A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage.

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase. One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.

Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.

The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Bidirectional uncoating of the genomic RNA of a helical virus.

An essential step in the initiation of a virus infection is the release of the viral genome from the other constituents of the virus particle, a process referred to as uncoating. We have used reverse transcription and polymerase chain reaction amplification procedures to determine the rate and direction of in vivo uncoating of the rod-shaped tobacco mosaic virus. The virus particles contain a single 6.4-kb RNA molecule that lies between successive turns of a helical arrangement of coat protein subunits. When the particles are introduced into plant cells, the subunits are removed via a bidirectional uncoating mechanism. Within 2-3 min, the part of the viral RNA from the 5' end to a position >70% toward the 3' end has been freed of coat protein subunits. This is followed by removal of subunits from the 3' end of the RNA and sequential uncoating of the RNA in a 3'-to-5' direction. An internal region of the viral RNA is the final part to be uncoated. Progeny virus particles are detected in the cells 35-40 min after inoculation.

Inhibition of a plant virus infection by analogs of melittin.

An approach that enables identification of specific synthetic peptide inhibitors of plant viral infection is reported. Synthetic analogs of melittin that have sequence and structural similarities to an essential domain of tobacco mosaic virus coat protein were found to possess highly specific antiviral activity. This approach involves modification of residues located at positions analogous to those that are critical for virus assembly. The degree of inhibition found correlates well with sequence similarities between the viral capsid protein and the melittin analogs studied as well as with the induced conformational changes that result upon interaction of the peptides and ribonucleic acid.

Towards the preparative and large-scale precision manafacture of virus-like particles

Virus-like particles (VLPs) are of interest in vaccination, gene therapy and drug delivery, but their potential has yet to be fully realized. This is because existing laboratory processes, when scaled, do not easily give a compositionally and architecturally consistent product. Research suggests that new process routes might ultimately be based on chemical processing by self-assembly, involving the precision manufacture of precursor capsomeres followed by in vitro VLP self-assembly and scale-up to required levels. A synergistic interaction of biomolecular design and bioprocess engineering (i.e. biomolecular engineering) is required if these alternative process routes and, thus, the promise of new VLP products, are to be realized.

Forced retroevolution of an RNA bacteriophage

The operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes. Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation. The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages. The evolutionary reconstruction of the operator failed in the majority of mutants. Instead, a poor imitation developed containing only some of the recognition signals for the coat protein. Three mutants were of particular interest in that they contained double nonsense codons in the lysis reading frame that runs through the operator hairpin. The simultaneous reversion of two stop codons into sense codons has a very low probability of occurring. Therefore the phage solved the problem by deleting the nonsense signals and, in fact, the complete operator, except for the initiation codon of the replicase gene. Several revertants were isolated with activities ranging from 1% to 20% of wild type. The operator, long thought to be a critical regulator, now appears to be a dispensable element. In addition, the results indicate how RNA viruses can be forced to step back to an attenuated form.

Desempenho de cabras leiteiras alimentadas com diferentes espécies de cactáceas

The objective of this research was to evaluate the production of dairy goats fed different species of cactus. Pluriparous five Saanen goats were used, with nine weeks of lactation, and average live weight of 50 kg ± 4 kg. The animals were distributed in latin square design (5x5) with 5 diets and 5 periods. No differences (P>0,05) were observed in the DM of the experimental diets by getting average values of 2.251,84 g dia-1 , 4,46 %PV e 118,91g kg0,75 . The DM contents of the diets were 50,55 to 55,92% by presenting a maximum variation of 10% between them. A significant difference (P<0,05) water consumption way tendered, between diets with different cactus species. The treatments cactus “Orelha de Elefante Mexicana” and Facheiro had lower water consumption compared to cactus “Palma Miúda. For milk yield no significant difference (P> 0,05) between diets formulated with cactus species, with an average of 1,90 kg/day treatments. The analysis of variance show a significant difference (P<0,05) among treatments for milk corrected to 4% fat and fat production. There was an effect (P<0,05) of the diets with different cactus on the crude protein (CP) and lactose in milk. All treatments with different cactus species can be used for dairy goats in view consumption have afforded sufficient to meet the nutritional requirements for milk nutrients, besides presenting the higher than levels of physical and chemical composition to minimum levels established by current legislation

Desempenho agronômico, bromatológico e estabilidade fenotípica de sorgo silageiro em Uberlândia - MG

