ABSTRACT. Current literature is lacking regarding the vegetative propagation of the cambuci tree (Campomanesia phaea O. Berg Landrum). This study aimed to verify the efficiency of cutting techniques via the assessment of cuttings of varying types and sizes, sampling dates, and the influence of treatments containing plant growth regulator indolebutyric acid (IBA) combined with the antioxidant ascorbic acid across different immersion times. The species was found not to develop adventitious roots easily, and hence, the herbaceous and woody stem materials were subjected to histochemical studies to elucidate their anatomical and physiological processes. To this end, plant material was polymerized in glycol methacrylate resin, cut into sections, and stained using toluidine blue (0.05%) or via histochemical staining with PAS/naphthol blue black. Anatomical analysis of plant structures revealed the presence of phenolic compounds and sclerenchyma tissue, which in turn are expected to negatively impact the development of adventitious roots.

Resumo O objetivo deste trabalho foi avaliar os aspectos qualitativos, na colheita e na pós-colheita, de mirtilos 'Emerald' cultivados em região de clima subtropical, sem frio hibernal, no Brasil. Os frutos foram colhidos no município de Piracicaba, no estado de São Paulo, em dois picos de colheita, em agosto e outubro, e avaliados quanto aos seus atributos qualitativos na colheita e aos seus atributos físicos, químicos e bioquímicos na pós-colheita. Os frutos foram mantidos em condições de temperatura ambiente, a 22°C e 70% de umidade relativa, e analisados no dia da colheita e a cada três dias até o décimo-segundo dia de armazenamento. Os mirtilos colhidos em agosto foram maiores e mais arredondados, e os colhidos em outubro apresentaram coloração azul mais intensa e maiores concentrações de compostos fenólicos, pH e sólidos solúveis, além de maior relação sólidos solúveis totais e acidez titulável. Durante o armazenamento, observou-se aumento no conteúdo de antocianinas, quercetinas e compostos fenólicos totais, bem como na atividade antioxidante, além de redução na acidez e na firmeza dos frutos. Independentemente do mês de colheita, os mirtilos cultivados em região subtropical no Brasil apresentam boa durabilidade pós-colheita por até 12 dias em temperatura ambiente, com níveis satisfatórios de qualidade dos frutos.

Abstract The objective of this work was to evaluate the qualitative attributes, at harvest and postharvest, of 'Emerald' blueberries grown in a subtropical climate region, without winter chilling, in Brazil. Fruit were harvested in the municipality of Piracicaba, in the state of São Paulo, in two harvest peaks, in August and October, and evaluated for their qualitative attributes at harvest and for their physical, chemical, and biochemical attributes at postharvest. The fruit were maintained at ambient conditions, at 22°C and 70% relative humidity, and evaluated on the day of harvest and every three days until the twelfth day of storage. The blueberries harvested in August were larger and rounder, and those harvested in October had a more intense blue coloration and higher concentrations of phenolic compounds, pH, and soluble solids, in addition to a higher total soluble solids and titratable acidity ratio. During storage, an increase was observed in the contents of anthocyanins, quercetins, and total phenolic compounds, as well as in antioxidant activity, besides a decrease in fruit acidity and firmness. Regardless of the harvest month, blueberries grown in a subtropical region of Brazil have a good postharvest shelf life up to 12 days at ambient temperature, with satisfactory fruit quality levels.

