Characterization of maize sorbitol dehydrogenase

Orientadores: Jose Andres Yunes, Paulo ArrudaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A análise do banco de ESTs de endosperma de milho (MAIZEST) revelou que o gene da sorbitol desidrogenase (SDH) é o transcrito mais abundante no início do desenvolvimento da semente (aos 10 dias após a polinização - DAP). A SDH cataliza a redução NADH-dependente da frutose em sorbitol ou a oxidação do sorbitol em frutose. Em Rosaceae esta enzima tem um importante papel na translocação do sorbitol das folhas para os frutos e no armazenamento da frutose nos frutos. A semente de milho, todavia, não acumula frutose nem sorbitol, que portanto, deve ser um metabólito intermediário. A atividade bioquímica da SDH de milho já havia sido caracterizada, porém não havia informações sobre sua estrutura genômica. Visando estudar o gene da SDH, a seqüência genômica completa foi seqüenciada. A região codificante da SDH mostrou-se muito conservada, o que não ocorre com as regiões não codificantes. Inclusive, ocorrendo uma perda de introns entre as diferentes espécies de plantas. Análises de Southem blot e isoenzima indicaram que há apenas um loeus de SDH em milho. As análises de Northem blot e atividade enzimática confirmaram que a expressão está restrita ao endosperma, e que começa logo após a polinização, I atingindo o ápice aos 15 DAP e caindo a níveis baixos aos 25 DAP. A localização enzimática in situ mostrou que a atividade está restrita ao endosperma amiláceo, mais especificamente na região basal da semente, próxima ao embrião. Os mutantes sugary1 e shrunken2, que acumulam mais açúcares, têm uma maior atividade de SDH. A injeção de até 150 mM de sacarose na semente de milho causou um aumento de atividade de SDH, confirmando os resultados obtidos com os mutantes. Esse aumento parece ser ao nível transcricional e ser regulado pelo intron 1. O intron 1 parece controlar não apenas à reposta a sacarose, mas também a resposta à hipoxia. O papel do sorbitol e da SDH nas sementes de milho em desenvolvimento parece ser crucial, porém permanece indefinido. O sorbitol é encontrado nos embriões de milho, apesar de não ter sido encontrada atividade da SDH. Além disso, foi demonstrado que os embriões conseguem se desenvolver tendo como única fonte de carbono o sorbitol. Foram encontrados ESTs no MAIZEST de um transportador de sorbitol no endosperma e de aldose redutase (AR), capaz de converter sorbitol em glicose, no embrião. Foi mostrado também que o sorbitol pode ser transportado do endosperma para o embrião. Desta forma, podemos sugerir a partir dos resultados que o sorbitol pode ser translocado do endosperma para o embrião pelo transportador de sorbitol e ser convertido em glicose pela AR no interior do embrião. Nossos resultados indicam que o sorbitol pode atuar de três maneiras; primeiro como um açúcar que é transportado de modo não vascular do endosperma para o embrião; segundo, como um metabólito intermediário; e terceiro, servindo como um escape para o excesso de NAD(P)H formado no interior hipóxico da semente. Os resultados apresentados nesta tese mostram um mecanismo inédito de interação metabólica entre o embrião e o endosperma, mediado pelo sorbitol, cuja síntese na semente, até então, era tida como um caminho "sem saída"Abstract: At 10 DAP (days after pollinization) sorbitol dehydrogenase (SDH) was found to be the most abundant transcript, as depicted by the number of reads in the MAIZEST database. SDH catalyzes the NADH-dependent reduction of fructose to sorbitol or the oxidation of sorbitol to fructose. In Rosaceae, this enzyme has an important role in the sorbitol translocation from leaves to fruits and fructose storage in fruits. Maize endosperm, however, does not store fructose or sorbitol. The biochemical activity of SDH was already characterized, but there is no information about SDH genomic structure. Trying to understand better this gene we sequenced a complete genomic SDH sequence. Amino acid sequence comparisons showed SDH to be highly eonserved, but the non-coding sequences were not, and an intron loss has occurred among plant species. Southern blot and isoenzyme analyses indicated that there is only one SDH loeus in maize. Northern blot and enzyme activity analyses confirmed that SDH expression is restricted to the endosperm, starting early after pollinization, reaehing a peak at 15 DAP and decreasing to low levels after 25 DAP. The in situ loealization of SDH activity revealed that the enzyme is expressed ali over the amilaceous endosperm, more especifically in the basal region. Sugary1 and Shrunken2 mutants, that store more sugar, had a higher SDH activity. Upon injection of up to 150 mM of sucrose in maize ear SDH activity incresead, confirming the mutant's results. Sueh increase seemed to be at the transcriptionallevel and regulated by the first intron. The first intron seems to control not only sucrose, but also hypoxia response. The role of sorbitol in developing maize kernels is potentially pivotal, but remains undefined. Suerose arriving at the kernel base is metabolized into fructose, which can be converted to sorbitol by SDH. This is a highly active enzyme in maize endosperm, but not in embryos. Still, there is considerable sorbitol in maize embryos. We verified that embryos eould grow having only sorbitol as a carbon source. We have found sorbitol transporter ESTs in endosperm and aldose-reductase, which can convert sorbitol into glueose, ESTs in embryo. We also demonstrated that sorbitol can be transported into the embryo. So, we hypothesize that sorbitol can be translocated into the embryo by sorbitol transporters, and inside the embryo, AR may convert sorbitol into glucose. In summary, SDH activity and sorbitol synthesis may contribute in three critica I ways during seed development; first as a non-vascular transport sugar moving from endosperm to embryo; second, as an intermediate metabolite largely isolated frem sugar-signaling paths; and third, as a much-needed shuttle for excess NAD(P)H forming in the hypoxic kernel interior. The presented results showed a new mechanism of embryo-endosperm interaction, mediated by sorbitol, which was considered a dead end, until nowDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecula

Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported

Beta-thalassemia Intermedia In A Brazilian Patient With-101 (c > T) And Codon 39 (c > T) Mutations.

We verified molecular alterations in a 72-year-old Brazilian male patient with a clinical course of homozygous beta-thalassemia intermedia, who had undergone splenectomy and was surviving without regular blood transfusions. The blood cell count revealed microcytic and hypochromic anemia (hemoglobin = 6.5 g/dl, mean cell volume = 74 fl, mean cell hemoglobin = 24 pg) and hemoglobin electrophoresis showed fetal hemoglobin = 1.3%, hemoglobin A2 = 6.78% and hemoglobin A = 79.4%. To identify mutations in a patient with the symptoms of beta-thalassemia intermedia. Molecular inquiry into the mutations possibly responsible for the clinical picture described. The structural molecular biology and genetic engineering center of the Universidade Estadual de Campinas, Campinas, Brazil. DNA extraction was performed on the patient's blood samples. The polymerase chain reaction (PCR) was done using five specific primers that amplified exons and the promoter region of the beta globin gene. The samples were sequenced and then analyzed via computer programs. Two mutations that cause the disease were found: -101 (C > T) and codon 39 (C > T). This case represents the first description of -101 (C > T) mutation in a Brazilian population and it is associated with a benign clinical course.12128-3

INSATISFAÇÃO CORPORAL, NÍVEL DE ATIVIDADE FÍSICA E COMPORTAMENTO SEDENTÁRIO EM ADOLESCENTES DO SEXO FEMININO

RESUMO Objetivo: Avaliar a associação da imagem corporal com o nível de atividade física, composição corporal e o comportamento sedentário de adolescentes do sexo feminino. Métodos: Estudo transversal e exploratório com 120 adolescentes do sexo feminino, de 14 a 19 anos, de Viçosa, MG. A imagem corporal foi avaliada por uma Escala de Silhuetas e pelo Body Shape Questionnaire (BSQ). O peso, a estatura e o perímetro da cintura foram aferidos, avaliando-se a relação cintura estatura e o percentual de gordura corporal. O nível de atividade física (NAF) foi avaliado pelo Recordatório de Atividade Física de 24h (R24h) e o comportamento sedentário, pelo tempo em frente à tela de uma TV, jogando videogame, ao computador e usando tablets e, separadamente, o tempo de tela ao celular. Resultados: A média de idade foi 16,5±1,5 anos, sendo a maioria das participantes eutróficas (77,6%), sedentárias/baixo NAF (84,2%), com tempo de tela (85,2%) e tempo de celular (58,7%) elevados. A insatisfação corporal manifestou-se em 40,6% pelo BSQ e em 45,8% pela Escala de Silhuetas. A distorção corporal manifestou-se em 52,9% das participantes. As medidas de composição corporal, juntamente com o tempo ao celular e o NAF, mostraram associação com a insatisfação corporal, sendo as adolescentes mais ativas as mais insatisfeitas. Conclusões: O estudo mostrou que as adolescentes com maior tempo ao celular tinham maior insatisfação corporal, assim como as fisicamente mais ativas. Todas as medidas de avaliação da composição corporal mostraram-se associadas ao nível de insatisfação, principalmente o índice de massa corporal, perímetro da cintura e relação cintura-estatura

beta-thalassemia intermedia in a Brazilian patient with - 101(C > T) and codon 39 (C > T) mutations

