Xylella fastidiosa gene expression analysis by DNA microarrays

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Aspectos atuais sobre aminoácidos de cadeia ramificada e exercício físico

Em humanos saudáveis, nove aminoácidos são considerados essenciais, uma vez que não podem ser sintetizados endogenamente e, portanto, devem ser ingeridos por meio da dieta. Dentre os aminoácidos essenciais, se incluem os três aminoácidos de cadeia ramificada, ou seja, leucina, valina e isoleucina. Esses aminoácidos participam da regulação do balanço protéico corporal além de serem fonte de nitrogênio para a síntese de alanina e glutamina. No tocante à regulação da síntese protéica muscular, verifica-se que a leucina age estimulando a fase de iniciação da tradução do RNA-mensageiro em proteína, por mecanismos tanto dependentes quanto independentes de insulina. No que concerne ao exercício físico, supõe-se que esses aminoácidos estejam envolvidos na fadiga central, no balanço protéico muscular, na secreção de insulina, na modulação da imunocompetência, no aumento da performance de indivíduos que se exercitam em ambientes quentes e na diminuição do grau de lesão muscular. Nesse contexto, essa revisão aborda os aspectos atuais do metabolismo e da suplementação de aminoácidos de cadeia ramificada no exercício físico.

Suplementação enteral e parenteral com glutamina em neonatos pré-termo e com baixo peso ao nascer

A glutamina é o aminoácido livre mais abundante no sangue e no músculo esquelético, bem como é o principal substrato energético para células de elevado turnover, como enterócitos e leucócitos. Adicionalmente, a glutamina representa o principal aminoácido transferido para o feto pela placenta e, juntamente com o glutamato, constituem os aminoácidos mais abundantes no leite materno. Todavia, bebês nascidos prematuramente sofrem interrupção abrupta do fornecimento placentário de glutamina, o que acarreta em dependência exclusiva da síntese endógena ou do fornecimento exógeno deste aminoácido. Aliado a isso, neonatos pré-termo (PT) e com baixo peso ao nascer (BPN), freqüentemente, recebem apenas nutrição parenteral total nas primeiras semanas de vida, a qual não contém glutamina. Cabe ainda destacar que esses bebês possuem pouca massa muscular e, portanto, seus estoques de glutamina são limitados. Uma vez que neonatos PT e com BPN estão sujeitos a intenso crescimento e a inúmeros estresses fisiológicos, é possível que a glutamina seja um nutriente condicionalmente essencial nessa fase da vida, fato que estimulou a realização de estudos com a finalidade de avaliar os possíveis benefícios clínicos da suplementação enteral e parenteral com glutamina em neonatos PT e com BPN.

Composição química do feijão caupi (Vigna unguiculata L. Walp), cultivar BRS-Milênio

O feijão caupi é uma das principais culturas alimentares do Nordeste brasileiro. Para melhorar sua resistência contra pragas e produtividade, a nova cultivar (BRS-Milênio) foi obtida por melhoramento genético. Este trabalho objetivou caracterizar o potencial nutricional desta variedade, determinando-se a composição centesimal, o perfil de aminoácidos e ácidos graxos, o conteúdo de minerais e a atividade inibitória de tripsina. A semente contém (g.100 g-1): 24,5 de proteínas; 51,4 de carboidratos; 16,6 de fibra insolúvel e 2,7 de fibra solúvel; 2,6 de cinzas, tendo como principais minerais (mg.100 g-1): ferro - 6,8; zinco - 4,1; manganês - 1,5; fósforo - 510,0 e potássio - 1430,0. O teor de lipídios foi de 2,2%, e seu perfil de ácidos graxos apresenta 29,4% de saturados e 70,7% de insaturados. Foi estimado valor calórico de 323,4 kcal.100 g-1 de semente. Encontrou-se baixa atividade inibitória de tripsina (8,0 UIT.mg-1 de amostra). O perfil de aminoácidos foi favorável ao padrão de referência, exceto pela deficiência de sulfurados, sugerindo a necessidade da combinação desta leguminosa com outras fontes alimentares. Os resultados indicam que o feijão caupi possui atributos desejáveis como altos teores de energia, proteínas, fibras e minerais, baixa atividade inibitória de tripsina e, apesar de reduzido conteúdo lipídico, contém alta proporção de ácidos graxos insaturados.

Apparent digestibility of diets with different concentrations of lysine and energy in piglets with different body weights and post-weaning age

The effects of body weight or age and dietary digestible lysine and metabolizable energy on apparent digestibility of energy and dry matter were evaluated in piglets after weaning. The animals were weaned at 21 days of age and distributed in two groups: 8.68 ± 0.76 kg at 28 days of age (weaned 7 days earlier); and 12.73 ± 0.99 kg at 35 days of age (weaned 14 days earlier). The pigs were allotted in digestibility cages in a completely randomized block design with the following factorial arrangements: 2 × 4 composed of two weight categories and four levels of digestible lysine (1.222; 1.305; 1.390 and 1.497%); and 2 × 3 composed of two weight categories and three levels of metabolizable energy (3,510; 3,700 and 3,830 kcal/kg rations). Digestible lysine was evaluated in six replications and metabolizable energy in eight replications and each animal constituted an experimental unit. Piglets with higher body weight and age were more efficient in nitrogen retention and energetic balance, compared to lighter and younger piglets, particularly those given lower concentration of lysine in the diet. The energy increase favored nitrogen retention by the heavier and older piglets. However, coefficients of dry matter and energy apparent digestibility did not differ among weight categories. Older and heavier piglets were more efficient in nitrogen retention, although this efficacy depended on concentration of the energy in the diet. This better use of protein and energy suggest differences on nutritional requirements.

Dietary levels of lysine for male broilers from 23 to 36 days of age: performance and body composition

One thousand and fifty (1,050) Ross male broilers from 23 to 36 days of age were used to evaluate the dietary effects of levels of lysine. It was used a random block design with 5 levels of digestible lysine (0.95; 1.00; 1.05; 1.10 and 1.15%). Each level was applied to seven replicates and the experimental unit was composed of 30 birds. Diets were offered in seven replicates and the experimental unit was represented by 30 birds. Weight gain, feed intake, feed conversion, carcass traits, cut yields, composition and deposition of body nutrients were evaluated. The concentration of 1.09% of digestible lysine optimized performance without significant effects on feed intake. Carcass traits and cut yields had no significant differences among levels of lysine. On the other hand, there was a quadratic effect on carcass weight, which was the best at the level of 1.07% of digestible lysine. On crude protein content of the empty body, the best response was observed at the level of 1.10% of dietary lysine. This same level corresponded to a quadratic response in the deposition of water from carcass and empty body. The level of digestible lysine for better performance, chemical composition and body nutrient deposition in broilers from 23 to 36 days of age is between 1.07 and 1.10%.

Lisina digestível para frangos de corte machos entre 12 e 22 dias de idade

Avaliou-se nível de lisina digestível para 1050 frangos de corte dos 12 aos 22 dias de idade. O delineamento experimental foi inteiramente ao acaso, com cinco tratamentos, sete repetições e 30 aves por unidade experimental. Os tratamentos foram: 1,05; 1,10; 1,15; 1,20 e 1,25% de lisina digestível. Avaliaram-se ganho de peso, consumo de ração, conversão alimentar, composição e deposição de nutrientes corporais. Foram constatados efeitos quadráticos de lisina digestível no consumo de ração e resposta linear ascendente no peso da carcaça. Na composição química da carcaça, houve resposta quadrática do nível de lisina na concentração de proteína. As taxas de deposição proteica, deposição de água, da carcaça e do corpo total tiveram aumento linear em resposta ao acréscimo de lisina na dieta. O aumento da concentração de lisina, todavia, coincidiu com a redução da matéria mineral nas vísceras e sangue e no corpo total. Considerado o desempenho, o nível 1,1% de lisina digestível atendeu às necessidades do frango de corte entre o 12º e o 22º dia de idade. Consideradas a composição química e as taxas de deposição dos nutrientes corporais, a demanda pelo aminoácido digestível torna-se igual ou maior que 1,25%

Digestible lysine and organic zinc for male broiler from 1 to 11 days of age: performance and body composition

This study evaluated levels of digestible lysine and organic zinc for male Ross strain broilers from 1 to 11 days of age. It was used 1,050 chicks distributed in randomized block design, in 5 × 2 factorial scheme, with seven repetitions of 15 birds per experimental unit. The dietary concentrations of digestible lysine were 0.90; 1.00; 1.10; 1.20; and 1.40% combined with 43 and 253 ppm zinc chelate. The diets contained 2,965 ± 18 kcal/kg of apparent metabolizable energy (AME) and 21.48 ± 0.18% of CP. It was determined chemical composition, protein, lipid, mineral and water depositions on carcass and empty body. There was no interaction among the factors digestible lysine and organic zinc. Effect of zinc concentration increase was observed on greatest deposition of body fat, indicating that there is interference in lipid metabolism of the birds in the studied phase. The most pronounced effects resulted from the dietary inclusion of lysine. There was a linear effect on reconstituted body weight as a response to the increase of dietary lysine, which suggests equal or superior requirement to the greatest studied level. However, the deposition of water and protein on the carcass had a quadratic increase, characterizing higher muscle mass accumulation up to the levels 1.25 and 1.27 of this amino acid in the diet. Considering the studied strain, broiler chickens from the first to the 11th days of age require 1.28 ± 0.01% of digestible lysine, according to the deposition of muscle mass.

