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1.
J Enzyme Inhib Med Chem ; 34(1): 1439-1450, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409157

RESUMO

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).


Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania infantum/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração Inibidora 50 , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismo
2.
Cancer Radiother ; 23(5): 449-465, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400956

RESUMO

Nowadays, ionizing radiations have numerous applications, especially in medicine for diagnosis and therapy. Pharmacological radioprotection aims at increasing detoxification of free radicals. Radiomitigation aims at improving survival and proliferation of damaged cells. Both strategies are essential research area, as non-contained radiation can lead to harmful effects. Some advances allowing the comprehension of normal tissue injury mechanisms, and the discovery of related predictive biomarkers, have led to developing several highly promising radioprotector or radiomitigator drugs. Next to these drugs, a growing interest does exist for biotherapy in this field, including gene therapy and cell therapy through mesenchymal stem cells. In this review article, we provide an overview of the management of radiation damages to healthy tissues via gene or cell therapy in the context of radiotherapy. The early management aims at preventing the occurrence of these damages before exposure or just after exposure. The late management offers promises in the reversion of constituted late damages following irradiation.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Genética/métodos , Lesões por Radiação/prevenção & controle , Proteção Radiológica/métodos , Amifostina/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Ensaios Clínicos como Assunto , Fracionamento da Dose de Radiação , Edição de Genes , Vetores Genéticos/uso terapêutico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Camundongos , Oxirredutases/genética , Oxirredutases/uso terapêutico , Lesões por Radiação/etiologia , Lesões por Radiação/terapia , Lesões Experimentais por Radiação/prevenção & controle , Lesões Experimentais por Radiação/terapia , Protetores contra Radiação/farmacologia , Protetores contra Radiação/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Fatores de Tempo
3.
J Agric Food Chem ; 67(26): 7399-7409, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244203

RESUMO

Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. We isolated OsFLS from the rice ( Oryza sativa) cultivar "Ilmi" OsFLS includes highly conserved 2-ODD-specific motifs and FLS-specific regions. Recombinant OsFLS exhibited both FLS and flavanone 3ß-hydroxylase (F3H) activities, converting dihydroflavonols into flavonols and flavanones into dihydroflavonols, respectively, and more efficiently used dihydrokaempferol than dihydroquercetin as a substrate. OsFLS was expressed in both nonpigmented and pigmented rice seeds and was developmentally regulated during seed maturation. Transgenic tobacco ( Nicotiana tabacum) plants expressing OsFLS produced pale pink or white flowers with significantly increased levels of kaempferol-3- O-rutinoside and dramatically reduced levels of anthocyanin in their petals. Additionally, pod size and weight were reduced compared to the wild type. Several early and late biosynthetic genes of flavonoid were downregulated in the transgenic flowers. We demonstrated that OsFLS is a bifunctional 2-ODD enzyme and functions in flavonol production in planta.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Oryza/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antocianinas/biossíntese , Cor , Flavonóis/biossíntese , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Oryza/genética , Tabaco/genética , Tabaco/metabolismo
4.
Plant Physiol Biochem ; 141: 415-422, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31229926

RESUMO

This study aimed to investigate the possible alleviating effect of chitosan on salt-induced growth retardation and oxidative stress and to elucidate whether this effect is linked to activation of mitochondrial respiration on the basis of alternative respiration in maize seedlings. Salt stress significantly reduced root length and plant height in comparison to the control, whereas foliar application of chitosan ameliorated the adverse effect of salinity to a certain degree. Moreover, chitosan resulted in plant growth promotion as compared to unstressed seedlings. The separate applications of chitosan and salt had a stimulatory effect on the activities of antioxidant enzymes; however, combined application of chitosan and salt were more effective than that of chitosan or salt alone. Similarly, mitochondrial total respiration rate (Vt) and alternative respiration capacity (Valt) were increased by separate applications of chitosan and salt; however, the combination of chitosan and salt gave the highest values for these parameters. The highest values of Valt/Vt was recorded at seedlings treated with salt plus chitosan. Similarly, cytochrome respiration capacity was also increased by chitosan in both stress-free and stressed conditions. In addition, AOX1, encoding alternative oxidase, was significantly upregulated by chitosan and/or salt. The maximum transcript level was recorded at seedlings treated with salt plus chitosan. Chitosan also significantly decreased superoxide anion and hydrogen peroxide contents and lipid peroxidation level under normal and the stressed conditions. These results suggest that the mitigating effect of chitosan on salt stress is linked to activation of alternative respiration at biochemical and molecular level.


