nÃo hà / A acerola Malpiguia emarginata DC à uma fruta dotada de qualidades nutricionais invejÃveis, sendo consumida tanto in natura quanto processada. A principal caracterÃstica que destaca a acerola à sua enorme capacidade em sintetizar o Ãcido ascÃrbico. A Embrapa desenvolveu quatro clones comerciais com caracterÃsticas fenotÃpicas e genÃticas bem definidas denominado BRS 235 (Apodi), BRS 236 (Cereja), BRS 237 (Roxinha) e BRS 238 (Frutacor). Os clones BRS 236 (Cereja) e BRS 237 (Roxinha) foram escolhidos devido a enorme diferenÃa de Ãcido ascÃrbico sintetizada entre eles, aproximadamente 50% a mais no clone Cereja. Para esse estudo foi analisada a expressÃo gÃnica das enzimas pertencentes a via Wheeler/Smirnoff, reconhecida como a principal via biossintÃtica do Ãcido ascÃrbico em plantas bem como a Oxidase alternativa (AOX) uma enzima desacopladora, nÃo fosforilante e insensÃvel ao cianeto, presente entre os complexos II e III da membrana mitocondrial interna, responsÃvel pela via alternativa de elÃtrons. O objetivo desse trabalho foi identificar quais enzimas sÃo determinantes na diferenÃa do conteÃdo de vitamina C entre os clones, bem como avaliar a expressÃo da AOX nos diferentes tecidos. De aceroleiras com cinco anos de idade foram colhidos quatro tecidos (Flores, Frutos verdes, Frutos semimaduros e Frutos maduros. Inicialmente os teores de Ãcido ascÃrbico foram dosados nos frutos verdes, semimaduros e maduros dos dois clones por titulometria de Tillman. Em seguida foi realizada a caracterizaÃÃo gÃnica da AOX para definiÃÃo de suas isoformas. ApÃs o isolamento do DNA e executadas as reaÃÃes de PCR com um par de primers degenerados os amplicons foram purificados e submetidos a clonagem, transformaÃÃo e seqÃenciamento. As dosagens de Ãcido ascÃrbico mostraram que ambos os clones decrescem seus nÃveis de Ãcido ascÃrbico a medida que os frutos se desenvolvem, alÃm do que em todos os trÃs estÃdios de desenvolvimento o clone Cereja apresenta quantidades de Ãcido ascÃrbico superiores ao clone Roxinha. Os nÃveis de Ãcido ascÃrbico entre os frutos verdes e maduros de ambos os clones revelaram que o clone Roxinha possui uma menor diferenÃa entre esses dois estÃdios de desenvolvimento. As anÃlises de expressÃo gÃnica revelaram que trÃs enzimas possuem sua expressÃo destacada das demais, sendo que essas mostraram um sinergismo de expressÃo com o padrÃo decrescente dos nÃveis de vitamina C contida no tecido, apresentando ainda diferenÃas de expressÃo favorÃveis ao clone Cereja. SÃo elas: Manose pirofosforilase, GDP-Manose 3â5â epimerase e GDP Galactose fosforilase. Com maior destaque para a GDP-Manose 3â5â epimerase e GDP Galactose fosforilase. NÃo existiam diferenÃas nas expressÃes gÃnicas das demais enzimas da via Wheeler/Smirnoff que justificassem as diferenÃas nos teores de Ãcido ascÃrbico presentes nos tecidos e tambÃm entre os clones. Quanto a AOX os resultados revelaram duas seqÃÃncias, uma relativa a uma AOX1 e outra a uma AOX2. A anÃlise da expressÃo gÃnica das isoformas da AOX demonstrou que a AOX1 eleva sua expressÃo gÃnica em ambos os clones a medida que os frutos amadurecem entretanto, o clone Roxinha possui uma expressÃo gÃnica mais elevada que o clone Cereja, em todos os trÃs estÃdios de desenvolvimento. A AOX2 possui diferenÃas de expressÃo gÃnica onde no clone Cereja ela se mostrou decrescente, jà no clone Roxinha ocorreu a elevaÃÃo da expressÃo nos trÃs estÃdios de desenvolvimento. TrÃs enzimas sÃo essenciais a biossÃntese do Ãcido ascÃrbico em Malpiguia emarginata DC, a Manose pirofosforilase, GDP-Manose 3â5â epimerase e GDP Galactose fosforilase, sendo a via Wheeler/Smirnoff determinante na quantidade de Ãcido ascÃrbico produzido. As expressÃes gÃnicas da AOX1 e AOX2 favorecem o clone Roxinha, aparentemente como um mecanismo compensatÃrio por esse clone sintetizar menos Ãcido ascÃrbico que o clone Cereja.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A Bothrops leucurus (jararaca do rabo branco) à uma serpente peÃonhenta que habita a regiÃo Nordeste do Brasil. Os efeitos biolÃgicos devido ao envenenamento por B. leucurus tÃm perfil semelhante Ãqueles apresentados por outras serpentes do gÃnero Bothrops, tais como, importantes efeitos locais e sistÃmicos graves como a InsuficiÃncia Renal Aguda (IRA). O veneno de Bothrops leucurus induziu nefrotoxicidade em sistema de perfusÃo de rim isolado de rato, associado com citotoxicidade em cÃlulas tubulares epiteliais renais. Neste trabalho foi avaliado o efeito nefrotÃxico direto da enzima L-aminoÃcido oxidase isolada do veneno de Bothrops leucurus (LAAO-Bl) sobre cÃlulas epiteliais renais (MDCK e HK2) e o seu potencial nefrotÃxico em rim isolado de rato. O tratamento com LAAO-Bl, 1.56 â 100 Âg/mL induziu significativa morte celular de maneira concentraÃÃo dependente em ambas Ãs linhagens celulares apÃs 12 horas de tratamento. Nas cÃlulas MDCK nÃo foi observada liberaÃÃo de LDH apÃs 12 horas de exposiÃÃo à LAAO-Bl, enquanto nas cÃlulas HK2 LAAO-Bl induziu ruptura de membrana nas maiores concentraÃÃes estudadas quando comparado ao controle nÃo tratado. Nas cÃlulas MDCK o tratamento com LAAO-Bl aumentou significativamente a porcentagem de cÃlulas em apoptose (Anexina-V+, IP-), necrose (Anexina-V-, IP+) e necrose secundÃria (Anexina-V+, IP+). Nas cÃlulas HK2 LAAO-Bl induziu um aumento na porcentagem de cÃlulas em necrose (IP+) e necrose secundÃria (Anexina-V+, IP+) de maneira concentraÃÃo dependente. A induÃÃo de apoptose nas cÃlulas MDCK foi acompanhada de liberaÃÃo de Ca2+ do retÃculo endoplasmÃtico, aumento de espÃcies reativas de oxigÃnio, disfunÃÃo mitocondrial e aumento de expressÃo de Bax. O tratamento com LAAO-Bl induziu ativaÃÃo de caspase 3 e 7 em ambas as linhagens celulares. LAAO-Bl (10 Âg/mL) exerce efeitos significativos no rim isolado de rato aumentando a pressÃo de perfusÃo e o fluxo urinÃrio e diminuindo a taxa de filtraÃÃo glomerular e os transportes tubulares de sÃdio, potÃssio e cloreto. Os nossos resultados sugerem que LAAO-Bl contribui para nefrotoxicidade observada no envenenamento por Bothorps leucurus. AlÃm disso, os efeitos citotÃxicos de LAAO-Bl nas cÃlulas epiteliais renais podem ser responsÃveis, pelo menos em parte, pela nefrotoxicidade observada no rim isolado. / The pit viper Bothrops leucurus (White-tailed-jararaca) is a poisonous snake habituating area in the northeast of Brazil. The biological effects due envenomation have similar profile than those observed with other Bothrops, such as important severe local and systemic effects such as Acute Renal Failure (ARF). Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney of rats associated with cytotoxicity against renal tubular epithelia cells. In this study, it was evaluated the direct nefrotoxicity of L-aminoacid oxidase isolated of B. leucurus venom (LAAO-Bl) on renal epithelial cells (MDCK and HK2) and their potential nephrotoxic in isolated rat kidney. In these cells treated with LAAO-Bl, 1.56 â 100 Âg/mL for 12 h, there was a decrease in their viability in a concentration-dependent manner after 12 hours of treatment. In MDCK cells LDH release was not observed after 12 h of LAAO-Bl exposure while LAAO-Bl induced membrane rupture in HK-2 cells at the highest concentrations studied when compared with untreated cells. In MDCK cells, LAAO-Bl significantly increased the percentage of early apoptotic (Annexin-V+, PI-), necrotic (Annexin-V-, PI+) and secondary necrotic cells (Annexin-V+, PI+) when compared with control untreated cells. In HK-2 cells LAAO-Bl induced an increase in necrotic (PI+ cells) and secondary necrotic cells (Annexin-V+, PI+) in a concentration-dependent manner. MDCK apoptosis induction was accompanied with Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation, mitochondria dysfunction with enhanced expression of Bax protein levels. LAAO-Bl induced caspase-3 and caspase-7 activation in both cell lines. LAAO-Bl (10 Âg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl contributes for the nephrotoxicity observed in the envenomation by Bothrops leucurus. Moreover, the cytotoxic of LAAO-Bl to renal epithelial cells might be responsible, least in part for the nephrotoxicity observed in isolated kidney.

No description available.

Mitochondria are involved in numerous cellular processes such as respiration, ATP production, calcium signalling and apoptosis. About 99% of mitochondrial proteins are nuclear-encoded and need to be imported into mitochondria for their function. The MIA pathway is used by many cysteine-containing proteins for their import into the mitochondrial intermembrane space (IMS). The pathway comprises two essential proteins: the disulphide carrier and import receptor Mia40, and the FAD-dependent sulphydryl oxidase Erv1. Together these proteins form a disulphide relay system inside the IMS. Initially, substrate proteins are imported in their cysteine-reduced form, which is oxidised by Mia40 in the IMS. Then, the now reduced Mia40 is in turn re- oxidised by Erv1. Finally, reduced Erv1 can transfer the electrons to oxygen directly, or via cyt c, to the respiratory chain. The overall aim of this study is to understand the structural and functional mechanisms of Erv1, from the effect of single mutations (R182H) to its quaternary structure and thermodynamic properties. The results are described in three chapters. First, biophysical techniques were used to evaluate the oligomerisation state of Erv1. Contrary to general belief, the results show that Erv1 adopts a tetramer conformation in solution. Tetramerisation provides Erv1 with a higher thermal stability, though it does not affect its oxidase activity. The second result chapter focuses on understanding the effects of a medically relevant mutant, Erv1 R182H, on the structure and function of the protein. The results show that at the physiological temperature of 37°C the mutant is less stable and becomes completely inactive after a few enzymatic cycles. The activity defect is linked to a weaker binding of the FAD cofactor in the mutant. Lastly, the third result chapter looks at the electron transfer within Erv1 from a thermodynamic perspective. Standard reduction potentials were determined for two of the three redox centres in Erv1. The results differ from those previously published, but are consistent with the current model of electron transfer in Erv1. Taken together, the results presented here offer an insightful perspective into the molecular mechanisms regulating Erv1.

