tissues, and ii) their expression was higher in grade III HCC tissues with respect to grade I-II samples. Therefore, we propose to use GPX4 and GPX7 as possible markers for improving HCC diagnosis/prognosis.


Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma Hepatocelular/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/biossíntese , Neoplasias Hepáticas/enzimologia , Proteínas de Neoplasias/biossíntese , Peroxidases/biossíntese , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade
13.
PLoS Genet ; 11(10): e1005617, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26496194

RESUMO

Natural disasters, including drought and salt stress, seriously threaten food security. In previous work we cloned a key zinc finger transcription factor gene, Drought and Salt Tolerance (DST), a negative regulator of drought and salt tolerance that controls stomatal aperture in rice. However, the exact mechanism by which DST regulates the expression of target genes remains unknown. In the present study, we demonstrated that DST Co-activator 1 (DCA1), a previously unknown CHY zinc finger protein, acts as an interacting co-activator of DST. DST was found to physically interact with itself and to form a heterologous tetramer with DCA1. This transcriptional complex appears to regulate the expression of peroxidase 24 precursor (Prx 24), a gene encoding an H2O2 scavenger that is more highly expressed in guard cells. Downregulation of DCA1 significantly enhanced drought and salt tolerance in rice, and overexpression of DCA1 increased sensitivity to stress treatment. These phenotypes were mainly influenced by DCA1 and negatively regulated stomatal closure through the direct modulation of genes associated with H2O2 homeostasis. Our findings establish a framework for plant drought and salt stress tolerance through the DCA1-DST-Prx24 pathway. Moreover, due to the evolutionary and functional conservation of DCA1 and DST in plants, engineering of this pathway has the potential to improve tolerance to abiotic stress in other important crop species.


Assuntos
Adaptação Fisiológica/genética , Peroxidases/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Secas , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Oryza , Peroxidases/biossíntese , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Tolerância ao Sal/genética , Dedos de Zinco/genética
14.
Int J Cardiol ; 197: 182-91, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26142204

RESUMO

BACKGROUND: Reactive oxygen species (ROS) is thought as a major reason of vascular injury in diabetes. Vascular peroxidase 1 (VPO1) is a newly found peroxidase playing an important role in inducing oxidative stress. In the present experiment, we tested the role of VPO1 in senescence of endothelial cells in streptozotocin (STZ)-induced diabetic rats and cultured endothelial cells. METHODS: Blood samples were collected from carotid arteries. Vasodilator responses to acetylcholine (Ach) in the isolated aortic rings were measured, serum concentration of glucose, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) and the expression of VPO1 in the aorta were determined. Endothelial cells were treated with high glucose or H2O2, the concentrations of MCP-1, TNF-α and hypochlorous acid (HOCl) and the expression of VPO1 were determined. shRNA of VPO1 was used for mechanism research in cultured cells. RESULTS: Vasodilator responses to Ach were impaired markedly and the serum concentrations of glucose, TNF-α and MCP-1 were significantly increased in diabetic rats. The expression of VPO1 in the aorta was upregulated in diabetic rats. High glucose treatment significantly decreased cell viability and elevated the levels of MCP-1, TNF-α and HOCl and upregulated the expression of VPO1. H2O2 treatment significantly induced cellular senescence, inhibited eNOS expression and NO production. The effects of high glucose and H2O2 were attenuated by shRNA interference of VPO1. CONCLUSIONS: VPO1 plays an important role in senescence of endothelial cells and endothelial dysfunction by induction of oxidative stress and inflammatory reaction in type 2 diabetic rats.


Assuntos
Diabetes Mellitus Experimental/genética , Endotélio Vascular/enzimologia , Regulação da Expressão Gênica , Hemeproteínas/genética , Estresse Oxidativo , Peroxidases/genética , RNA Mensageiro/genética , Vasodilatação , Animais , Western Blotting , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/fisiopatologia , Hemeproteínas/biossíntese , Humanos , Masculino , Peroxidases/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
16.
PLoS Negl Trop Dis ; 9(2): e0003411, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25692783

RESUMO

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.