Assuntos
Quitosana/química , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Tolerância ao Sal , Plântula/genética , Zea mays/genética , Antioxidantes/metabolismo , Citocromos/metabolismo , Perfilação da Expressão Gênica , Peroxidação de Lipídeos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Estresse Oxidativo , Oxirredutases/genética , Proteínas de Plantas/genética , RNA/metabolismo , Estresse Fisiológico , Zea mays/enzimologia
5.
Plant Sci ; 285: 1-13, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203874

RESUMO

Bioactive gibberellins (GAs) play multiple roles in plant development and stress responses. GA2-oxidases (GA2oxs) are a class of 2-oxoglutarate-dependent dioxygenases that regulate the deactivation of bioactive GAs. In this study, we investigated the phylogeny and domain structures of the seven GA2ox genes present in the Arabidopsis thaliana genome. Comprehensive expression analysis using translational reporter lines showed that the seven GA2ox genes are differentially expressed during Arabidopsis growth and development: GA2ox1 is specifically expressed in the hypocotyl and lateral root primordium; GA2ox2 is highly expressed in aboveground tissues; GA2ox3 is expressed in the chalazal endosperm of the early embryo sac and inflorescences; GA2ox4 is expressed in the shoot apical meristem and during lateral root initiation; GA2ox6 is expressed in the maturation zone, but not in the meristem or elongating zone of the root; GA2ox7 is constitutively expressed during almost all developmental stages; and GA2ox8 is exclusively expressed in stomatal cells. Overexpression of each of these GA2ox genes inhibited high temperature-induced hypocotyl elongation in both wild-type and elongated hypocotyl 5 plants, which have an elongated hypocotyl phenotype, suggesting that these genes negatively regulate hypocotyl elongation by reducing bioactive GA levels. This study provides a valuable resource for further elucidating the roles of GA2ox genes during different stages of development.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes de Plantas/fisiologia , Giberelinas/metabolismo , Oxirredutases/genética , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Giberelinas/fisiologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Oxirredutases/metabolismo , Oxirredutases/fisiologia , Filogenia , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Transcriptoma
6.
Food Chem ; 293: 285-290, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151613

RESUMO

Exogenous adenosine triphosphate (ATP) treatment at 0, 250, 500, 750, and 1000 µM retarded cap browning in mushrooms by 0, 34, 26, 51 and 32 %, respectively, during storage at 4 °C for 18 days. Triggering signaling H2O2 accumulation arising from elevating NADPH oxidase enzyme activity during 6 days of storage at 4 °C may be pivotal for promoting shikimate dehydrogenase enzyme activity in mushrooms treated with ATP during 18 days of storage at 4 °C. Promoting melatonin accumulation (390 µg kg-1 FW vs. 160 µg kg-1 FW) in mushrooms treated with ATP during cold storage may attribute to signaling H2O2 accumulation. Higher DPPH scavenging capacity (72 % vs. 65 %) in mushrooms treated with ATP may attribute to higher phenols accumulation arising from higher phenylalanine ammonialyase/polyphenol oxidase enzymes activity concomitant with higher alternative oxidase gene expression during 18 days of storage at 4 °C.