The generation of oxygen free radicals by the cytosolic enzyme, xanthine oxidase (XO), has been implicated in post-ischemic or reperfusion damage in several organs. XO catalyzes the conversion of hypoxanthine to urate with the concomitant production of superoxide anion free radical (0₂̅˙) and hydrogen peroxide (H₂O₂). Oxygen free radical-mediated injury has also been demonstrated in inflammatory lung disease. The possible involvement of XO in oxidative injury in the lung has not yet been studied. Therefore, this research project was designed to determine whether XO is present in the lung and to investigate its characteristics in porcine, bovine, rat and human lung and other tissues. Immunochemical analysis of xanthine oxidase in the tissues employed on polyclonal antibody raised to bovine milk XO. Proteins were separated by SDS-polyacrylamide gel electrophoresis of tissue homogenates. Proteins were transfered from the gels to nitrocellulose filters by Western blotting. After incubating the filters with a antisera containing the antibody to the purified bovine XO. XO on the filter was detected by its reaction with an enzyme-conjugated second antibody. XO was immunologically detectable in bovine lung and milk. Rat lung, kidney and liver all showed XO reactivity. XO was detectable in porcine liver but not detectable in porcine lung or kidney. Thus, the antibody to bovine XO was cross-reactive with porcine and rat XO. XO protein was not immunologically detectable in human lung possibly because the antibody was not cross reactive with the bovine antibody. In vivo, xanthine oxidase exists predominantly as a dehydrogenase rather than an oxidase. In this form as xanthine dehydrogenase (XDH) the enxyme does not produce either 0₂̅˙ or H₂O₂. The activity of both XDH and XO was measured in several tissues using a fluorometric assay which uses an artifical substrate, pterin which is catalytically converted to the fluorescent product isoxanthopterin (IXP). XO activity in porcine liver was of 1.1 x 10⁻³ µg IXP/mg protein/min although XO activity was not detectable in porcine lung and kidney, in rat lung of 1.7 x 10⁻² µg IXP/mg protein/min, rat kidney of 1.5 x 10⁻² µg IXP/mg protein/min, and rat liver of 2.2 x 10⁻² µg IXP/mg protein/min. Seven human lung biopsy samples were obtained after lung resection and initially tested for viability by determination of NADH oxidase activity and then assayed for XO-XDH. Three of these samples showed NADH oxidase activity indicating tissue viability, but only one of these three showed measurable XO activity of 5.35 x 10⁻⁶ µg IXP/mg protein/min. Irreversible conversion of XDH to XO is thought to be the result of limited proteolysis by a Ca²⁺/calmodulin activated protease, whereas reversible conversion of the enzyme occurs by oxidation of critical thiol groups. Studies on the rate and nature of fluorescence assay to detect catalytic activities of both enzyme forms. Incubation of lung homogenates with trypsin for 60 min caused irreverisble conversion of 90% of the XDH to XO. In contrast, incubation of homogenates at 15°C for 10 hours caused conversion of 100% of the XDH to XO. This conversion was reversible to the extent of 80% by reduction of thiol groups with dithiothreitol (DTT). The effects of free Ca²⁺ on the conversion of XDH to X0 was examined by using EDTA, a chelator of Ca²⁺ and other divalent cations; and EGTA, a more specific chelator of Ca²⁺. The presence of these chelating agents during homogenization of either normoxic or ischemic rat lung tissue did not inhibit reversible enzyme conversion. Increased XO activity was reversible by DTT. In the normoxic rat lung, homogenates prepared with EDTA and EGTA showed a similar conversion of 95% of XDH to XO which was reversible to 70% with DTT. In the ischemic rat lung, samples prepared with EDTA and EGTA showed a'conversion of 80% and 95% XDH to XO which was similar to control samples. The extent of reversibility to XDH was 75% with DTT incubation. In addition, perfusion of rat lungs with EDTA and DTT via a pulmonary artery cannula prior to 60 min of ischemia and homogenization did not affect the extent of XDH to XO conversion. These results indicate that irreversible Ca²⁺-mediated proteolytic conversion of XDH to XO does not occur to a great extent in the rat lung during either normoxia or ischemia. However, reversible conversion of XDH to XO does occur, suggesting that reversible thiol dependent conversion may play a role in the lung under both physiological and pathophysiological states. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

Inhibition of blueberry PPO activity by sodium benzoate, potassium sorbate and potassium metabisulfite and their influence on degradation of individual anthocyanins in an extract was studied. Maceration of blueberries was carried out at 55ºC for 1h with the addition of 0.1% sodium benzoate or with blanching pretreatment at 90ºC for 1min. After maceration pretreatments the extracted juice was processed with traditional hot fill pasteurization, high hydrostatic pressure (HHP) and pulsed electric field (PEF). Sodium benzoate and potassium metabisulfite were very effective PPO inhibitors in concentrations of 0.1% and 10ppm, respectively. Potassium sorbate was the weakest inhibitor, with 50% PPO remaining. Degradation of anthocyanins by PPO was dependent on their structure. Tri-phenolic anthocyanins experienced the most degradation, followed by diphenolic and monophenolic compounds, respectively. Sodium benzoate was the most effective at preventing anthocyanin degradation; potassium metabisulfite did not have any protective effect, while potassium sorbate increased anthocyanin degradation Blanching of blueberries inactivated native PPO, but also increased the degradation of anthocyanins, especially malvidin glycosides. Addition of 0.1% sodium benzoate decreased PPO activity when compared to frozen blueberries but not in respect to control maceration. Only 12% of anthocyanins and 33-41% of phenolics were extracted into juice from the frozen fruit. Hot fill pasteurization, high hydrostatic pressure and pulsed electric field did not significantly influence anthocyanins, phenolics and antioxidant activity in blueberry juice.

No description available.

No description available.

No description available.