Análise quantitativa de rna mensageiros, proteínas e atividades enzimáticas no estudo da rede de regulação controlada pelo gene Opaco-2

The Opaque-2 (O2) gene encodes a transcriptional activator specifically expressed for grain development of maize. o2 mutants have an opaque and chalky kernel, with a decrease in zein storage protein content, and an increase in the proportions of lysine and tryptophan. In this review, we present recent results investigating genetic properties of the O2 network, using transcriptome and proteome approaches, associated with measurements of activities of enzymes of the aspartate pathway and lysine degradation. The structural polymorphism at the O2 locus was investigated by RFLP in a collection of 51 maize inbred lines. Most polymorphic sites were found outside the coding regions. We then searched for relationships between RFLP polymorphism and (i) mRNA abundance of O2 and of known or suspected target genes, (ii) activity of SDH and (iii) amount of zein isoforms. Polymorphic restriction sites in the 5' upstream regions of the O2 gene were found associated with O2 mRNA abundance (three sites) and the amount of two 19 kDa alpha-zein isoforms (two sites). One restriction site on the 3' side of the O2 gene was found associated with Lor/Sdh mRNA abundance. Our results indicate relationships between polymorphism at the O2 locus and the expression of some of its target genes. Evidence of these associations has to be confirmed on larger samples, and the analysis of the O2 gene sequence should allow more precise testing of the actual involvement of O2 polymorphism in its own transcriptional expression, and in the expression of its target genes.O gene Opaco-2 (O2), expresso especificamente no grão de milho, transcreve para um fator de transcrição da família "leucine-zipper". Mutantes o2 apresentam grãos opacos, redução na quantidade de zeínas e aumento na proporção de lisina e triptofano. Genes cuja expressão é controlada diretamente pelo O2 são conhecidos (alfa-zeínas de 22 kDa, beta-zeínas de 14 kDa, b-32 e cyPpdk1). Nesta revisão, nós apresentamos resultados da caracterização genética de genes relacionados com o O2, através de abordagens de transcritoma, proteoma e de atividades enzimáticas da via metabólica do aspartato e da degradação da lisina. O polimorfismo do locus O2 foi avaliado utilisando-se a técnica de RFLP em 51 linhagens de milho. A maioria dos polimorfismos foi observada nas regiões não codificadoras da proteína. Análises de correlação foram realizadas entre os polimorfismos de RFLP e (i) quantidade de RNAm do O2, cyPpdk, Lor/Sdh e Ahas (ii) quantidade de isoformas de zeínas e (iii) atividade da enzima SDH. Sítios polimórficos foram correlacionados com a quantidade de RNAm do próprio O2, do gene Lor/Sdh e com a quantidade de duas isoformas de a-zeinas de 19 kDa. Nossos resultados indicam a presença de relações entre o polimorfismo do locus O2 e o nível de expressão de genes sob o seu controle. A utilização de um maior número de linhagens e o uso de dados de seqüência do O2 permitirá uma análise precisa da conseqüência do polimorfismo deste fator de transcrição sobre o controle do seu próprio nível de expressão e dos genes por ele controlados

Tracking the evolution of transiently transfected individual cells in a microfluidic platform

Transient gene expression (TGE) technology enables the rapid production of large amount of recombinant proteins, without the need of fastidious screening of the producing cells required for stable transfection (ST). However, several barriers must be overcome before reaching the production yields using ST. For optimizing the production yields from suspended cells using TGE, a better understanding of the transfection conditions at the single cell level are required. In this study, a universal droplet microfluidic platform was used to assess the heterogeneities of CHO-S population transiently transfected with cationic liposomes (CL) (lipoplexes) complexed with GFP-coding plasmid DNA (pDNA). A single cell analysis of GFP production kinetics revealed the presence of a subpopulation producing higher levels of GFP compared with the main population. The size of high producing (HP) cells, their relative abundance, and their specific productivity were dependent on the charge and the pDNA content of the different lipoplexes: HPs showed increased cell size in comparison to the average population, lipoplexes with positive charge produced more HPs, and lipoplexes carrying a larger amount of pDNA yielded a higher specific productivity of HPs. This study demonstrates the potential for time-resolved single-cell measurements to explain population dynamics from a microscopic point of view8CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP202086/2015-12014/24797-2; 2014/10557-5; 2015/26701-