Assuntos
Trifosfato de Adenosina/farmacologia , Agaricus/efeitos dos fármacos , Temperatura Baixa , Armazenamento de Alimentos , Reação de Maillard , Trifosfato de Adenosina/administração & dosagem , Agaricus/enzimologia , Agaricus/fisiologia , Oxirredutases do Álcool/metabolismo , Compostos de Bifenilo/química , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/metabolismo , Melatonina/metabolismo , Proteínas Mitocondriais/genética , NADPH Oxidases/metabolismo , Oxirredutases/genética , Fenóis/metabolismo , Picratos/química , Proteínas de Plantas/genética , Transdução de Sinais
7.
Pol J Microbiol ; 68(1): 43-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050252

RESUMO

Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Prodigiosina/biossíntese , Serratia marcescens/genética , Serratia marcescens/metabolismo , Fatores de Transcrição/genética , Transcrição Genética/genética , Aciltransferases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Temperatura Alta , Oxirredutases/genética , Ativação Transcricional/genética
8.
Plant Cell Rep ; 38(9): 1039-1051, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31144112

RESUMO

KEY MESSAGE: Overexpression of FvC5SD improves drought tolerance in soybean. Drought stress is one of the most important abiotic stress factors that influence soybean crop quality and yield. Therefore, the creation of drought-tolerant soybean germplasm resources through genetic engineering technology is effective in alleviating drought stress. FvC5SD is a type of C-5 sterol desaturase gene that is obtained from the edible fungus Flammulina velutipes. This gene has good tolerance to the effects of stresses, including drought and low temperature, in yeast cells and tomato. In this study, we introduced the FvC5SD gene into the soybean variety Shennong9 through the Agrobacterium-mediated transformation of soybean to identify drought-tolerant transgenic soybean varieties. PCR, RT-PCR, and Southern blot analysis results showed that T-DNA was inserted into the soybean genome and stably inherited by the progeny. The ectopic expression of FvC5SD under the control of a CaMV 35S promoter in transgenic soybean plants enhanced the plant's tolerance to dehydration and drought. Under drought conditions, the transgenic plants accumulated lower levels of reactive oxygen species and exhibited higher activities and expression levels of enzymes and cell than wild-type soybean. iTRAQ analysis of the comparative proteomics showed that some exogenous genes coding either functional or regulatory proteins were induced in the transgenic lines under drought stress. FvC5SD overexpression can serve as a direct and efficient target in improving drought tolerance in soybean and may be an important biotechnological strategy for trait improvement in soybean and other crops.


Assuntos
Flammulina/genética , Depuradores de Radicais Livres/metabolismo , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Espécies Reativas de Oxigênio/metabolismo , Secas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Oxirredutases/metabolismo , Plantas Geneticamente Modificadas , Soja/genética , Estresse Fisiológico , Transgenes
9.
Plant Cell Physiol ; 60(8): 1829-1841, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31119292

RESUMO

Alternative oxidase (AOX) has been reported to be involved in mitochondrial function and redox homeostasis, thus playing an essential role in plant growth as well as stress responses. However, its biological functions in nonseed plants have not been well characterized. Here, we report that AOX participates in plant salt tolerance regulation in moss Physcomitrella patens (P. patens). AOX is highly conserved and localizes to mitochondria in P. patens. We observed that PpAOX rescued the impaired cyanide (CN)-resistant alternative (Alt) respiratory pathway in Arabidopsis thaliana (Arabidopsis) aox1a mutant. PpAOX transcription and Alt respiration were induced upon salt stress in P. patens. Using homologous recombination, we generated PpAOX-overexpressing lines (PpAOX OX). PpAOX OX plants exhibited higher Alt respiration and lower total reactive oxygen species accumulation under salt stress condition. Strikingly, we observed that PpAOX OX plants displayed decreased salt tolerance. Overexpression of PpAOX disturbed redox homeostasis in chloroplasts. Meanwhile, chloroplast structure was adversely affected in PpAOX OX plants in contrast to wild-type (WT) P. patens. We found that photosynthetic activity in PpAOX OX plants was also lower compared with that in WT. Together, our work revealed that AOX participates in plant salt tolerance in P. patens and there is a functional link between mitochondria and chloroplast under challenging conditions.