Resumo O objetivo deste trabalho foi avaliar a alporquia, a estaquia e a enxertia, com diferentes doses de ácido indolbutírico (AIB), para a propagação vegetativa de Eugenia involucrata. A alporquia foi realizada por dois anos consecutivos, com cinco diferentes doses de AIB. A estaquia foi testada com estacas lenhosas e herbáceas, quatro doses de AIB, em três estações do ano. Os métodos de enxertia – garfagem em fenda cheia e inglês simples – foram avaliados em duas estações do ano. Para os ensaios de alporquia, estaquia e enxertia, os delineamentos experimentais foram em blocos ao acaso, inteiramente casualizado em arranjo fatorial 3x4 e inteiramente casualizado, respectivamente. As plantas foram avaliadas quanto ao enraizamento, à percentagem de sobrevivência e ao desenvolvimento da parte aérea. Na alporquia, foram observadas baixas taxas de enraizamento foram observadas na alporquia de 0 a 50%, o uso de AIB não apresentou resultados consistentes e nenhum propágulo resultante sobreviveu após o transplante. A técnica de estaquia mostrou baixas taxas de sobrevivência, enraizamento e retenção foliar, sem efeito consistente das doses de AIB. A enxertia mostrou de 35 a 50% de fixação do enxerto, sem diferença significativa entre as técnicas de enxerto de emenda e de fenda. As técnicas de alporquia e estaquia não são eficientes para a propagação de E. involucrata, pois não favorecem a sobrevivência e o enraizamento das mudas. A enxertia, por ambos os métodos, é a técnica mais eficiente para a propagação de E. involucrata.

Abstract The objective of this work was to evaluate air layering, cutting, and grafting, with different indolebutyric acid (IBA) rates, for the vegetative propagation of Eugenia involucrata. Air layering was carried out for two consecutive years with five different IBA rates. Cutting was tested for woody and herbaceous cuttings, using four IBA rates, in three seasons. The grafting methods – cleft graft and splice graft – were tested in two seasons. For the air layering, cutting, and grafting trials the experimental designs were randomized complete blocks, completely randomized in a 3x4 factorial arrangement, and completely randomized, respectively. Plants were evaluated for rooting, survival percentage, and shoot development. In air layering, low rooting rates of 0 to 50% were observed, the use of IBA showed no consistent results, and no resulting propagules survived after being transplanted. The cutting technique showed low survival, rooting, and leaf retention rates, with no consistent effect of IBA rates. Grafting showed 35 to 50% graft fixation, with no significant difference between the splice and cleft grafting techniques. The air layering and cutting techniques are not efficient for the propagation of E. involucrata, as they do not favor the survival and rooting of seedlings. Grafting, by both techniques, is the most efficient method for E. involucrata propagation.

Resumo: O objetivo deste trabalho foi avaliar as técnicas de alporquia e enxertia na produção de plantas de cambucizeiro (Campomanesia phaea). Foram conduzidos dois experimentos em blocos ao acaso para alporquia, com plantas adultas de cambuci, e dois inteiramente casualizados para a enxertia, utilizando mudas. A alporquia foi avaliada com diferentes doses de ácido indolbutírico, e as técnicas do tipo inglês simples e fenda lateral foram utilizadas para a enxertia. O enraizamento dos alporques não foi favorecido pela aplicação de doses de ácido indolbutírico, enquanto a enxertia do tipo fenda lateral garantiu a fixação de 6% dos enxertos. Análises histológicas dos alporques revelaram atividade das células parenquimáticas do xilema na formação de estruturas similares a calos. Nas plantas enxertadas, essas células garantiram a união e a regeneração dos tecidos. A técnica da alporquia foi inviável, mas a enxertia é recomendada para fixação de genótipos de cambucizeiro.

Abstract: The objective of this work was to evaluate the air-layering and grafting techniques in the production of cambuci (Campomanesia phaea) plants. Two experiments were conducted in randomized complete blocks for air-layering, using adult cambuci plants, and two completely randomized for the grafting, using seedlings. Air layering was evaluated with different rates of indolebutyric acid; the splice, side-veneer, and chip-budding techniques were used for grafting. Air-layering rooting was not favored by the application of indolebutyric acid rates, whereas side-veneer grafts ensured the fixation of 6% of the grafts. Histological analyses of the air layers revealed the action of parenchyma cells from the xylem in the formation of structures similar to calluses. In the grafted plants, these cells ensured tissue union and regeneration. The air-layering technique was unfeasible, but grafting is recommended to establish cambuci tree genotypes.