CONTEXT: We verified molecular alterations in a 72-year-old Brazilian male patient with a clinical course of homozygous beta-thalassemia intermedia, who had undergone splenectomy and was surviving without regular blood transfusions. The blood cell count revealed microcytic and hypochromic anemia (hemoglobin = 6.5 g/dl, mean cell volume = 74 fl, mean cell hemoglobin = 24 pg) and hemoglobin electrophoresis showed fetal hemoglobin = 1.3%, hemoglobin A2 = 6.78% and hemoglobin A = 79.4%. OBJECTIVE: To identify mutations in a patient with the symptoms of beta-thalassemia intermedia. DESIGN: Molecular inquiry into the mutations possibly responsible for the clinical picture described. SETTING: The structural molecular biology and genetic engineering center of the Universidade Estadual de Campinas, Campinas, Brazil. PROCEDURES: DNA extraction was performed on the patient's blood samples. The polymerase chain reaction (PCR) was done using five specific primers that amplified exons and the promoter region of the beta globin gene. The samples were sequenced and then analyzed via computer programs. RESULTS: Two mutations that cause the disease were found: -101 (C > T) and codon 39 (C > T). CONCLUSIONS: This case represents the first description of 101 (C > T) mutation in a Brazilian population and it is associated with a benign clinical course

BODY DISSATISFACTION, PHYSICAL ACTIVITY, AND SEDENTARY BEHAVIOR IN FEMALE ADOLESCENTS

<p></p><p>ABSTRACT Objective: To evaluate the association of body image with physical activity level, body composition, and sedentary behavior (SB) of female adolescents. Methods: Exploratory cross-sectional study conducted with 120 female adolescents aged between 14-19 years, from the city of Viçosa, Minas Gerais, Southeast Brazil. Body image was evaluated with a Body Silhouette Scale (BSS) and a Body Shape Questionnaire (BSQ). Weight, height, and waist circumference values were analyzed, as well as the waist-to-height ratio and body fat percentage. The physical activity level (PAL) was assessed by 24-hour Physical Activity Recall and SB by screen time, that is, time spent in front of a TV, playing video game, on the computer and using tablets, and, separately, the cell phone time. Results: Mean age was 16.5±1.5 years, and most adolescents were eutrophic (77.6%), sedentary/low PAL (84.2%), with high screen time (85.2%) and cell phone time (58.7%). Body dissatisfaction was stated in 40.6% of BSQ and 45.8% of BSS evaluations. Body distortion was identified in 52.9% of participants. All body composition measures, along with cell phone time and PAL, were associated with body dissatisfaction, the more active adolescents presenting higher levels of dissatisfaction. Conclusions: This study concluded that female adolescents with higher cell phone time also present higher body dissatisfaction, as well as the most physically active ones. All body composition measurements were associated with body dissatisfaction, mainly body mass index, waist circumference, and waist-to-height ratio.</p><p></p

Structural And Kinetic Characterization Of A Maize Aldose Reductase.

The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from prokaryotes to eukaryotes. The AKR superfamily consists of more than 120 proteins that are distributed throughout 14 families. Very few plant AKRs have been characterized and their biological functions remain largely unknown. Previous work suggests that AKRs may participate in stress tolerance by detoxifying reactive aldehyde species. In maize endosperm, the presence of an aldose reductase (AR; EC 1.1.1.21) enzyme has also been hypothesized based on the extensive metabolism of sorbitol. This manuscript identifies and characterizes an AKR from maize (Zea mays L.) with features of an AR. The cDNA clone, classified as AKR4C7, was expressed as a recombinant His-tag fusion protein in Escherichia coli. The product was purified by immobilized metal affinity chromatography followed by anion exchange chromatography. Circular dichroism spectrometry and SAXS analysis indicated that the AKR4C7 protein was stable, remained folded throughout the purification process, and formed monomers of a globular shape, with a molecular envelope similar to human AR. Maize AKR4C7 could utilize dl-glyceraldehyde and some pentoses as substrates. Although the maize AKR4C7 was able to convert sorbitol to glucose, the low affinity for this substrate indicated that AKR4C7 was probably a minimal contributor to sorbitol metabolism in maize seeds. Polyclonal antisera raised against AKR4C7 recognized at least three AR-like polypeptides in maize kernels, consistent with the presence of a small gene family. Diverse functions may have evolved for maize AKRs in association with specific physiological requirements of kernel development.4798-10