Assuntos
Bryopsida/metabolismo , Cloroplastos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Tolerantes a Sal/metabolismo , Bryopsida/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Mitocondriais/genética , Oxirredução , Oxirredutases/genética , Proteínas de Plantas/genética , Plantas Tolerantes a Sal/genética
10.
Plant Cell Rep ; 38(8): 965-980, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31055623

RESUMO

KEY MESSAGE: In citrus, abscisic acid-deficiency was associated with a dwarfing phenotype, slow growth, small leaves, decreased fresh weight, and faster water loss. ABA supplementation reversed the dwarfing phenotype and enhanced growth. Abscisic acid (ABA) is a ubiquitously distributed phytohormone, which is almost produced by all living kingdoms. In plants, ABA plays pleiotropic physiological roles in growth, development, and stress responses. We explored the hidden relationship between ABA deficiency, and citrus dwarfing. We used targeted-HPLC, targeted-GC-MS, molecular genetics, immunoassays, and gene expression techniques to investigate the effects of the silencing of phytoene desaturase (PDS) gene on the ABA-biosynthetic pathway, endogenous ABA content, and other phytohormones. Silencing of PDS directly suppressed the carotenoids compounds involved in ABA biosynthesis, altered phytohormonal profile, and caused phytoene accumulation and ABA deficiency. The reduction of ABA presumably due to the limited availability of its precursor, zeaxanthin. The ABA-deficient citrus cuttings displayed photobleaching, a dwarf phenotype with impaired growth characteristics that included slow growth, small leaves, decreased fresh weight, and faster water loss. ABA supplementation enhanced the growth and reversed the dwarfing phenotype of the ABA-deficient cuttings. Our data demonstrate that ABA-deficiency may lead to dwarfing phenotype and impaired growth in citrus cuttings. The negative influence of ABA-deficiency on growth rate is the result of altered water relations. Addition of ABA to the CTV-tPDS roots restored shoot growth and reversed the dwarfing phenotype.


Assuntos
Ácido Abscísico/metabolismo , Citrus/metabolismo , Oxirredutases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oxirredutases/genética , Doenças das Plantas/genética , Zeaxantinas/metabolismo
11.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088920

RESUMO

The concept that anaerobic microorganisms can directly accept electrons from Fe(0) has been controversial because direct metal-microbe electron transfer has previously only been indirectly inferred. Fe(0) oxidation was studied with Geobacter sulfurreducens strain ACL, an autotrophic strain that was previously shown to grow with electrons derived from a graphite cathode as the sole electron donor. Strain ACL grew with Fe(0) as the sole electron donor and fumarate as the electron acceptor. However, it appeared that at least a portion of the electron transfer was via H2 produced nonenzymatically from the oxidation of Fe(0) to Fe(II). H2, which accumulated in abiotic controls, was consumed during the growth of strain ACL, the cells were predominately planktonic, and genes for the uptake hydrogenase were highly expressed. Strain ACLHF was constructed to prevent growth on H2 or formate by deleting the genes for the uptake of hydrogenase and formate dehydrogenases from strain ACL. Strain ACLHF also grew with Fe(0) as the sole electron donor, but H2 accumulated in the culture, and cells heavily colonized Fe(0) surfaces with no visible planktonic growth. Transcriptomics suggested that the outer surface c-type cytochromes OmcS and OmcZ were important during growth of strain ACLHF on Fe(0). Strain ACLHF did not grow on Fe(0) if the gene for either of these cytochromes was deleted. The specific attachment of strain ACLHF to Fe(0), coupled with requirements for known extracellular electrical contacts, suggest that direct metal-microbe electron transfer is the most likely option for Fe(0) serving as an electron donor.IMPORTANCE The anaerobic corrosion of iron structures is expensive to repair and can be a safety and environmental concern. It has been known for over 100 years that the presence of anaerobic respiratory microorganisms can accelerate iron corrosion. Multiple studies have suggested that there are sulfate reducers, methanogens, and acetogens that can directly accept electrons from Fe(0) to support sulfate or carbon dioxide reduction. However, all of the strains studied can also use H2 as an electron donor for growth, which is known to be abiotically produced from Fe(0). Furthermore, no proteins definitely shown to function as extracellular electrical contacts with Fe(0) were identified. The studies described here demonstrate that direct electron transfer from Fe(0) can support anaerobic respiration. They also map out a simple genetic approach to the study of iron corrosion mechanisms in other microorganisms. A better understanding of how microorganisms promote iron corrosion is expected to lead to the development of strategies that can help reduce adverse impacts from this process.