O hiperadrenocorticismo é uma das endocrinopatias mais comuns em cães, sendo caracterizado pela exposição excessiva de glicocorticóides secretados pelas adrenais. A hipercortisolemia crônica pode promover várias complicações, incluindo hipertensão sistêmica e glomerulonefrite. A glomerulonefrite pode desencadear variáveis graus de proteinúria e uma tendência de evolução para doença renal crônica. A perda de proteínas na urina, principalmente da albumina, é uma característica das doenças glomerulares e a determinação de variáveis laboratoriais, como a razão proteína:creatinina urinária (RPC), albuminúria (teste de ELISA) e eletroforese das proteínas urinárias, são recomendadas para a elucidação do diagnóstico. Assim, o objetivo do estudo é avaliar a relação entre proteinúria e hipertensão arterial sistêmica em cães com hiperadrenocorticismo e verificar, pela avaliação da albuminúria e do peso molecular das proteínas urinárias, o segmento do néfron que foi comprometido ou lesado. Foram avaliados 30 cães com diagnóstico de hiperadrenocorticismo, subdivididos em 13 cães com hipertensão arterial sistêmica (grupo I) e 17 cães normotensos (grupo II). Foram determinados a RPC; a albuminúria pela avaliação da albumina normalizada e razão albumina:creatinina urinária (RAC) e a eletroforese de proteínas pela técnica em gel de poliacrilamida, contendo dodecil sulfato de sódio (SDS-PAGE). Os resultados foram comparados com os dados obtidos de 30 cães clinicamente saudáveis. Foi constatado que não houve influência da hipertensão arterial sistêmica nos cães com hiperadrenocorticismo em relação à quantificação da albuminúria, determinada pelo método ELISA, e nem na qualidade e quantidade das bandas de proteínas de baixo (<60 kDa) e de alto peso molecular (>60 kDa). No entanto foi determinado que cães com hiperadrenocorticismo podem desenvolver lesões glomerulares e tubulares, caracterizadas pela presença de albuminúria e de proteínas de alto e de baixo pesos moleculares, independentemente da presença de hipertensão arterial sistêmica. Conclui-se que a avaliação quantitativa (RPC e RAC) e qualitativa (SDS-PAGE) das proteínas urinárias traz informações adicionais que indicam os possíveis segmentos comprometidos dos néfrons que causaram as perdas de proteínas na urina.

Hyperadrenocorticism is one of the commonest endocrinopathies in dogs, and it is characterized by the excessive exposure of glucocorticoids excreted by adrenals. Chronic hypercortisolemia may promote several complications, including systemic hypertension and glomerulonephritis. Glomerulonephritis may initiate several variable degrees of proteinuria and leading to the development of chronic kidney disease. The loss of proteins through urine, mainly predominant albumin, is a characteristic of glomerular diseases and the determination of laboratorial variables, such as the urinary protein-to- creatinine ratio (UPC), urinary albumin-to-creatinine ratio (UAC; ELISA test) and electrophoresis of urinary proteins are recommended to elucidate the diagnosis. Therefore, the goal of this study is to evaluate the relationship between proteinuria and systemic arterial hypertension in dogs with hyperadrenocorticism and to determine through evaluation of albuminuria and molecular weight of urinary proteins, the segment of the nephron that could be damaged. Thirty dogs with hyperadrenocorticism were evaluated and subdivided into groups; 13 dogs with systemic arterial hypertension (group I) and 17 normotensive (group II). The UPC was determined, as well as UAC and the urine protein electrophoresis by polyacrylamide gel technique, containing dodecyl sodium sulphate (SDS-PAGE). The results were compared with data obtained from 30 clinically healthy dogs. No association between systemic arterial hypertension and albuminuria was detected in dogs with hyperadrenocorticism as well as no alterations of proteins patterns or molecular weights bands of low (<60 kDa) or high molecular weight (> 60 kDa) was found. However, dogs with hyperadrenocorticism may develop glomerular and tubular injuries that were characterized by the presence of albuminuria and proteins of low and high molecular weights, independently of systemic arterial hypertension. In conclusion, the quantitative (UPC and UAC) and qualitative (SDS-PAGE) evaluation of urinary proteins could add information to indicate the possible segments of the nephrons that caused the loss of those proteins.