Análise dialélica para conteúdos de lisina e óleo em grãos de milho

Six S5 lines of maize, with differences for lysine and oil contents in grains, were used to carry out a complete series of diallelic crosses. The resulting 15 hybrids were grown in a field at two nitrogen levels (10 and 130 kg N ha-1). The general combining ability (GCA) and specific combining ability (SCA) were obtained by using the method 4, model I of Griffing for grain yield, and grain lysine and oil contents. Significant (p < 0.001) interaction was observed between GCA and N levels for grain yield, indicating the selection of different lines for each N level. This interaction was not significant for lysine content, but there were significant effects of GCA for this trait at both N levels (p < 0.1). Significant effects were not observed for GCA or SCA for oil content, however a positive correlation was observed between lysine and oil contents in the hybrids, in the lines and even in the control cultivars. The results indicate the effectiveness of selection for lysine content, irrespective of N level, in the studied non-opaque maize lines, and the possibility of achieving both high lysine and oil content in grains.Seis linhagens S5 de milho, com diferenças para conteúdos de óleo e de lisina nos grãos, foram usadas para realizar uma série completa de cruzamentos dialélicos. Os 15 híbridos resultantes foram cultivados em campo com dois níveis de nitrogênio (10 e 130 kg N ha-1). A capacidade geral de combinação (CGC) e a capacidade específica de combinação (CEC) foram obtidas com o método 4, modelo I de Griffing para produtividade de grãos e concentração de lisina e óleo nos grãos. Interação significativa (p < 0.001) foi observada entre CGC e níveis de N para produtividade de grãos, indicando seleção de diferentes linhagens para cada nível de N. Esta interação não foi significativa para conteúdo de lisina, mas existiu efeito significativo (p < 0.1) da CGC nos dois níveis de N para esta variável. Os efeitos de CGC e CEC foram não significativos para conteúdo de óleo, contudo, foi observada correlação positiva entre conteúdos de lisina e óleo nos híbridos, nas linhagens e mesmo nas cultivares utilizadas como controle. Os resultados indicam a efetividade da seleção para conteúdo de lisina nas linhagens não opaco de milho estudadas, independente dos níveis de N, e a possibilidade de atingir concomitantemente maiores níveis de óleo e lisina nos grãos

Caracterização do desenvolvimento de cultivares de feijão-caupi e do conteúdo e da qualidade proteica dos seus grãos

The objective of this work was to evaluate cowpea (Vigna unguiculata) cultivars regarding plant development and the quantity and quality of soluble proteins in their grains, for breeding purposes. The experiment was conducted in a greenhouse, in a completely randomized experimental design, with the Paulistinha, BRS Novaera, Epace 10, and BR 17-Gurguéia cultivars. Leaf area, shoot fresh and dry matter, leaf protein content, number of nodules, and nodule and root dry matter were evaluated. In mature grains, soluble fractions and soluble amino acids were also quantified, and the electrophoretic analysis was performed with protein denaturation. There were no differences between cultivars for the plant development variables. However, protein quantity and quality in the grain differed between cultivars. 'BRS Novaera' and 'BR 17-Gurguéia' showed a higher soluble protein content in their grains. 'BRS Novaera' exhibited higher contents of two soluble sulfur amino acids – methionine and cysteine – , not differing from 'BR 17-Gurguéia' regarding methionine content. Both cultivars presented protein band polymorphism, but BRS Novaera had an extra band for albumins. The BRS Novaera and BR 17-Gurguéia cultivars have the highest content of soluble proteins in their grains and the greatest protein polymorphism, which makes them suitable for improving the nutritional quality of cowpea.O objetivo deste trabalho foi avaliar cultivares de feijão-caupi (Vigna unguiculata) quanto ao desenvolvimento da planta e à quantidade e à qualidade de proteínas solúveis em seus grãos, com vistas ao melhoramento. O experimento foi conduzido em casa de vegetação, em delineamento experimental inteiramente casualizado, com as cultivares Paulistinha, BRS Novaera, Epace 10 e BR 17-Gurguéia. Avaliaram-se área foliar, peso fresco e seco da parte aérea, conteúdo de proteína foliar, número de nódulos, e massa seca de nódulos e de raiz. Nos grãos maduros, também quantificaram-se as frações solúveis e os aminoácidos solúveis, e a análise eletroforética foi realizada com desnaturação das proteínas. Não houve diferenças entre as cultivares quanto às variáreis do desenvolvimento da planta. No entanto, a quantidade e a qualidade das proteínas nos grãos diferiram entre as cultivares. 'BRS Novaera' e 'BR 17-Gurguéia' apresentaram maior teor de proteína solúvel nos seus grãos. 'BRS Novaera' apresentou maior teor de dois aminoácidos sulfurados solúveis – metionina e cisteína – , não tendo diferido de 'BR 17-Gurguéia' quanto ao conteúdo de metionina. Ambas as cultivares apresentaram polimorfismo de bandas, mas BRS Novaera apresentou uma banda extra para albuminas. As cultivares BRS Novaera e BR 17-Gurguéia apresentam o maior conteúdo de proteínas solúveis nos seus grãos e o maior polimorfismo de proteínas, o que as torna adequadas para o melhoramento nutricional do feijão-caupi