Assuntos
Geobacter/genética , Geobacter/metabolismo , Ferro/metabolismo , Anaerobiose , Corrosão , Citocromos/genética , Transporte de Elétrons , Formiato Desidrogenases/genética , Oxirredução , Oxirredutases/genética , Transcriptoma
12.
Environ Pollut ; 250: 944-952, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31085481

RESUMO

The Minamata Convention entered into force in 2017 with the aim to phase-out the use of mercury (Hg) in manufacturing processes such as the chlor-alkali or vinyl chloride monomer production. However, past industrial use of Hg had already resulted in extensive soil pollution, which poses a potential environmental threat. We investigated the emission of gaseous elemental mercury (Hg0) from Hg polluted soils in settlement areas in the canton of Valais, Switzerland, and its impact on local air Hg concentrations. Most soil Hg was found as soil matrix-bound divalent Hg (HgII). Elemental mercury (Hg0) was undetectable in soils, yet we observed substantial Hg0 emission (20-1392 ng m-2 h-1) from 27 soil plots contaminated with Hg (0.2-390 mg Hg kg-1). The emissions of Hg0 were calculated for 1274 parcels covering an area of 8.6 km2 of which 12% exceeded the Swiss soil remediation threshold of 2 mg Hg kg-1. The annual Hg0 emission from this area was approximately 6 kg a-1, which is almost 1% of the total atmospheric Hg emissions in Switzerland based on emission inventory estimates. Our results show a higher abundance of Hg resistance genes (merA) in soil microbial communities with increasing soil Hg concentrations, indicating that biotic reduction of HgII is likely an important pathway to form volatile Hg0 in these soils. The total soil Hg pool in the top 20 cm of the investigated area was 4288 kg; hence, if not remediated, these contaminated soils remain a long-term source of atmospheric Hg, which is prone to long-range atmospheric transport.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/genética , Monitoramento Ambiental/métodos , Mercúrio/análise , Mercúrio/toxicidade , Oxirredutases/genética , Poluentes do Solo/análise , Solo/química , Farmacorresistência Bacteriana/genética , Poluição Ambiental/análise , Indústrias , Conceitos Meteorológicos , Suíça
13.
Parasit Vectors ; 12(1): 236, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097010

RESUMO

BACKGROUND: Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor. METHODS: Using the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations. RESULTS: Adult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti. As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids. CONCLUSIONS: In view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.


Assuntos
Aedes/enzimologia , Aedes/genética , Resistência a Inseticidas/genética , Mosquitos Vetores/genética , Alelos , Animais , Dengue/transmissão , Feminino , Genótipo , Glutationa Transferase/genética , Proteínas de Insetos/genética , Inseticidas , Malation , Malásia , Mosquitos Vetores/enzimologia , Mutação , Oxirredutases/genética , Reação em Cadeia da Polimerase , Piretrinas , Análise de Sequência de DNA , Canais de Sódio/genética
14.
Planta ; 250(1): 347-366, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31037486

RESUMO

MAIN CONCLUSION: The involvement of a WRKY transcription factor in the regulation of lignan biosynthesis in flax using a hairy root system is described. Secoisolariciresinol is the main flax lignan synthesized by action of LuPLR1 (pinoresinol-lariciresinol reductase 1). LuPLR1 gene promoter deletion experiments have revealed a promoter region containing W boxes potentially responsible for the response to Fusarium oxysporum. W boxes are bound by WRKY transcription factors that play a role in the response to stress. A candidate WRKY transcription factor, LuWRKY36, was isolated from both abscisic acid and Fusarium elicitor-treated flax cell cDNA libraries. This transcription factors contains two WRKY DNA-binding domains and is a homolog of AtWRKY33. Different approaches confirmed LuWRKY36 binding to a W box located in the LuPLR1 promoter occurring through a unique direct interaction mediated by its N-terminal WRKY domain. Our results propose that the positive regulator action of LuWRKY36 on the LuPLR1 gene regulation and lignan biosynthesis in response to biotic stress is positively mediated by abscisic acid and inhibited by ethylene. Additionally, we demonstrate a differential Fusarium elicitor response in susceptible and resistant flax cultivars, seen as a faster and stronger LuPLR1 gene expression response accompanied with higher secoisolariciresinol accumulation in HR of the resistant cultivar.


