Identification of factors limiting the allotopic production of the Cox2 subunit of yeast cytochrome c oxidase.

Mitochondrial genes can be artificially relocalized in the nuclear genome in a process known as allotopic expression, such is the case of the mitochondrial cox2 gene, encoding subunit II of cytochrome c oxidase (CcO). In yeast, cox2 can be allotopically expressed and is able to restore respiratory growth of a cox2-null mutant if the Cox2 subunit carries the W56R substitution within the first transmembrane stretch. However, the COX2W56R strain exhibits reduced growth rates and lower steady-state CcO levels when compared to wild-type yeast. Here, we investigated the impact of overexpressing selected candidate genes predicted to enhance internalization of the allotopic Cox2W56R precursor into mitochondria. The overproduction of Cox20, Oxa1, and Pse1 facilitated Cox2W56R precursor internalization, improving the respiratory growth of the COX2W56R strain. Overproducing TIM22 components had a limited effect on Cox2W56R import, while overproducing TIM23-related components showed a negative effect. We further explored the role of the Mgr2 subunit within the TIM23 translocator in the import process by deleting and overexpressing the MGR2 gene. Our findings indicate that Mgr2 is instrumental in modulating the TIM23 translocon to correctly sort Cox2W56R. We propose a biogenesis pathway followed by the allotopically produced Cox2 subunit based on the participation of the 2 different structural/functional forms of the TIM23 translocon, TIM23MOTOR and TIM23SORT, that must follow a concerted and sequential mode of action to insert Cox2W56R into the inner mitochondrial membrane in the correct Nout-Cout topology.

Sistematización de una sistematización: aprendizajes sobre el método / Systematization of a systematization: learning about the method / Sistematização de uma sistematização: aprendizagem sobre o método

Resumen Objetivo: presentar los aprendizajes de una sistematización como método, para la construcción de conocimiento basado en la experiencia vivida por los propios investigadores de varios proyectos sobre la educación en crianza, en el campo de la salud pública, llevado a cabo en la vereda Granizal, Antioquia, durante el período 2013-2019. Materiales y método: se empleó la sistematización de experiencias y los participantes fueron los mismos investigadores. Se inició con la organización de los materiales, los diarios de campo de los círculos de investigación temática y relatorías de las reuniones del equipo de investigación y de los actores comunitarios. Se siguió con la elaboración de una matriz con la recuperación histórica. Y, finalmente, de las reflexiones grupales, surgieron categorías y relaciones para la construcción teórica de visión de conjunto. Resultados: se presenta la propuesta teórica de la sistematización y los procedimientos para implementarla, orientada por cinco momentos: vivir la experiencia, formular un plan de sistematización, recuperar el proceso vivido, las reflexiones de fondo y los puntos de llegada. Se destacan cuatro rasgos de la sistematización: una investigación creativa; basada en un proceso de construcción colectiva; para aprender de la práctica y construir conocimiento; y como escenario transformador de esta y de quienes la realizaron. Conclusión: la "sistematización de la sistematización" permitió hacer conciencia sobre la propia praxis investigativa. Muestra la comprensión del grupo acerca de la conceptualización y metodología de esta perspectiva investigativa. Trasciende la recopilación y organización de la información del proceso vivido en la medida en que la reconstrucción de la experiencia es una comprensión más amplia que transforma la práctica de los sujetos involucrados.

Abstract Objective: to present the learning of a systematization as a method, for the construction of knowledge based on the experience lived by the researchers of several projects on parenting education in the field of public health carried out in the rural settlement of Granizal, Antioquia, during the period 2013-2019. Materials and method: the systematization of experiences was used and the participants were the researchers. It began with the organization of the materials, the field diaries of the thematic research circles and reports of the meetings of the research team and community actors. It was continued with the elaboration of a matrix with the historical recovery. And, finally, reflections, categories and relationships emerged from the group for the theoretical construction of the overall vision. Results: the theoretical proposal of the systematization and the procedures to implement it are presented, guided by five moments: living the experience, formulating a systematization plan, recovering the lived process, the background reflections and the arrival points. Four features of systematization stand out: creative research; research based on a process of collective construction; learning from practice and building knowledge; and as a transforming scenario of this research and of those who carried it out. Conclusion: the "systematization of the systematization" allowed raising awareness of the research praxis itself. It shows the understanding of the group on conceptualization and methodology of this research perspective. It transcends the collection and organization of the information of the process lived to the extent that the reconstruction of the experience is a broader understanding that transforms the practice of the subjects involved.