Manipulação de cereais para acúmulo de lisina em sementes

A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido aspártico, pela qual são também sintetizados os aminoácidos treonina, metionina e isoleucina. Além disso, a lisina é o principal aminoácido limitante em todos os cereais e por cerca de 30 anos a via do ácido aspártico tem sido estudada em plantas, com o intuito de desvendar e caracterizar os principais pontos chave na regulação das vias de biossíntese desses aminoácidos. Duas etapas distintas, uma primeira originada a partir do desenvolvimento da cultura de tecidos (anos 70-80) e a segunda a partir do desenvolvimento de técnicas para a transformação de plantas (anos 90), permitiram que mutantes bioquímicos e plantas trangênicas fossem produzidos com alterações específicas em passos metabólicos chave, levando à superprodução e acúmulo de treonina em vários tecidos das plantas. Entretanto, a acumulação de lisina em sementes não foi obtida. Tal fato, associado a estudos bioquímicos da via de degradação da lisina em cereais e em leguminosas, indicou que a manipulação da degradação seria tão ou mais importante que a manipulação da biossíntese de lisina para o acúmulo deste aminoácido em sementes dos cereais . Em milho, o uso e estudo de outros mutantes tais como o opaco-2 e variedades QPM (Quality Protein Maize) contribuíram significativamente para a compreensão dos eventos regulatórios. As estratégias para a obtenção de materiais ricos em lisina e sua relevância à manipulação de outros aminoácidos são revisados.The nutrition value of a protein is directly related to its amino acid composition. Some of these amino acids, termed essential amino acids, cannot be synthesized by humans and therefore must be supplied in the diet for adults and in particular for infants and children. Lysine is an essential amino acid synthesized via the aspartic acid metabolic pathway, in which threonine, methionine and isoleucine are also endproducts. Moreover, lysine is the first limiting amino acid in all cereal grains. For over 30 years, the aspartic acid metabolic pathway has been studied in higher plants with the aim of identifying and characterizing the key regulatory points controlling the biosynthetic pathway. Two clear distinct time periods, one begining with the development of tissue culture techniques (1970-80's) and the second with the development of plant transformation techniques (90's), has encouraged the production of biochemical mutants and transgenic plants with specific alterations in key enzymes of the pathway, leading to the overproduction and accumulation of threonine in all plant tissues. However, the accumulation of lysine in seeds has been particularly difficult to achieve. Such an observation, associated with the recent biochemical studies on lysine degradation in cereal and legume plant species, has indicated that the manipulation of lysine degradation is as important as the manipulation of lysine synthesis, if the goal of accumulating this amino acid in cereal seeds is to be achieved. In maize, the study and use of other mutants such as the opaque-2 and QPM (Quality Protein Maize) varieties, has contributed significantly to our understanding of the regulatory aspects of the aspartate pathway. The strategies of obtaining cereals rich in lysine and their relevance to the manipulation of other amino acids have been discussed

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

<p>Abstract</p> <p>Background</p> <p>The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally.</p> <p>Results</p> <p>We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 μg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-γ and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 μg).</p> <p>Conclusion</p> <p>Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.</p

LKR/SDH Plays Important Roles throughout the Tick Life Cycle Including a Long Starvation Period

BACKGROUND:Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. CONCLUSIONS/SIGNIFICANCE:Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods