No description available.

No description available.

No description available.

This thesis was undertaken to determine if there is any interrelationship between the two terminal enzymes of the haem biosynthetic pathway, protoporphyrinogen oxidase (PPO) and ferrochelatase, with particular reference to porphyria variegata (PV) and erythropoietic protoporphyria (EPP)• It has previously been found that both enzymes were deficient in PV and EPP, there being a qualitative difference in so far as ferrochelatase deficiency is concerned. / IT2018

by Suen Tsang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 96-103). / Abstract also in Chinese. / Chapter Chapter 1: --- Literature review --- p.1 / Chapter 1.1 --- "Straw mushroom, Volvariella volvacea" --- p.1 / Chapter 1.2 --- Problems which restrict the market of straw mushroom --- p.3 / Chapter 1.3 --- Non-enzymatic browning --- p.5 / Chapter 1.4 --- Enzymatic browning --- p.7 / Chapter 1.5 --- Impact of browning --- p.12 / Chapter 1.6 --- Mechanism of inhibition of PPO --- p.13 / Chapter 1.7 --- Sulfites --- p.13 / Chapter 1.8 --- Classification of PPO inhibitors based on chemical property --- p.14 / Chapter 1.9 --- Classification of PPO inhibitors based on inhibitory mechanism --- p.17 / Chapter 1.10 --- Physical methods for prolonging shelf-life --- p.18 / Chapter 1.11 --- Significance of this research --- p.20 / Chapter Chapter2: --- Characterization of PPO in straw mushroom --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- PPO content in straw mushroom compared to other food sources: potato and pear --- p.22 / Chapter 2.2.2 --- "Optimal pH, enzyme kinetics and localization of PPO in straw mushroom" --- p.24 / Chapter 2.2.3 --- PPO isoenzymes in straw mushroom --- p.25 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- PPO content in straw mushroom compared to other food sources: potato and pear --- p.29 / Chapter 2.3.2 --- "Optimal pH, enzyme kinetics and localization of PPO in straw mushroom" --- p.29 / Chapter 2.3.3 --- PPO isoenzymes in straw mushroom --- p.32 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter3: --- Several attempts to solve browning problem of straw mushroom --- p.55 / Chapter 3.1 --- Inhibitors of PPO in straw mushroom --- p.55 / Chapter 3.1.1 --- Investigation of inhibitors of PPO in straw mushroom --- p.55 / Chapter 3.1.1.1 --- Materials and methods --- p.55 / Chapter 3.1.1.2 --- Results --- p.56 / Chapter 3.1.2 --- The potential of using a combination of different PPO inhibitors --- p.58 / Chapter 3.1.2.1 --- Materials and methods --- p.58 / Chapter 3.1.2.2 --- Results --- p.59 / Chapter 3.1.3 --- Direct application of PPO inhibitors --- p.61 / Chapter 3.1.3.1 --- Materials and methods --- p.61 / Chapter 3.1.3.2 --- Results --- p.62 / Chapter 3.1.4 --- PPO and lipase content in straw mushroom under post harvest storage --- p.62 / Chapter 3.1.4.1 --- Materials and Methods --- p.74 / Chapter 3.1.4.2 --- Results --- p.75 / Chapter 3.2 --- Vacuum packaging --- p.75 / Chapter 3.2.1 --- Materials and methods --- p.75 / Chapter 3.2.2 --- Results --- p.78 / Chapter 3.4 --- Discussion --- p.78 / Chapter Chapter 4: --- Future work --- p.87 / Chapter 4.1 --- Suggested improvements of experiments --- p.87 / Chapter 4.2 --- Suggested experiment in future: application of calcium chloride --- p.88 / Chapter Chapter 5: --- Conclusion --- p.94 / References --- p.96

Cu+-ATPases are transmembrane enzymes that couple the efflux of cytoplasmic Cu+ to the hydrolysis of ATP. It is well established that Cu+-ATPases control cytoplasmic Cu+ levels. However, bacterial genomes, particularly those of symbiotic/pathogenic organisms, contain multiple copies of genes encoding Cu+-ATPases, challenging the idea of a singular role for these enzymes. Our lab has demonstrated that one of the two Cu+-ATPases in Pseudomonas aeruginosa, a FixI-type ATPase, has an alternative role, most likely Cu+ loading of cytochrome c oxidase (Cox). To further study alternative roles of Cu+-ATPases, we study the symbiont Sinorhizobium meliloti. Rhizobia are soil-dwelling bacteria that interact with legumes, forming plant root nodules that actively fix N2. The S. meliloti genome contains five Cu+-ATPases, two of which are FixI-type. Both of these enzymes, termed FixI1 and FixI2, are downstream of Cox operons. We hypothesized that the presence of multiple FixI-type ATPases was not an example of redundancy, but rather is an evolutionary adaptation that allows rhizobia to survive under the wide variety of adverse conditions faced during early infection and establishment of symbiosis. Towards this goal, this work focused on examining the effects of mutation of each ATPase on both free-living bacteria and on the ability of rhizobia to establish an effective symbiosis with its host legume. Each of these mutants presents a different phenotype at varying points of the nodulation process, and only the fixI2 mutation produces a respiratory-deficient phenotype during aerobic growth. These results are consistent with our hypothesis that the two proteins have non-redundant physiological functions. Understanding the factors that contribute to an effective symbiosis is beneficial, since N2 fixation in legumes is important to both agriculture and industry.

Resumo: A presente dissertação permitiu quantificar a presença de glicerol-3-fosfato oxidase (GPO, sn-glicerol-3-fosfato: oxigênio 2-oxidorredutase, EC 1.1.3.21) em extratos de levedura seca de panificação por dois métodos: polarográfico e colorimétrico. A melhor metodologia de purificação da GPO foi obtida por rompimento celular com esferas de vidro, em homogenizador do tipo Bead Beater (Biospec products, USA), por 15 minutos, com 27,6% de eficiência de lise celular. O extrato celular bruto foi tratado com 1% de sulfato de estreptomicina antes da precipitação com igual volume de solução 30% (p/v) de polietilenoglicol 3350, dialisado e a sua atividade otimizada por método colorimétrico. A determinação das características da GPO foi possível em ensaios contendo: 250 mM de glicerol-3-fosfato em tampão 0,1 M Tris-HCl pH 8,0 contendo 0,1% Triton X-100; 0,0133% de 4-aminoantipirina; 0,0266% de fenol; cerca de 0,40 unidade de peroxidase (PO) e água destilada para completar o volume de ensaio. A reação foi iniciada pela adição de 15 æL de extrato enzimático diluído 10 vezes seguido de uma incubação de 2 horas a 60°C e interrompida pela adição de solução 10% de SDS e a coloração desenvolvida foi medida a 500 nm. A GPO apresentou alta estabilidade térmica, pH de estabilidade entre 7,0 - 8,0 e a presença de azida de sódio na concentração de 0,05% manteve a atividade da enzima por 21 dias a 40°C. Este método permitiu também quantificar glicerol-3- fosfato, importante metabólito intermediário da biossíntese lipídica e glicolítica, na faixa de 56 - 250 mM. / Abstract: The present dissertation allowed to quantify the presence of glycerol-3- phosphate oxidase (GPO, sn-glycerol-3-phosphate: oxygen 2-oxidoreductase, EC 1.1.3.21) in baker’s yeast extract by two methods: polarographic and colorimetric. The best methodology of purification of GPO was obtained by cell debris with glass beads, in a Bead Beater homogenizator (Biospec products, USA), for 15 minutes, with 27.6% of efficiency of cellular lysis. The crude cellular extract was treated with 1% of streptomycin sulfate before the precipitation, with equal volume of a solution of 30% (w/v) polyethylene glycol 3350, dialysed and its activity was optimized by colorimetric method. The determination of the characteristics of GPO was possible in assays containing: 250 mM of glycerol-3-phosphate in 0.1 M Tris-HCl buffer, pH 8.0 containing 0.1% Triton X-100; 0.0133% of 4-aminoantipyrine; 0.0266% of phenol; about 0.40 unit of peroxidase (PO) and water distilled to complete the volume. The reaction was started by the addition of 15 ìL of enzymatic extract diluted 10 times, followed by incubation of 2 hours at 60°C, interrupted by the addition of solution 10% of SDS, and the developed coloration was measured at 500 nm. GPO presented high thermal stability, pH of stability among 7.0 - 8.0, and the presence of sodium azide in the concentration of 0.05% maintained the activity of the enzyme for 21 days at 40°C. This method also allowed to quantify glicerol-3- phosphate, important intermediate metabolite of lipid biosynthesis and glycolysis, in the range 56 - 250 mM. / Orientador: Edwil Aparecida de Lucca Gattás / Coorientador: Maristela de Freitas Sanches Peres / Banca: Valdir Augusto Neves / Banca: Maria de Lourdes T. de Moraes Polizeli / Mestre