Lysine and zinc chelate in diets for brown laying hens: effects on egg production and composition

It was evaluated the effects of levels of digestible lysine and chelate zinc combined in the diet for laying on the egg quality. It was used 720 birds, from 48 to 60 weeks of age, distributed in a completely randomized design in a 3 × 5 factorial scheme with three levels of zinc and five levels of lysine, applied into six replicates in the experimental units of eight birds per plot. The levels were: 137, 309 and 655 ppm zinc and 0.482, 0.527, 0.582, 0.644 and 0.732% digestible lysine. It was not observed any interaction among digestible lysine and zinc for the primary variables of fractions and egg composition. Levels of zinc reduced egg weight, suggesting the lowest efficiency in nutrient intake. At the highest dietary concentration of zinc, the addition of digestible lysine coincided with a linear increase in shell weight. However, zinc addition, regardless of lysine level in the diet, resulted in the reduction of egg weight and of the percentage of mineral matter in the yolk, limiting the efficiency of mineral deposition in this fraction of the egg. Concentration of zinc that produced the best results was 137 ppm inasmuch as higher quantities limit the use of digestible lysine, with effects harming composition and egg quality. The study indicates the following requirement for digestible lysine: 0.639% from the 48th to the 52nd week, 0.679% from the 52nd to 56th week, and 0.635% from the 56th to the 60th week. Considering the total period from 48th to the 60th week, the level 0.638% of lysine or the daily intake of 707 mg of the amino acid met the requirement for egg quality of semi-heavy layers used in this study.

Níveis de lisina digestível e zinco quelato sobre os parâmetros produtivos de poedeiras marrons

Objetivou-se avaliar a produção de ovos em galinhas Isa Brown alimentadas com dietas com diferentes níveis de lisina digestível e zinco quelato. Utilizaram-se 720 poedeiras marrons, em dois períodos de avaliação, de 24 a 36 e de 48 a 60 semanas de idade. As dietas foram avaliadas em delineamento inteiramente casualizado, em esquema fatorial 5 × 3, composto de 5 níveis de lisina digestível (0,482; 0,527; 0,582; 0,644 e 0,732%) e 3 de zinco quelato (20, 40 e 80 ppm), de modo que cada combinação foi testada em 6 repetições de 8 aves por unidade experimental. Na fase de 24 a 36 semanas de idade, houve influência da interação de lisina digestível e zinco no consumo médio diário de ração e na porcentagem de postura. Na fase de 48 a 60 semanas, a interação foi evidenciada somente no consumo médio diário de ração. O aumento da concentração de lisina digestível favoreceu o consumo de ração em todos os níveis de zinco. Em ambas as fases, a menor concentração de zinco atende às necessidades de produção das aves. O valor médio estimado de lisina digestível ideal foi de 0,732% para a fase de 24 a 36 semanas e de 0,578% para a fase de 48 a 60 semanas de idade.

Effect of protein intake on bone and muscle mass in the elderly

The aging process is frequently characterized by an involuntary loss of muscle (sarcopenia) and bone (osteoporosis) mass. Both chronic diseases are associated with decreased metabolic rate, increased risk of falls fracture, and, as a result, increased morbidity and loss of independence in the elderly. The quality and quantity of protein intake affects bone and muscle mass in several ways and there is evidence that increased essential amino acid or protein availability can enhance muscle protein synthesis and anabolism, as well as improve bone homeostasis in older subjects. A thorough evaluation of renal function is important, since renal function decreases with age. Finally, protein and calcium intake should be considered in the prevention or treatment of the chronic diseases osteoporosis and sarcopenia

Changes in Glucose and Glutamine Lymphocyte Metabolisms Induced by Type I Interferon alpha

In lymphocytes (LY), the well-documented antiproliferative effects of IFN-alpha are associated with inhibition of protein synthesis, decreased amino acid incorporation, and cell cycle arrest. However, the effects of this cytokine on the metabolism of glucose and glutamine in these cells have not been well investigated. Thus, mesenteric and spleen LY of male Wistar rats were cultured in the presence or absence of IFN-alpha, and the changes on glucose and glutamine metabolisms were investigated. The reduced proliferation of mesenteric LY was accompanied by a reduction in glucose total consumption (35%), aerobic glucose metabolism (55%), maximal activity of glucose-6-phosphate dehydrogenase (49%), citrate synthase activity (34%), total glutamine consumption (30%), aerobic glutamine consumption (20.3%) and glutaminase activity (56%). In LY isolated from spleen, IFN alpha also reduced the proliferation and impaired metabolism. These data demonstrate that in LY, the antiproliferative effects of IFN alpha are associated with a reduction in glucose and glutamine metabolisms.

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.