Resumo Objetivo: apresentar as aprendizagens de uma sistematização como método, para a construção de conhecimento baseado na experiência vivida pelos próprios pesquisadores de vários projetos sobre a educação em criação, no campo da saúde pública, levado a cabo no povoado Granizal, Antioquia, durante o período 2013-2019. Materiais e método: utilizou-se a sistematização de experiências os participantes foram os mesmos pesquisadores. Iniciou-se com a organização dos materiais, os diários de campo dos círculos de pesquisa temática e relatorias das reuniões da equipe de pesquisa e dos atores comunitários. Conseguiu-se com a elaboração de uma matriz com a recuperação histórica. E, finalmente, das reflexões grupais, surgiram categorias e relações para a construção teórica de visão de conjunto. Resultados: apresenta-se a proposta teórica da sistematização e os procedimentos para implementá-la, orientada por cinco momentos: viver a experiência, formular um plano de sistematização, recuperar os processos vividos, as reflexões de fundo e os pontos de chegada. Destacam-se quatro rasgos da sistematização: uma pesquisa criativa; baseada em um processo de construção coletiva; para aprender da prática e construir conhecimento; e como cenário transformador desta e de quem a realizaram. Conclusão: a "sistematização da sistematização" permitiu fazer consciência sobre a própria práxis pesquisaria. Amostra a compreensão do grupo acerca da conceptualização e metodologia desta perspectiva pesquisaria. Trascende a recopilação e organização da informação do processo vivido na medida em que a reconstrução da experiência é uma compreensão mais ampla que transforma a prática dos sujeitos envolvidos.

The plastid proteome of the nonphotosynthetic chlorophycean alga Polytomella parva.

The unicellular, free-living, nonphotosynthetic chlorophycean alga Polytomella parva, closely related to Chlamydomonas reinhardtii and Volvox carteri, contains colorless, starch-storing plastids. The P. parva plastids lack all light-dependent processes but maintain crucial metabolic pathways. The colorless alga also lacks a plastid genome, meaning no transcription or translation should occur inside the organelle. Here, using an algal fraction enriched in plastids as well as publicly available transcriptome data, we provide a morphological and proteomic characterization of the P. parva plastid, ultimately identifying several plastid proteins, both by mass spectrometry and bioinformatic analyses. Data are available via ProteomeXchange with identifier PXD022051. Altogether these results led us to propose a plastid proteome for P. parva, i.e., a set of proteins that participate in carbohydrate metabolism; in the synthesis and degradation of starch, amino acids and lipids; in the biosynthesis of terpenoids and tetrapyrroles; in solute transport and protein translocation; and in redox homeostasis. This is the first detailed plastid proteome from a unicellular, free-living colorless alga.

Education, child rearing and social justice / Educación, crianza infantil y justicia social / Educação, educação infantil e justiça social

Abstract Health professionals often teach about child rearing based on traditional and transmissionist educational models that prescribe knowledge and meanings without considering whether families have the means to carry out the type of child rearing they value. This article discusses the meanings that child rearing has for a group of caregivers in a settlement inhabited mainly by people forcibly displaced by the armed conflict in Colombia as a way to go forward in understanding child rearing education. This work is a partial product of an action research and education project based on the strategy of thematic investigation circles. The analysis of the findings from the perspective of justice identified three areas of injustice that caregivers face as they undertake child rearing: structural-material, symbolic and cognitive. Addressing health education initiatives, including child rearing education, from a social justice perspective, entails helping people to overcome the unjust conditions faced by the subaltern population. It is also essential that health professionals learn from the students with whom they interact in order to develop more relevant education that aims to promote individual and social transformation.