A vitamina A (retinol) e seus derivados, os retinóides, são importantes reguladores do ciclo celular, exercendo um papel central na proliferação, apoptose e diferenciação de diversos tipos celulares. Atualmente, o papel dos retinóides na proliferação e diferenciação celular tem sido bastante investigado. Nesse contexto, diversos estudos têm demonstrado que formas ativas de retinóides como o ácido retinóico inibem a proliferação em modelos de células tumorais e não-tumorais, caracterizando-os como potenciais agentes quimioterápicos. Entretanto, um crescente número de estudos tem sugerido que os retinóides apresentam propriedades pró-oxidantes em sistemas biológicos, as quais podem induzir dano celular, ativação de proto-oncogenes e, até mesmo, transformação neoplásica. Essas contradições estimularam-nos a investigar as propriedades proliferativas/antiproliferativas e antioxidantes/pró-oxidantes dos principais retinóides presentes no meio intracelular - retinol e ácido retinóico- e os mecanismos envolvidos nesses efeitos em células de Sertoli, um dos principais alvos fisiológicos da vitamina A em mamíferos. A vitamina A (retinol) e seus derivados, os retinóides, são importantes reguladores do ciclo celular, exercendo um papel central na proliferação, apoptose e diferenciação de diversos tipos celulares. Atualmente, o papel dos retinóides na proliferação e diferenciação celular tem sido bastante investigado. Nesse contexto, diversos estudos têm demonstrado que formas ativas de retinóides como o ácido retinóico inibem a proliferação em modelos de células tumorais e não-tumorais, caracterizando-os como potenciais agentes quimioterápicos. Entretanto, um crescente número de estudos tem sugerido que os retinóides apresentam propriedades pró-oxidantes em sistemas biológicos, as quais podem induzir dano celular, ativação de proto-oncogenes e, até mesmo, transformação neoplásica. Essas contradições estimularam-nos a investigar as propriedades proliferativas/antiproliferativas e antioxidantes/pró-oxidantes dos principais retinóides presentes no meio intracelular - retinol e ácido retinóico- e os mecanismos envolvidos nesses efeitos em células de Sertoli, um dos principais alvos fisiológicos da vitamina A em mamíferos. A diminuição na viabilidade foi mediada pela ativação da xantina oxidase, uma vez que concentrações elevadas de retinol induziram ativação desta enzima e aumentaram a produção de espécies reativas, e a inibição da xantina oxidase com alopurinol atenuou tanto a produção de espécies reativas quanto o dano oxidativo a proteínas e a citotoxicidade induzida pelo retinol. Além disso, demonstramos que a incubação da xantina oxidase com retinol promove a produção de superóxido in vitro, e investigamos alguns fatores envolvidos nesse evento. Por outro lado, o principal derivado ativo do retinol, o ácido retinóico, não apresentou qualquer efeito pró-oxidante ou proliferativo, nem ativou MAPKs. Ao contrário do retinol, o ácido retinóico apresentou efeitos antiproliferativos, diminuindo os níveis do inibidor de CDKs p21 e induzindo parada no ciclo celular. Os dados apresentados nesse trabalho podem contribuir para a elucidação dos efeitos pró-oxidantes, proliferativos/antiproliferativos e citotóxicos da vitamina A e seus derivados em sistemas biológicos. / Vitamin A (Retinol) and its derivatives, retinoids, are important regulators of the cell cycle, playing a role on proliferation, apoptosis and differentiation of diverse cell types. In recent years, the influence of retinoids on cell growth and differentiation has been investigated. There is a growing body of in vitro data demonstrating that active retinoids as retinoic acid antagonize cell growth in a variety of normal and tumour cells, characterizing them as potential chemotherapeutic agents. On the other hand, a growing body of evidence has suggested that retinoids present pro-oxidant properties in biological systems, which might induce cell damage, proto-oncogene activation and neoplasic transformation. These discordances stimulated us to investigate the proliferative/antiproliferative properties of two major intracellular retinoids, retinol and retinoic acid, and its mechanisms in a model of Sertoli cells, a major vitamin A physiological target. Our data showed that supra-physiological concentrations of retinol, but not retinoic acid, are able to increase reactive species production in Sertoli cells. These pro-oxidant effects may induce different cellular fates depending on of retinol concentrations. At low pro-oxidant levels (7 μM), retinol induced an oxidant-dependent proliferation, which was mediated by a rapid, nonclassic and redox-sensitive activation of the MAPKs JNK1/2, p38 and ERK1/2. The investigation of potential sites of reactives species production indicated that mitochondria act as a primary source of reactive species involved in retinol redox signaling. Retinol 7 μM induces dysfunction in the mitochondrial electron transfer system leading to increases in superoxide anion production. Inhibition of the mitochondrial-dependent superoxide formation blocked both reactive species production and MAPKs activation, characterizing a new mechanism of nongenomic action of the vitamin A. MAPKs inhibition blocked retinol-induced proliferation. On the other hand, at high concentrations (10 to 20 μM) the pro-oxidant effects of retinol were significantly high to induce extensive oxidative damage and decreases in cell viability. At high concentrations, retinol-induced decreases in cell viability were mediated by stimulation of the xanthine oxidase activity in Sertoli cells, since retinol treatment increased xanthine oxidase activity and reactive species production, and inhibition of the enzyme with allopurinol attenuated retinol-induced reactive species, protein oxidative damage and cytotoxicity. In addition, we showed that incubation of xanthine oxidase with retinol promotes superoxide generation in vitro, and investigated the potential factors involved in this effect. On the other hand, the principal retinol derivative, retinoic acid, induced neither reactive species production or proliferation nor MAPKs activation. In contrast to retinol, retinoic acid presented its well documented antiproliferative effects by decreasing DNA synthesis and increasing the level of the CDK inhibitor p21 leading to cell arrest. Data presented in this work, may contribute to elucidation of the mechanisms involved in pro-oxidants, proliferatives/antiproliferatives and cytotoxic actions of vitamin A and its derivatives in biologycal systems.