Sorghum (Sorghum bicolor (L.) Moench) is a good alternative to be used as silage, especially in places with water scarcity and high temperatures, due to their morphological and physiological characteristics. The appropriate management, as the ideal seeding time, interferes both productivity and the quality of silage. The work was conducted with the objective of evaluating the agronomic and bromatological performance of varieties and hybrids of silage sorghum and their phenotypic stability in two seasons, season and off-season, in the city of Uberlândia, Minas Gerais. The experiments were performed at Capim Branco Experimental Farm of Federal University of Uberlândia (UFU), located in the referred city. There were two sowing dates in the same experimental area, off-season (March to June 2014) and season (November 2014 to March 2015), and the varieties and hybrids were evaluated in both situations. The design was a randomized block with 25 treatments (hybrids and varieties of sorghum) and three replications. Agronomical and bromatological data were subjected to an analysis of variance; averages were grouped by Scott-Knott test at 5% of probability, through Genes computer program; and to estimate the stability, it was opted for Annicchiarico method. The flowering of cultivars, dry matter productivity, plant height, Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF) and Crude Protein (CP) are affected by the environment and the variety. Regarding productivity and quality of the fiber, SF11 variety was superior, independent of the rated environment. In relation to the performance stability of dry matter, the varieties SF15, SF11, SF25, PROG 134 IPA, 1141572, 1141570 and 1141562 were highlighted. For the stability of the quality of fibers (FDA and FDN), the variety 1141562 stood out. The environment reduces the expression of characters “days of flowering”, “plant height” and “productivity of dry matter of hybrids”. From the 25 hybrids analyzed for productivity and stability of dry matter performance, seven were highlighted, regardless of the rated environment: Volumax commercial hybrid and experiments 12F39006, 12F39007, 12F37014, 12F39014, 12F38009 and 12F02006.

Silagem do bagaço de sorgo sacarino

Brazilian sweet sorghum is used to generate ethanol and the bagasse is burned in industrial boilers or deposited on soil polluting the environment. This study evaluated the performance of sorghum plants and its bagasse silage nutritional value aiming to use it in the ruminant nutrition. Experiments were set up on the UTFPR campus at Dois Vizinhos-PR. The first trial was established on October 2nd, 2012 using the genotypes ADV 2010, Hunnigreen, Sugargraze, Volumax, BR 505, 503, 501 and the second trial at 2013 on November 27th assessing the materials ADV 2010, Sugargraze, Hunnigreen, EX 5110, BR 506, 508, 509 and 511. Experimental was laid out as a randomized block design with three replications. Results were analyzed through ANOVA comparing the averages by Duncan test at 5% error probability. As field variables were evaluated: plant height (Pl hei), green mass production (GM Prod), percentage of leaves, stems and panicles in relation to the plant, stems production without straw (Prod stems with straw), whole stems production (Who stems Prod), stem diameter (Stem diam), juice production (Juice prod) and Brix degree (oBrix). After juice plant extraction, forage bagasse was crushed and packed in silos for 60 days and green matter yield was estimated (GM). In bagasse silage gauged to buffer capacity (BC), dry matter (DM), mineral matter (MM), crude protein (CP), ether extract (EE), total carbohydrates (TC), non-fibrous carbohydrates (NFCH), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin (LIG ) and digestibility "in vitro" (DIGIV). There was no significant difference (P> 0.05) between genotypes regarding to green matter production and on average, the second crop yield was lower and reflected in the reduction of bagasse production. Bagasse silage DM was of 32.3% and 33.1%; NDF 73% and 65.8%, crude protein 3.8% and 5.9; pH 3.7 and 3.7; TC and 9.8 and 10.7. mg MS-1; the amount of NFCH was 11.1 and 13.5%; DIG of DM 36.9 and 62.4% for the respectively to the bagasse produced from materials grown in the 2012/2013 and 2013/2014 seasons. Hybrid genotypes had a better agronomic performance while the varieties were more efficient in bromatological indexes. And despite the high percentage of NDF and the low protein level, it is possible to feed ruminants with this coproduct.

Grazing behavior of lactating Murrah buffalo cows supplemented with cupuassu byproduct.

The goal of this work was to evaluate grazing behavior of lactating Murrah buffalo cows supplemented with cupuassu byproduct containing 16.8, 51.4, 8.3, 83.5 and 3.4% of ether extract (EE), neutral detergent fiber (NDF), crude protein (CP), total digestible nutrients (TDN) and nonfibrous carbohydrate (NFC), respectively. Experimental rations were balanced for reaching levels of 22.0 % of crude protein (CP) and 80.1 % of total digestible nutrient (TDN).

Milk composition of Murrah buffalo cows supplemented with cupuassu byproduct in Western Amazon.

The aim of this study was to evaluate milk composition of Murrah buffalo cows supplemented with rations containing cupuassu byproduct in substitution of ground corn. Isoprotein and isoenergy rations were balanced with 22.0% of crude protein (CP) and 80.1% of total digestible nutrient (TDN).

Detection of Rupestris stem pitting associated virus in seedlings of virus - infected maternal grapevine plants.

Rupestris stem pitting associated virus (RSPaV) is a species in the genus Foveavirus (Martelli and Jelkman, 1998) and the family Flexiviridae. The virion has a positive sense, single stranded, polyadenylated RNA genome of 8.7kb in size and a coat protein of 28kD (Martelli and Jelkman, 1998). The virus has been reported to be present in pollen (Rowhani et aI., 2000) and seeds (Stewart and Nassuth, 2001), however, it has not been proved to be seed-transmitted. In our investigation reported here we have proven that RSPaV transmits by seed from RSPaV-infected mother plants to their siblings.

Desempenho produtivo de Pseudoplatystoma corruscans estocados em sistemas de criação: semi-intensivo (viveiro escavado) e intensivo (tanque-rede)

Objetivou-se comparar o desempenho produtivo e custos de produção de exemplares de pintado, Pseudoplatystoma corruscans, estocados em dois sistemas de criação: semi-intensivo (viveiro escavado, VE) e intensivo (tanque-rede, TR). Trezentos (300) peixes, com um ano de idade, foram estocados, sendo 150 em um VE (médias de peso e comprimento: 1,48±0,46kg e 57,31±6,42cm) e 150 divididos em três TR (médias de peso e comprimento: 1,27±0,34kg e 55,05±4,11cm). Foram alimentados com ração extrusada de 15mm (diâmetro) 40% PB e 3110kcal ED kg-1, ajustada mensalmente à quantidade de ração. Os parâmetros físico-químicos da água, observados durante o experimento, foram temperatura = 24,08°C±3,23; pH=6,89±0,39 e oxigênio dissolvido = 7,57±0,97mg L-1. Os reultados obtidos dos valores médios finais dos comprimentos (VE=74,07±4,34cm; TR=70,33±5,02cm) e pesos dos peixes (VE=3,41±0,58kg e TR=2,94±0,60kg) indicaram desempenho semelhante nos dois sistemas. As médias do fator de condição (0,09-0,036); ganho em peso diário (9,29g dia-1 - 8,95g dia-1); conversão alimentar (3,09-4,15); consumo total de ração (29,60g dia-1 - 74,16g dia-1); índice de crescimento (0,219-0,215) e sobrevivência (97,33-90,67%) para VE e TR, respectivamente. Houve interação significativa entre os sistemas de criação e mês (P<0,05). O quilo de peixe produzido foi de R$ 8,76 (US$ 2,85) e R$ 8,73 (US$ 2,33) para o VE e TR, respectivamente. Embora o VE tenha demonstrado melhor desenvolvimento durante o período e uma vantagem econômica, o índice de crescimento mostrou que ambos os sistemas tiveram desempenhos semelhantes.

Processo de maturação das gônadas de pintado (Pseudoplatystoma corruscans) alimentado com dois níveis proteicos e suplementados com óleo de milho

Investigou-se o efeito da adição de C18:2n6, por meio da inclusão de óleo de milho em dietas com dois níveis de proteína bruta, sobre o processo de maturação de gametas de pintado, Pseudoplatystoma corruscans, mantidos em tanques-rede. Foram avaliados: taxa de sobrevivência, relação peso x comprimento, fator de condição (K) e índice gonadossomático (IGS). O experimento foi realizado entre março de 2004 e fevereiro de 2006, em 12 tanques-rede, distribuídos em seis viveiros-escavados de 600m² e densidade de estocagem de 20 peixes/tanque-rede. Utilizaram-se três tratamentos (T) com duas repetições/viveiro: T1 com 28% de PB; T2 com 28% de PB + 5% óleo de milho e T3 com 40% de PB. O crescimento foi ligeiramente mais alto nos peixes do T3. As taxas de sobrevivência foram acima de 77%. Pode-se inferir que as rações ofertadas não causaram alterações histomorfológicas durante o processo de maturação gonadal dessa espécie. O IGS e o K foram ligeiramente mais altos nos animais alimentados com a ração enriquecida com óleo de milho.