94 research outputs found
Analysis of the Organic Hydroperoxide Response of Chromobacterium violaceum Reveals That OhrR Is a Cys-Based Redox Sensor Regulated by Thioredoxin
Organic hydroperoxides are oxidants generated during bacterial-host interactions. Here, we demonstrate that the peroxidase OhrA and its negative regulator OhrR comprise a major pathway for sensing and detoxifying organic hydroperoxides in the opportunistic pathogen Chromobacterium violaceum. Initially, we found that an ohrA mutant was hypersensitive to organic hydroperoxides and that it displayed a low efficiency for decomposing these molecules. Expression of ohrA and ohrR was specifically induced by organic hydroperoxides. These genes were expressed as monocistronic transcripts and also as a bicistronic ohrR-ohrA mRNA, generating the abundantly detected ohrA mRNA and the barely detected ohrR transcript. The bicistronic transcript appears to be processed. OhrR repressed both the ohrA and ohrR genes by binding directly to inverted repeat sequences within their promoters in a redox-dependent manner. Site-directed mutagenesis of each of the four OhrR cysteine residues indicated that the conserved Cys21 is critical to organic hydroperoxide sensing, whereas Cys126 is required for disulfide bond formation. Taken together, these phenotypic, genetic and biochemical data indicate that the response of C. violaceum to organic hydroperoxides is mediated by OhrA and OhrR. Finally, we demonstrated that oxidized OhrR, inactivated by intermolecular disulfide bond formation, is specifically regenerated via thiol-disulfide exchange by thioredoxin (but not other thiol reducing agents such as glutaredoxin, glutathione and lipoamide), providing a physiological reducing system for this thiol-based redox switch.INCT de Processos Redox em Biomedicina-Redoxoma (FAPESP/CNPq/CAPES) [2008/57721-3, 2008/573530]INCT de Processos Redox em BiomedicinaRedoxoma (FAPESP/CNPq/CAPES)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [07/58147-6]CNPqCNP
Reduction of sulfenic acids by ascorbate in proteins, connecting thiol-dependent to alternative redox pathways
Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 Ă 103 Mâ1 sâ1 through a competition assay developed here, employing 2,6âdichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4â2.2 Ă 103 Mâ1 sâ1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4â2.2 Ă 103 Mâ1 sâ1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The was 7.4 ± 0.07 Ă 103 Mâ1 sâ1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high
As atitudes dos alunos para com a MatemĂĄtica: Um estudo em duas turmas do 6.Âș ano do Ensino BĂĄsico
O presente artigo irĂĄ incidir sobre um estudo realizado ao longo do ano letivo de 2017/2018,
no Ăąmbito da âPrĂĄtica de Ensino Supervisionada IIâ do Mestrado em Ensino do 1.Âș Ciclo do
Ensino BĂĄsico e de MatemĂĄtica e CiĂȘncias Naturais no 2.Âș Ciclo do Ensino BĂĄsico. Este estudo
teve como objetivo conhecer as atitudes dos alunos de uma turma do 6.Âș ano do ensino
bĂĄsico para com a MatemĂĄtica antes e apĂłs o perĂodo de intervenção, de modo a comparĂĄ-las
e a compreender que aspetos da prĂĄtica interventiva podem ter influenciado eventuais
mudanças de atitude. Para tal, optou-se por uma metodologia quantitativa e, como método de
recolha de dados, foram aplicados questionĂĄrios, com itens fechados e abertos, antes e apĂłs o perĂodo de intervenção, assim como foram realizadas entrevistas aos alunos que mais
diferenças mostraram entre estes dois perĂodos. Embora nĂŁo se tenha verificado uma
diferença significativa nas atitudes dos alunos para com a disciplina, em termos gerais o grupo
evoluiu no que diz respeito a este domĂnio. Os resultados, para alĂ©m de mostrarem que os
alunos se sentem mais apoiados quando estĂŁo mais professoras presentes na sala de aula,
tambĂ©m evidenciam que o tipo de atividades que foram desenvolvidas durante o perĂodo de
intervenção â atividades exploratĂłrias com recurso a materiais manipulĂĄveis â constitui uma
motivação extra para que os discentes se envolvam no trabalho matemåtico em sala de aula.Abstract
This article will focus on a study carried out during 2017/2018 academic year, within the
scope of the âSupervised Teaching Practice IIâ of the Master in Teaching in the 1st Cycle of
Basic Education and Maths and Science in the 2nd Cycle of Basic Education. This study aimed
to know the attitudes of students in a 6th grade class of basic education towards mathematics
before and after the intervention period, in order to compare them and to understand which
aspects of the interventional practice may have influenced possible changes in attitudes. To
this end, a quantitative methodology was chosen and, as a method of data collection,
questionnaires were applied, with closed and open items, before and after the intervention
period, as well as interviews with students that most showed differences between these two
periods. Although there was no significant difference in the students' attitudes towards the
discipline, in general terms the group has evolved with respect to this domain. The results, in
addition to showing that students feel more supported when there are more teachers present
in the classroom, also show that the type of activities that were developed during the
intervention period - exploratory activities using manipulable materials - constitutes extra
motivation for students to get involved in mathematical work in the classroom.Résumé
Cet article se concentrera sur une étude réalisée au cours de l'année académique 2017/2018,
dans le cadre de la «Pratique pédagogique supervisée II» du Master en enseignement du 1er
cycle de l'enseignement de base et des mathématiques et sciences naturelles du 2e Cycle
d'Ă©ducation de base. Cette Ă©tude visait Ă connaĂźtre les attitudes des Ă©lĂšves d'une classe de
base de 6e année à l'égard des mathématiques avant et aprÚs la période d'intervention, afin de
les comparer et de comprendre quels aspects de la pratique interventionnelle ont pu
influencer possibles changements d'attitude. à cette fin, une méthodologie quantitative a été
choisie et, comme méthode de collecte des données, des questionnaires ont été appliqués,
avec des éléments fermés et ouverts, avant et aprÚs la période d'intervention, ainsi que des
entretiens avec des étudiants qui ont le plus montré des différences entre cettes deux
périodes. Bien qu'il n'y ait pas de différence significative dans les attitudes des étudiants vis-à vis de la discipline, en général le groupe a évolué par rapport à ce domaine. Les résultats, en
plus de montrer que les Ă©lĂšves se sentent plus soutenus lorsqu'ils sont plus d'enseignants
présents dans la salle de classe, montrent également que le type d'activités développées au
cours de la période d'intervention - activités exploratoires utilisant du matériel manipulable -
constitue également motivation supplémentaire pour les élÚves à s'impliquer dans des travaux
mathématiques en classe.info:eu-repo/semantics/publishedVersio
Catalytic Thr or ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms
Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (C P ), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-Ï) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-Ï bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states
Oxidative modification of proteins: from damage to catalysis, signaling, and beyond
Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteo-toxicity and cellular homeostasis disruption.
Recent Advances: Previously, protein oxidation was associated exclusively to damage. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, and signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions.
Critical Issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data have been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies, and the fate of the modified proteins is of clinical relevance.
Future Directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions
Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
umor cells), and its tumoricidal activity has been well established.-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules.We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity
Additive Contributions of Two Manganese-Cored Superoxide Dismutases (MnSODs) to Antioxidation, UV Tolerance and Virulence of Beauveria bassiana
The biocontrol potential of entomopathogenic fungi against arthropod pests depends on not only their virulence to target pests but tolerance to outdoor high temperature and solar UV irradiation. Two Beauveria bassiana superoxide dismutases (SODs), BbSod2 and BbSod3, were characterized as cytosolic and mitochondrial manganese-cored isoenzymes (MnSODs) dominating the total SOD activity of the fungal entomopathogen under normal growth conditions. To probe their effects on the biocontrol potential of B. bassiana, ÎBbSod2, ÎBbSod3, and three hairpin RNA-interfered (RNAi) mutants with the transcripts of both BbSod2 and BbSod3 being suppressed by 91â97% were constructed and assayed for various phenotypic parameters in conjunction with ÎBbSod2/BbSod2, ÎBbSod3/BbSod3 and wild-type (control strains). In normal cultures, the knockout and RNAi mutants showed significant phenotypic alterations, including delayed sporulation, reduced conidial yields, and impaired conidial quality, but little change in colony morphology. Their mycelia or conidia became much more sensitive to menadione or H2O2-induced oxidative stress but had little change in sensitivity to the hyperosmolarity of NaCl and the high temperature of 45°C. Accompanied with the decreased antioxidative capability, conidial tolerances to UV-A and UV-B irradiations were reduced by 16.8% and 45.4% for ÎBbSod2, 18.7% and 44.7% for ÎBbSod3, and âŒ33.7% and âŒ63.8% for the RNAi mutants, respectively. Their median lethal times (LT50s) against Myzus persicae apterae, which were topically inoculated under a standardized spray, were delayed by 18.8%, 14.5% and 37.1%, respectively. Remarkably, the effects of cytosolic BbSod2 and mitochondrial BbSod3 on the phenotypic parameters important for the fungal bioncontrol potential were additive, well in accordance with the decreased SOD activities and the increased superoxide levels in the knockout and RNAi mutants. Our findings highlight for the first time that the two MnSODs co-contribute to the biocontrol potential of B. bassiana by mediating cellular antioxidative response
Study protocol for the multicentre cohorts of Zika virus infection in pregnant women, infants, and acute clinical cases in Latin America and the Caribbean: The ZIKAlliance consortium
Background: The European Commission (EC) Horizon 2020 (H2020)-funded ZIKAlliance Consortium designed a multicentre study including pregnant women (PW), children (CH) and natural history (NH) cohorts. Clinical sites were selected over a wide geographic range within Latin America and the Caribbean, taking into account the dynamic course of the ZIKV epidemic. Methods: Recruitment to the PW cohort will take place in antenatal care clinics. PW will be enrolled regardless of symptoms and followed over the course of pregnancy, approximately every 4 weeks. PW will be revisited at delivery (or after miscarriage/abortion) to assess birth outcomes, including microcephaly and other congenital abnormalities according to the evolving definition of congenital Zika syndrome (CZS). After birth, children will be followed for 2 years in the CH cohort. Follow-up visits are scheduled at ages 1-3, 4-6, 12, and 24 months to assess neurocognitive and developmental milestones. In addition, a NH cohort for the characterization of symptomatic rash/fever illness was designed, including follow-up to capture persisting health problems. Blood, urine, and other biological materials will be collected, and tested for ZIKV and other relevant arboviral diseases (dengue, chikungunya, yellow fever) using RT-PCR or serological methods. A virtual, decentralized biobank will be created. Reciprocal clinical monitoring has been established between partner sites. Substudies of ZIKV seroprevalence, transmissio
Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences
The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & NemĂ©sio 2007; Donegan 2008, 2009; NemĂ©sio 2009aâb; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported
by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on
18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based
researchers who signed it in the short time span from 20 September to 6 October 2016
Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes
Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions
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