No description available.

The biological membrane is composed of lipids and proteins that make up dynamic barriers around cells and organelles. Membrane-spanning proteins are involved in many key processes in the cell such as energy conversion, nerve conduction and signal transduction. These proteins interact closely with lipids as well as with other proteins in the membrane, which modulates and affects their structure and function. In the energy-conversion process, membrane-bound proton-transport proteins maintain an electrochemical proton gradient across the mitochondrial inner membrane or the cytoplasmic membrane of bacteria. This gradient is utilized for ATP synthesis or transport of ions and molecules across the membrane. Results from earlier studies have shown that proton transporters are influenced by their environment. Here, one of these proton transporters, cytochrome c oxidase, has been purified and reconstituted into liposomes or nanodiscs and membrane effects on specific proton-transfer processes were studied. In these studies we observed that the membrane accelerated proton transfer to the surface of cytochrome c oxidase and that there is a protonic link, via a Glu residue that mediates proton transfer from the membrane surface to a proton-transfer pathway in this protein. In addition, the membrane was shown to modulate specific internal electron and proton-transfer reactions. The results from these studies show that the membrane composition influences transmembrane transport. Consequently, our understanding of these processes requires investigation of these transporter proteins in different membrane-mimetic systems of variable and well-defined composition. Furthermore, the data show that membrane surfaces facilitate lateral proton transfer which is presumably essential for maintaining high efficiency in energy conversion. This is particular important in organisms such as alkaliphilic bacteria where the driving force of the electrochemical proton gradient, between the bulk solution on each side of the membrane is not sufficient for ATP synthesis.