Assuntos
Linho/genética , Fusarium/fisiologia , Lignanas/biossíntese , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/farmacologia , Fatores de Transcrição/metabolismo , Ácido Abscísico/farmacologia , Etilenos/farmacologia , Linho/metabolismo , Linho/microbiologia , Biblioteca Gênica , Modelos Biológicos , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Estresse Fisiológico , Fatores de Transcrição/genética
15.
Nat Commun ; 10(1): 1822, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015394

RESUMO

Large reservoirs of natural gas in the oceanic subsurface sustain complex communities of anaerobic microbes, including archaeal lineages with potential to mediate oxidation of hydrocarbons such as methane and butane. Here we describe a previously unknown archaeal phylum, Helarchaeota, belonging to the Asgard superphylum and with the potential for hydrocarbon oxidation. We reconstruct Helarchaeota genomes from metagenomic data derived from hydrothermal deep-sea sediments in the hydrocarbon-rich Guaymas Basin. The genomes encode methyl-CoM reductase-like enzymes that are similar to those found in butane-oxidizing archaea, as well as several enzymes potentially involved in alkyl-CoA oxidation and the Wood-Ljungdahl pathway. We suggest that members of the Helarchaeota have the potential to activate and subsequently anaerobically oxidize hydrothermally generated short-chain hydrocarbons.


Assuntos
Archaea/metabolismo , Proteínas Arqueais/metabolismo , Genoma Arqueal/genética , Hidrocarbonetos/metabolismo , Oxirredutases/metabolismo , Anaerobiose , Archaea/genética , Proteínas Arqueais/genética , Sedimentos Geológicos/microbiologia , Fontes Hidrotermais/microbiologia , Redes e Vias Metabólicas/genética , Metagenômica , Oceanos e Mares , Oxirredutases/genética , Filogenia
16.
PLoS Negl Trop Dis ; 13(4): e0007260, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30943203

RESUMO

BACKGROUND: Leishmania donovani is a protozoan parasite, a primary causative agent of visceral leishmaniasis. Sterol produced via the mevalonate pathway, show differences in composition across biological kingdoms. The specific occurrence of Δ22-unsaturated sterols, containing a double bond at the C-22 position in the side chain occurs in fungi as ergosterol and as stigmasterol in plants. In the present study, we report the identification and functional characterization of a plant-like Cytochrome P450 subfamily CYP710C1 in L. donovani as the Leishmania C-22 desaturase. METHODOLOGY: In silico analysis predicted the presence of a plant like CYP710C1 gene that encodes a sterol C-22 desaturase, a key enzyme in stigmasterol biosynthesis. The enzymatic function of recombinant CYP710C1 as C-22 desaturase was determined. To further study the physiological role of CYP710C1 in Leishmania, we developed and characterized an overexpressing strain and a gene deletion mutant. C-22 desaturase activity and stigmasterol levels were estimated in the wild-type, overexpressing promastigotes and heterozygous mutants. CONCLUSION: We for the first time report the presence of a CYP710C1 gene that encodes a plant like sterol C-22 desaturase leading to stigmasterol biosynthesis in Leishmania. The recombinant CYP710C1 exhibited C-22 desaturase activity by converting ß-sitosterol to stigmasterol. Axenic amastigotes showed higher expression of CYP710C1 mRNA, protein and stigmasterol levels compared to the promastigotes. Sterol profiling of CYP710C1 overexpressing L. donovani and heterozygous mutant parasites demonstrated that CYP710C1 was responsible for stigmasterol production. Most importantly, we demonstrate that these CYP710C1 overexpressing promastigotes are resistant to amphotericin B, a drug of choice for use against leishmaniasis. We report that Leishmania sterol biosynthesis pathway has a chimeric organisation with characteristics of both plant and fungal pathways.


Assuntos
Anfotericina B/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Resistência a Medicamentos/genética , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Genes de Plantas , Leishmania donovani/enzimologia , Leishmaniose Visceral , Oxirredutases/genética , Deleção de Sequência , Sitosteroides/metabolismo , Esteróis/biossíntese , Estigmasterol/metabolismo
17.
Chem Biol Interact ; 305: 163-170, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30928400

RESUMO

Mammalian steroid 5ß-reductases belong to the Aldo-Keto Reductase 1D sub-family and are essential for the formation of A-ring 5ß-reduced steroids. Steroid 5ß-reduction is required for the biosynthesis of bile-acids and the metabolism of all steroid hormones that contain a Δ4-3-ketosteroid functionally to yield the 5ß-reduced metabolites. In mammalian AKR1D enzymes the conserved catalytic tetrad found in all AKRs (Y55, H117, K84 and D50) has changed in that the conserved H117 is replaced with a glutamic acid (E120). E120 may act as a "superacid" to facilitate enolization of the Δ4-ketosteroid. In addition, the absence of the bulky imidazole side chain of histidine in E120 permits the steroid to penetrate deeper into the active site so that hydride transfer can occur to the steroid C5 position. In murine steroid 5ß-reductase AKR1D4, we find that there is a long-form, with an 18 amino-acid extension at the N-terminus (AKR1D4L) and a short-form (AKR1D4S), where the latter is recognized as AKR1D4 by the major data-bases. Both enzymes were purified to homogeneity and product profiling was performed. With progesterone and cortisol, AKR1D4L and AKR1D4S catalyzed smooth conversion to the 5ß-dihydrosteroids. However, with Δ4-androstene-3,17-dione as substrate, a mixture of products was observed which included, 5ß-androstane-3,17-dione (expected) but 3α-hydroxy-5ß- androstan-17-one was also formed. The latter compound was distinguished from its isomeric 3ß-hydroxy-5ß-androstan-17-one by forming picolinic acid derivatives followed by LC-MS. These data show that AKR1D4L and AKR1D4S also act as 3α-hydroxysteroid dehydrogenases when presented with Δ4-androstene-3,17-dione and suggest that E120 alters the position the steroid to enable a correct trajectory for hydride transfer and may not act as a "superacid".


Assuntos
Ácido Glutâmico/química , Oxirredutases/metabolismo , Androstanos/análise , Androstanos/química , Androstanos/metabolismo , Animais , Biocatálise , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Ácido Glutâmico/metabolismo , Humanos , Isomerismo , Cinética , Fígado/metabolismo , Camundongos , Oxirredução , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esteroides/química , Esteroides/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
18.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940709

RESUMO

Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization to the biofilm community such that biofilm residents can benefit from the production of common goods while being protected from exogenous insults. Spatial organization is driven by the presence of chemical gradients, such as oxygen. Here we show that two quinol oxidases found in Escherichia coli and other bacteria organize along the biofilm oxygen gradient and that this spatially coordinated expression controls architectural integrity. Cytochrome bd, a high-affinity quinol oxidase required for aerobic respiration under hypoxic conditions, is the most abundantly expressed respiratory complex in the biofilm community. Depletion of the cytochrome bd-expressing subpopulation compromises biofilm complexity by reducing the abundance of secreted extracellular matrix as well as increasing cellular sensitivity to exogenous stresses. Interrogation of the distribution of quinol oxidases in the planktonic state revealed that ∼15% of the population expresses cytochrome bd at atmospheric oxygen concentration, and this population dominates during acute urinary tract infection. These data point toward a bet-hedging mechanism in which heterogeneous expression of respiratory complexes ensures respiratory plasticity of E. coli across diverse host niches.IMPORTANCE Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization in the biofilm community. Here we demonstrate that oxygen gradients in uropathogenic Escherichia coli (UPEC) biofilms lead to spatially distinct expression programs for quinol oxidases-components of the terminal electron transport chain. Our studies reveal that the cytochrome bd-expressing subpopulation is critical for biofilm development and matrix production. In addition, we show that quinol oxidases are heterogeneously expressed in planktonic populations and that this respiratory heterogeneity provides a fitness advantage during infection. These studies define the contributions of quinol oxidases to biofilm physiology and suggest the presence of respiratory bet-hedging behavior in UPEC.


Assuntos
Biofilmes/crescimento & desenvolvimento , Variação Biológica da População , Heterogeneidade Genética , Oxirredutases/metabolismo , Oxigênio/metabolismo , Escherichia coli Uropatogênica/fisiologia , Aerobiose , Anaerobiose , Oxirredução , Oxirredutases/genética
19.
J Agric Food Chem ; 67(16): 4444-4452, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30939238

RESUMO

This study aimed to examine the effects of UV-B on AsA and gene expression in cucumber seedlings. Particular emphasis was placed on identifying genes that were responsive to UV-B to increase AsA levels and elucidate the key UV-B response pathway. We found that the activities of myo-inositol oxygenase (MIOX), galactono-1,4-lactone dehydrogenase (GLDH), ascorbate oxidase (AO), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and the transcript levels of CsMIOX1, CsGLDH, CsAO2, CsAO4, CsGR1, CsAPX5, and CsDHAR1 significantly increased with UV-B exposure. These observations indicate that UV-B induces the expression of genes involved in d-mannose/l-galactose and myo-inositol pathways and the ascorbate-glutathione system. Moreover, several genes related to the low and high UV-B fluence responses were considered. CsHY5 and CsMYB60 were involved with the low-fluence response and appeared to be responsive from 2 to 28 h. Together, these data show that these genes respond to UV-B to increase AsA levels through the low-fluence UV-B response pathway.


Assuntos
Ácido Ascórbico/metabolismo , Cucumis sativus/metabolismo , Cucumis sativus/efeitos da radiação , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Cucumis sativus/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Glutationa/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/efeitos da radiação , Raios Ultravioleta
20.
MBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015328

RESUMO

In Mycobacterium tuberculosis, recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances. Interpreting the functional importance of the new resistance-associated mutations has been complicated, however, by a lack of experimental validation and a poor understanding of the epistatic factors influencing these correlations, including strain background and programmatic variation in treatment regimens. Here we perform a genome-wide association analysis in a panel of Mycobacterium tuberculosis strains from China to identify variants correlated with resistance to the second-line prodrug ethionamide (ETH). Mutations in a bacterial monooxygenase, Rv0565c, are significantly associated with ETH resistance. We demonstrate that Rv0565c is a novel activator of ETH, independent of the two known activators, EthA and MymA. Clinically prevalent mutations abrogate Rv0565c function, and deletion of Rv0565c confers a consistent fitness benefit on M. tuberculosis in the presence of partially inhibitory doses of ETH. Interestingly, Rv0565c activity affects susceptibility to prothionamide (PTH), the ETH analog used in China, to a greater degree. Further, clinical isolates vary in their susceptibility to both ETH and PTH, to an extent that correlates with the total expression of ETH/PTH activators (EthA, MymA, and Rv0565c). These results suggest that clinical strains considered susceptible to ETH/PTH are not equally fit during treatment due to both Rv0565c mutations and more global variation in the expression of the prodrug activators.IMPORTANCE Phenotypic antibiotic susceptibility testing in Mycobacterium tuberculosis is slow and cumbersome. Rapid molecular diagnostics promise to help guide therapy, but such assays rely on complete knowledge of the molecular determinants of altered antibiotic susceptibility. Recent genomic studies of antibiotic-resistant M. tuberculosis have identified several candidate loci beyond those already known to contribute to antibiotic resistance; however, efforts to provide experimental validation have lagged. Our study identifies a gene (Rv0565c) that is associated with resistance to the second-line antibiotic ethionamide at a population level. We then use bacterial genetics to show that the variants found in clinical strains of M. tuberculosis improve bacterial survival after ethionamide exposure.


Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Etionamida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Protionamida/farmacologia , China , Estudo de Associação Genômica Ampla , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Oxirredutases/metabolismo
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