Resumen Es común que los profesionales de la salud realicen educación sobre la crianza desde modelos educativos tradicionales y transmisionistas, los cuales imponen conocimientos y significados sin tener en cuenta las oportunidades que tienen las familias para realizar la crianza que valoran. Este artículo presenta los significados que sobre la crianza tiene un grupo de cuidadoras de un asentamiento habitado principalmente por población en situación de desplazamiento forzado debido al conflicto armado que vive Colombia. Es un producto parcial de un proyecto de investigación acción/educación cuya estrategia central fueron los círculos de investigación temática. El análisis de los hallazgos, realizado desde una perspectiva de justicia, identificó tres ámbitos de injusticia que las cuidadoras afrontan a la hora de realizar sus crianzas: estructural-material, simbólica y cognitiva. Abordar las acciones en salud pública y la educación para la salud - en particular la educación sobre la crianza - desde una perspectiva de justicia social, se hace necesario para contribuir a superar las condiciones de injusticia de las poblaciones subalternas; además es fundamental para que los profesionales de salud aprendan de los educandos con los cuales interactúan, como requisito para el desarrollo de procesos educativos más pertinentes tendientes a promover la transformación individual y social.

Resumo É comum que os profissionais da saúde realizam educação sobre a criação desde modelos educativos tradicionais e os professores dedicados, os quais impõem conhecimentos e significados sem ter em conta as oportunidades que têm as famílias para realizar a criação que valoram. Este artigo apresenta os significados que sobre a criação tem um grupo de cuidadoras dum assentamento habitado principalmente por população em situação de êxodo forçado devido ao conflito armado que mora na Colômbia. É um produto parcial dum projeto de pesquisa ação/educação cuja estratégia central foram os círculos de pesquisa temática. O analise dos descobrimentos, realizado desde uma perspectiva de justiça, identificou três âmbitos de injustiça que as cuidadoras afrontam à hora de realizar sua criação: estrutural-material, simbólica e cognitiva. Abordar as ações em saúde pública e a educação para a saúde - em particular a educação sobre a criação - desde uma perspectiva de justiça social, se faz necessário para contribuir a superar as condições de injustiça da população subalterna; além é fundamental para que os professionais de saúde aprendam dos estudantes com os quais interatuam, como requisito para o desenvolvimento de processos educativos mais pertinentes tendentes a promover a transformação individual e social.

Subunit Asa3 ensures the attachment of the peripheral stalk to the membrane sector of the dimeric ATP synthase of Polytomella sp.

The mitochondrial ATP synthase of Polytomella exhibits a peripheral stalk and a dimerization domain built by the Asa subunits, unique to chlorophycean algae. The topology of these subunits has been extensively studied. Here we explored the interactions of subunit Asa3 using Far Western blotting and subcomplex reconstitution, and found it associates with Asa1 and Asa8. We also identified the novel interactions Asa1-Asa2 and Asa1-Asa7. In silico analyses of Asa3 revealed that it adopts a HEAT repeat-like structure that points to its location within the enzyme based on the available 3D-map of the algal ATP synthase. We suggest that subunit Asa3 is instrumental in securing the attachment of the peripheral stalk to the membrane sector, thus stabilizing the dimeric mitochondrial ATP synthase.

Oxidative phosphorylation supercomplexes and respirasome reconstitution of the colorless alga Polytomella sp.

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F1FO-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-ß-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V2 and V4) and different respiratory supercomplexes (I/IV6, I/III4, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V4 ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption.

Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome c oxidase.

Deletion of the yeast mitochondrial gene COX2, encoding subunit 2 (mtCox2) of cytochrome c oxidase (CcO), results in a respiratory-incompetent Δcox2 strain. For a cytosol-synthesized Cox2 to restore respiratory growth, it must carry the W56R mutation (cCox2W56R). Nevertheless, only a fraction of cCox2W56R is matured in mitochondria, allowing ∼60% steady-state accumulation of CcO. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2W56R. We generated a strain expressing the mutant protein mtCox2W56R inside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2. This strain exhibited growth rates, CcO steady-state levels, and CcO activity similar to those of the wild type; therefore, the efficiency of Cox2 biogenesis is the limiting step for successful allotopic expression. Upon coexpression of cCox2W56R and mtCox2, each protein assembled into CcO independently from its genetic origin, resulting in a mixed population of CcO with most complexes containing the mtCox2 version. Notably, the presence of the mtCox2 enhances cCox2W56R incorporation. We provide proof of principle that an allotopically expressed Cox2 may complement a phenotype due to a mutant mitochondrial COX2 gene. These results are relevant to developing a rational design of genes for allotopic expression intended to treat human mitochondrial diseases.

Cox2A/Cox2B subunit interaction in Polytomella sp. cytochrome c oxidase: role of the Cox2B subunit extension.

Subunit II of cytochrome c oxidase (Cox2) is usually encoded in the mitochondrial genome, synthesized in the organelle, inserted co-translationally into the inner mitochondrial membrane, and assembled into the respiratory complex. In chlorophycean algae however, the cox2 gene was split into the cox2a and cox2b genes, and in some algal species like Chlamydomonas reinhardtii and Polytomella sp. both fragmented genes migrated to the nucleus. The corresponding Cox2A and Cox2B subunits are imported into mitochondria forming a heterodimeric Cox2 subunit. When comparing the sequences of chlorophycean Cox2A and Cox2B proteins with orthodox Cox2 subunits, a C-terminal extension in Cox2A and an N-terminal extension in Cox2B were identified. It was proposed that these extensions favor the Cox2A/Cox2B interaction. In vitro studies carried out in this work suggest that the removal of the Cox2B extension only partially affects binding of Cox2B to Cox2A. We conclude that this extension is dispensable, but when present it weakly reinforces the Cox2A/Cox2B interaction.

Near-neighbor interactions of the membrane-embedded subunits of the mitochondrial ATP synthase of a chlorophycean alga.

Mitochondrial F1FO-ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600kDa. It is composed of eight highly conserved orthodox subunits (α, ß, γ, δ, ε, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6-Asa6, Asa6-Asa8, Asa6-Asa9, Asa8-Asa8 and Asa8-Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices.

Midwestern Latino caregivers' knowledge, attitudes and sense making of the oral health etiology, prevention and barriers that inhibit their children's oral health: a CBPR approach.

BACKGROUND: Using community-based participatory research, the Health Protection Model was used to understand the cultural experiences, attitudes, knowledge and behaviors surrounding caries etiology, its prevention and barriers to accessing oral health care for children of Latino parents residing in Central Indiana. METHODS: A community reference group (CBPR) was established and bi-lingual community research associates were used to conduct focus groups comprised of Latino caregivers. Transcripts were analyzed for thematic content using inductive thematic analysis. RESULTS: Results indicated significant gaps in parental knowledge regarding caries etiology and prevention, with cultural underlays. Most parents believed the etiology of caries was related to the child's ingestion of certain foods containing high amounts of carbohydrates. Fewer parents believed either genetics/biological inheritance or bacteria was the primary causative factor. Fatalism negatively impacted preventive practices, and a clear separation existed concerning the perceived responsibilities of mothers and fathers to provide for the oral needs of their children. Females were more likely to report they were primarily responsible for brushing their children's teeth, overseeing the child's diet and seeking dental care for the child. Fathers believed they were primarily responsible for providing the means to pay for professional care. Perceived barriers to care were related to finances and communication difficulties, especially communicating with providers and completing insurance forms. CONCLUSION: The main study implication is the demonstration of how the CBPR model provided enhanced understanding of Latino caregivers' experiences to inform improvements in oral prevention and treatment of their children. Current efforts continue to employ CBPR to implement programs to address the needs of this vulnerable population.

Dissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae.

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600 kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Subunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes.

La lactancia materna como un fenómeno singular y Sociocultural surcado por diferencias y tensiones / Breastfeeding as a singular and sociocultural phenomenon marked by differences and tensions

Se presentan los resultados de una investigación etnográfica dirigida a comprender los significados de la lactancia materna para las madres educadoras comunitarias en un programa de atención a la familia de orden nacional. Metodología: se realizaron entrevistas individuales y grupales a madres educadoras. Adicionalmente se realizaron observaciones de las sesiones educativas que las madres FAMI realizan con sus grupos de madres usuarias. El análisis se hizo simultáneo con la recolección de la información. Resultados: los resultados muestran la relación entre lactancia, crianza y sujeto, que remiten a la necesidad de comprender la lactancia materna como un fenómeno singular, pero al mismo tiempo social, surcado por diferencias y tensiones. Asimismo se describe el proceso de educación que realizan las madres FAMI como líderes comunitarias, una educación sensible y flexible. Conclusión: se presenta una ruta explicativa para dar cuenta de los hallazgos encontrados. Se proponen reflexiones derivadas de los resultados para programas y políticas, y en particular para la educación sobre lactancia materna.

The present paper is an ethnographic study with the objective of understanding the meanings of breastfeeding for community mother educators in a national program for family care. Methodology: individual and group interviews to mother educators were done. In addition observations were made of the educational sessions that the FAMI mothers performed with their mothers' groups. Data analysis and field work were done simultaneously. The results show the relationships between breastfeeding, child rearing and subject, which requires understanding breastfeeding as a singular and social phenomenon, involving differences and tensions. The education process undertaken by educator mothers as community leaders is described as a sensitive and flexible education. Conclusion: an explanatory path is presented to account for the findings. Reflections derived from the results for programs and policies are proposed, particularly for breastfeeding education.

In vitro import and assembly of the nucleus-encoded mitochondrial subunit III of cytochrome c oxidase (Cox3).

The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described.

Interactions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit α in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase.

Síndrome de Down: Experiencia maternal de crianza: Entre alergías y tristezas / Maternal experience of raising children with Down’s Syndrome: Between joys and sorrows

Las madres reconocen que la crianza de niños con Síndrome de Down tiene múltiples significados culturales, sociales y emocionales que se viven durante las etapas del ciclo vital de sus hijos y que cada una de ellas lleva a alegrías y tristezas. Objetivo: Conocer la experiencia maternal de crianza de niños con Síndrome de Down en la ciudad de Medellín-Colombia. Método: investigación etnográfica con postura ética, participación de 20 madres, previo consentimiento, mediante entrevistas semiestructuradas y observaciones de campo. Análisis concomitante con la recolección de datos. Resultados: La experiencia de crianza comineza, en la mayoría de las madres participantes, con una gran tristeza en el encuentro con su hijo con Síndrome de Down, dadas las preconcepciones sociales. Luego aparece la alegría y una crianza amorosa como una posibilidad de interrelación entre madres e hijos. Discusión: la tristeza inicial se transforma en alegría al ver las potencialidades y los logros de sus hijos, así como por la relación amorosa que se construye en la crianza. Las tristezas se deben más a la exclusión que las madres experimentan en los diferentes entornos de crianza, mientras que las alegrías están relacionadas con el niño mismo y sus logros. Conclusión: Las madres de este estudio consideran que la crianza de sus hijos esta surcada por alegrías y tristezas y que un acompañamiento adecuado en los diferentes entornos hace que esta experiencia sea positiva, tanto para sus hijos con Síndrome de Down como para las familias.