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Tendo como objetivo a obtenção de hidrolisados proteicos de farinha de trigo com baixo teor de fenilalanina (Phe), foram preparados, inicialmente, extratos proteicos da farinha de trigo, empregando-se método enzimático pela ação de protease de Bacillus licheniformis. Em seguida, esses extratos foram hidrolisados sob a ação do extrato enzimático bruto (EEB), obtido de casca de abacaxi, e de pancreatina comercial; e alguns parâmetros hidrolíticos foram avaliados, tais como temperatura (30; 35; 40; 50; e 70 °C), tempo (1 hora e 30 minutos; 2 horas e 30 minutos; 3 horas e 30 minutos), e pH de reação (6,0; 7,0; 8,0 e 9,0). Para a remoção de Phe, empregou-se o carvão ativado (CA) e a eficiência deste processo foi avaliada determinando-se o teor de Phe por espectrofotometria derivada segunda, na farinha de trigo, assim como nos hidrolisados após tratamento com CA. Para os três parâmetros estudados, observaram-se efeitos variados sobre a remoção de Phe, sendo que os melhores resultados foram encontrados ao se empregar a associação sucessiva de EEB (E:S 10:100, 1 hora e 30 minutos), com a pancreatina (E:S 4:100, 3 horas e 30 minutos), em pH 7,0 a 50 °C, tendo atingido 66,28% de remoção de Phe, o que corresponde a um teor final de Phe de 522,44 mg.100 g-1 de hidrolisado.

With the aim of obtaining wheat flour hydrolysates with low-phenylalanine (Phe) content, protein extracts were prepared using an enzymatic method through the action of a protease from Bacillus licheniformis. Next, these protein extracts were hydrolyzed by a crude enzymatic extract (CEE) obtained from pineapple peel followed by a commercial enzyme (pancreatin). Some parameters, such as temperature (30, 35, 40, 50 and 70 °C), time (1 hour and 30 minutes, 2 hours and 30 minutes, 3 hours and 30 minutes), and pH of the reaction (6.0, 7.0, 8.0 and 9.0) were evaluated. Activated carbon (AC) was used for removing Phe and the efficiency of this process was evaluated by second derivative spectrophotometry, measuring Phe content in wheat flour as well as in its hydrolysates after AC treatment. Varied effects were observed for the three parameters studied, and the best results were found using successive association of CEE (E: S 10:100, 1 hour and 30 minutes) with pancreatin (E:S 4:100, 3 hour and 30 minutes) pH 7.0, at 50°C having reached a final Phe content of 522.44 mg.100 g-1 of hydrolysate.

Inga cylindrica Trypsin Inhibitor (ICTI) was purified as a single polypeptide chain by one step anion-exchange chromatography from a crude extract of Inga cylindrica (Vell.) Mart. seeds. ICTI is a 19.5 kDa protein presenting a remarkable inhibitory activity against bovine trypsin (EC 3.4.21.4) (Ki = 4.3 nM). Circular dichroism analysis revealed that this inhibitor is a β type protein (40.4% of β-strand; 24.6% of β-turn and 6.7% of α-helix) in accordance with properties displayed in Kunitz type inhibitors. ICTI is a thermal stable protein within a wide range of pH (1.6 to 10.0) exhibiting highest stability at pH 7.0 as indicated by Tm of 70.0 ºC and ΔG25 of 48.5 ± 0.7 kJ.mol-1. The values of ΔG25 at pH 1.6 (22.5 ± 1.2 kJ.mol-1) and pH 10.0 (31.5 ± 1.0 kJ.mol-1) indicate a reduced structural stability of the protein under these conditions. This is likely to result from pKa differences of the acid and basic side chains reflecting the changes in the non-covalent interactions in the folded state.