Assuntos
Proteínas de Choque Térmico HSP70/biossíntese , Leishmania mexicana/metabolismo , Leishmaniose Tegumentar Difusa/parasitologia , Peroxidases/biossíntese , Proteínas de Protozoários/biossíntese , Serina Endopeptidases/biossíntese , Linfócitos T/imunologia , Animais , Antígenos de Protozoários/imunologia , Brasil , Modelos Animais de Doenças , Feminino , Humanos , Leishmania mexicana/isolamento & purificação , Leishmania mexicana/patogenicidade , Leishmaniose Tegumentar Difusa/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Isoformas de Proteínas/biossíntese
17.
Bioprocess Biosyst Eng ; 38(1): 57-68, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24996650

RESUMO

Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.


Assuntos
Reatores Biológicos , Lacase/biossíntese , Peroxidases/biossíntese , Avena/metabolismo , Cinética , Modelos Teóricos , Polyporaceae/metabolismo
18.
Biotechnol Appl Biochem ; 62(2): 173-85, 2015 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24953758

RESUMO

Coculturing of two white-rot fungi, Dichomitus squalens and Ceriporiopsis subvermispora, was explored for the optimization of cultivation media for simultaneous augmentation of laccase and peroxidase activities by response surface methodology (RSM). Nutrient parameters chosen from our previous studies with the monocultures of D. squalens and C. subvermispora were used to design the experiments for the cocultivation study. Glucose, arabinose, sodium nitrate, casein, copper sulfate (CuSO4 ), and manganese sulfate (MnSO4 ) were combined according to central composite design and used as the incubation medium for the cocultivation. The interaction of glucose and sodium nitrate resulted in laccase and peroxidase activities of approximately 800 U/g protein. The addition of either glucose or sodium nitrate to the medium also modifies the impact of other nutrients on the ligninolytic activity. Both enzyme activities were cross-regulated by arabinose, casein, CuSO4 , and MnSO4 as a function of concentrations. Based on RSM, the optimum nutrient levels are 1% glucose, 0.1% arabinose, 20 mM sodium nitrate, 0.27% casein, 0.31 mM CuSO4 , and 0.07 mM MnSO4 . Cocultivation resulted in the production of laccase of 1,378 U/g protein and peroxidase of 1,372 U/g protein. Lignin (16.9%) in wheat straw was degraded by the optimized enzyme mixture.


Assuntos
Técnicas de Cocultura/métodos , Coriolaceae/enzimologia , Meios de Cultura/metabolismo , Lacase/biossíntese , Peroxidases/biossíntese , Polyporales/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Coriolaceae/crescimento & desenvolvimento , Meios de Cultura/química , Ativação Enzimática , Estabilidade Enzimática , Lacase/química , Lacase/isolamento & purificação , Peroxidases/química , Peroxidases/isolamento & purificação , Polyporales/crescimento & desenvolvimento
19.
Curr Genet ; 61(2): 127-40, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25407463

RESUMO

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.


Assuntos
Calmodulina/antagonistas & inibidores , Peroxidase/biossíntese , Peroxidases/biossíntese , Pleurotus/enzimologia , Calmodulina/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Isoenzimas , Lignina/genética , Lignina/metabolismo , Peroxidase/antagonistas & inibidores , Pleurotus/efeitos dos fármacos , Pleurotus/genética , Sulfonamidas/administração & dosagem , Sulfonamidas/metabolismo
20.
Sheng Wu Gong Cheng Xue Bao ; 31(12): 1682-9, 2015 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-27093831

RESUMO

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.


Assuntos
Coprinus/enzimologia , Glicoproteínas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peroxidases/biossíntese , Pichia/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Meios de Cultura , Citoplasma , Fermentação , Espectrometria de Massas , Fator de Acasalamento , Peptídeos , Dobramento de Proteína , Saccharomyces cerevisiae
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