Mothers recognize that raising children with Down's syndrome has multiple cultural, social, and emotional meanings that are experienced during the different stages of their children’s life cycles, each of which leads to joys and sorrows. Objective: to know the maternal experience of raising children with Down’s syndrome in the city of Medellín-Colombia. Methodos: Ethnographic research with ethical posture, participation of 20 mothers, by means of semi-structured interviews and field observations. Analysis of data was performed concomitantly with collection. Results: Given social preconceptions The parenting experience starts in most mothers of this research with a great sadness in the initial meeting with their child with Down’s syndrome,. Later in this relationship appears the joy and loving nurture as a possibility of interaction between mothers and children. Discussion: The initial sadness turns into joy as the mothers see the potentialities and achievements of their children, as well as with the loving relationship that is built during parenting. Much sadness is due to the exclusion that mothers experience in different environments while joy is related with the child and his o her achievements. Conclusion: The mothers in this study believe that parenting of children with Down;s syndrome is grooved by joys and sadness; appropriate support in different environments renders this a positive experience for children with Down’s syndrome and their families.

The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase.

Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.

In Polytomella sp. mitochondria, biogenesis of the heterodimeric COX2 subunit of cytochrome c oxidase requires two different import pathways.

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.

During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway.

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.

La praxis como fundamento de una educación para la salud alternativa: estudio de investigación-acción en el Programa de Crecimiento y Desarrollo en Medellín, Colombia / Praxis as a foundation for an alternative health education: an action research study in the Growth and Development Monitoring Program in Medellin, Colombia

La propuesta fue reflexionar sobre la educación problematizadora en un ambiente de educación para la salud y contribuir a su desarrollo teórico, en un proceso de investigación-acción, teniendo como objeto el programa de crecimiento y desarrollo en Medellín. El estudio incluyó el uso de la observación participante del programa de desarrollo de los niños y análisis de la información centrada en un proceso reflexivo permanente. Los educadores-investigadores analizaron sus prácticas pedagógicas, intentando transformar la educación en salud en una práctica más dialógica y productiva. Se encontraron dificultades para cambiar la educación tradicional en el ámbito de los servicios de salud. Se evidenció la importancia de la reflexión en la transformación de la práctica. El educando como sujeto, con experiencias y condiciones particulares contribuyó a la comprensión de una educación centrada en el ser humano. Se resaltó la necesidad de considerar los adultos como agentes centrales y la educación infantil como eje del programa.

The aim of this study was to reflect on Freire's Education Model in a health education environment in order to promote its theoretical development. An action research was developed, whose object was the Growth and Development Monitoring Program of children in Medellin, Colombia. The study involved the use of participant observation in the meetings of this program, and analysis of the information focusing on a continuous reflective process. Educators-researchers analyzed their own pedagogical practices, aiming to transform health education into a more dialogic and productive practice. Findings showed difficulties to change traditional educational practices in the scope of health services, but from a praxis perspective, reflection was found to be an important process in transforming practice. The emergence of the student as a subject with specific experiences and conditions helped the educators-researches to understand the meaning of a human-based education. The need to consider caregivers as central subjects and child rearing as a central topic in health programs was highlighted.

Propondo a reflexão sobre a educação problematizadora, em um ambiente de educação para a saúde, e contribuir para seu desenvolvimento teórico, foi desenvolvida pesquisa-ação tendo como objeto o programa de crescimento e desenvolvimento em Medellín, Colômbia. O estudo envolveu o uso da observação participante das reuniões do programa de monitorização do crescimento e desenvolvimento de crianças, e a análise da informação focada em um processo reflexivo. Durante o processo, os educadores-pesquisadores analisaram suas próprias práticas pedagógicas, visando transformar a educação em saúde numa prática mais dialógica e produtiva. Encontraram-se dificuldades para mudar a educação tradicional no âmbito dos serviços de saúde, mas evidenciou-se a importância da reflexão na transformação da prática. O educando como sujeito, com experiências e condições particulares, contribuiu para a compreensão da educação centrada no ser humano. Ressalta-se a necessidade de se considerarem os adultos como atores centrais e a Educação Infantil como eixo do programa.