O microcalorímetro de condução pode ser usado para avaliar as taxas metabólicas do espermatozóide eqüino. Dois ejaculados de quatro garanhões foram avaliados quanto à motilidade progressiva pela microscopia, viabilidade espermática (eosina 3%), integridade funcional da membrana (teste hiposmótico) e produção de calor (microcalorimetria). Concentrações ótimas de glicose e de células espermáticas foram determinadas, para mensurar o calor liberado resultante do metabolismo espermático em relação à capacidade de detecção do calor pelo microcalorímetro. Não foi observada diferença da motilidade, viabilidade e integridade funcional de membrana espermática quando adicionada glicose nas três concentrações estudadas. No entanto a avaliação por microcalorimetria ressaltou um maior fluxo de calor a uma concentração de 6 mM de glicose e uma concentração espermática de 10(8) espermatozóides/mL. Portanto, a técnica de microcalorimetria oferece informações adicionais sobre o metabolismo tornando-se uma ferramenta importante no estudo do processo de preservação do sêmen eqüino.

The heat conduction microcalorimeter can be used to evaluate the metabolic rates of the sperm cell. Two ejaculates of four stallions were cooled to +5ºC and checked for sperm motility (bright field microscopy), viability (eosin 3%), functional membrane integrity (hyposmotic swelling test), and heat production (microcalorimetry). Glucose and sperm cell concentrations were determined in order to measure the heat outputs resulting from sperm metabolism. Sperm viability, membrane integrity and sperm motility did not differ among the different glucose concentrations tested. Nevertheless, the highest heat output detected by the microcalorimeter was obtained with 6 mM glucose and 10(8) spermatozoa/mL. Since conduction microcalorimetry offered additional information on equine sperm metabolism, it could be used as a method to study equine semen preservation.

In this article, we describe a novel methodology to extract semantic characteristics from protein structures using linear algebra in order to compose structural signature vectors which may be used efficiently to compare and classify protein structures into fold families. These signatures are built from the pattern of hydrophobic intrachain interactions using Singular Value Decomposition (SVD) and Latent Semantic Indexing (LSI) techniques. Considering proteins as documents and contacts as terms, we have built a retrieval system which is able to find conserved contacts in samples of myoglobin fold family and to retrieve these proteins among proteins of varied folds with precision of up to 80%. The classifier is a web tool available at our laboratory website. Users can search for similar chains from a specific PDB, view and compare their contact maps and browse their structures using a JMol plug-in.

O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.

The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.

Neste trabalho foram produzidos anticorpos policlonais anti-fator IX humano em coelhos New Zealand imunizados com FIX humano (hFIX) comercial puro (Octanyne®, Octapharma, EUA). O soro contendo as imunoglobulinas anti-hFIX foi útil para a detecção do antígeno hFIX em frações do plasma humano submetido a cromatografia de troca iônica. Imunoensaios (ELISA) usando soro-albumina bovina, tripsina e peptídeos gerados por ensaios de clivagem com tripsina com enzima de digestão foram realizados e revelaram especificidade adequada dos anticorpos policlonais produzidos.

In this work, polyclonal antibodies anti-human Factor IX were produced in New Zealand rabbits by immunization with commercial pure human FIX (hFIX) (Octanyne®, Octapharma, USA). The serum containing immunoglobulins anti-hFIX was useful to detect hFIX antigen in human plasma fractions submitted to anionic exchange chromatographic process and with a large yield. Immunoassays (ELISA) using bovine serum albumin, trypsin and peptides generated by cleavage assays with trypsin as digestion enzyme was performed and revealed adequate specificity of the polyclonal antibodies produced.

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.