Ureases (EC 3.5.1.5) são metaloenzimas que hidrolisam uréia para produzir amônia e dióxido de carbono. Estas enzimas, que são amplamente encontradas em fungos, bactérias e plantas, compartilham de estruturas similares. A presença de urease em várias bactérias patogênicas (Helicobacter pylori e Proteus mirabilis, p.e) está fortemente correlacionada com a patogênese em doenças humanas. Muitos fungos de importância médica possuem atividade ureásica, entre eles citamos Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii, e espécies de Trichosporon e Aspergillus. C. neoformans é uma levedura que produz vários fatores de virulência conhecidos, como presença de cápsula polisacarídica, produção de melanina e capacidade de desenvolvimento a 37ºC. A maioria de isolados clínicos produz grandes quantidades de urease e muitos autores sugerem que a urease de Cryptococcus exerça uma função importante na patogênese, porém com mecanismos ainda não esclarecidos. Cryptococcus gattii – sorotipo B, tipo molecular VGII, linhagem R265, com capacidade de infectar pacientes imunocompetentes, causou uma epidemia na Ilha de Vancouver (Canadá) entre 1999 e 2003. Neste trabalho desenvolvemos um procedimento de purificação e apresentamos a caracterização físico-química e cinética da urease de C. gattii, cepa R265, após ter sido purificada na razão de 539 vezes. A massa molecular estimada foi de 120 kDa, Km 2,0 mM para uréia, pH ótimo 8,0. O ácido acetohidroxâmico demonstrou ser um bom inibidor em concentrações micromolares, enquanto ρ-hidroximercuriobenzoato causou inibição em concentrações mais altas, comparado a outras ureases. Espera-se que estudos adicionais com essa urease purificada permitam investigar propriedades biológicas independentes da atividade ureolítica e estabelecer sua contribuição para a patogênese da criptococose. / Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide. These enzymes, which are found in fungi, bacteria, and plants show very similar structures. The presence of urease in many pathogenic bacteria (Helicobacter pylori and Proteus mirabilis) is strongly correlative with pathogenesis in human diseases. Many medically important fungi have urease activity, among which are Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii, and species of Trichosporon and Aspergillus. C. neoformans is a heterothallic yeast with several known virulence factors, including a polysaccharide capsule, melanin production, and the ability to grow at 37°C.The majority of clinical isolates produce large amounts of urease and several authors suggest that Cryptococcal urease play an important role in pathogenesis but with unclear mechanisms but probably by different routes dependent and independent of ureolytic activity, althought many questions are unclear. We wanted to further investigate the role of urease in biological properties by using Cryptococcus gattii as a pathogen model. C. gatti – serotype B, molecular type VGII, R265 strain, which has capacity to infect immunocompetent individuals, caused an outbreak on Vancouver Island in Canada from 1999 to 2003. This work presents the physicochemical characterization of a urease from C. gatti strain R265 after a 539 fold purification. The estimated native molecular mass was 120 kDa, Km 2.0 mM urea, pH optimum 8.0. Aceto hydroxamic acid was a strong inhibitor at low concentration while ρ-hidroxy mercuribenzoate needed higher concentrations for inhibition compared to other ureases.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvi, 134 p. : ill., (some col.). Includes abstract and vita. Advisor: Kathryn A. Eaton, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 107-134).

Current enzyme kinetic equations are inadequate for modelling enzymatic reactor systems because they fail to take into account the interactions between that various process parameters. They also are unable to predict reaction rates in complex solute systems. A quasi-native kinetic model was developed that predicts enzyme activity by examining the effect of solute addition on the overall protein structure. The theory was tested using the enzyme urease (urea aminohydrolase EC 3.5.1.5). / The quasi-native model was found to accurately predict both the activation and inhibition phenomena observed with urease and could also predict enzymatic activity in complex solute systems. The quasi-native isomerization constant was shown to be a function of hydrophobic effects characterized by the Sechenov theory and electrostatic effects characterized by the DeBye-Huckel theory. The Sechenov constant was found to be independent of temperature and pH. / The urease denaturation rate constant displayed a response to solute addition similar to that observed with the quasi-native isomerization equilibrium constant. However, the effect of pH on urease kinetics was a complex function of the ionization of active-site ligands and enzyme surface charge interactions.

The use of urea fertiliser has been associated with relatively poor nitrogen (N) use efficiency (NUE) due to heavy N losses such as gaseous emissions of ammonia (NH₃) and nitrous oxide (N₂O) and nitrate (NO₃⁻) leaching into surface and ground waters. Improving N use-efficiency of applied urea is therefore critical to maximise its uptake and to minimise its footprint on the environment. The study was conducted under laboratory-glasshouse conditions (Chapter 2-4)and lysimiter-field plot studies (Chapter 5). In chapter 2, Two glasshouse-based experimentswere conducted to investigate the potential of incorporating urea fertiliser with ureaseinhibitor, (N-(n-butyl) thiophosphoric triamide (nBTPT) or ‘Agrotain’) to enhance fertiliser N uptake efficiency. Urea, with or without Agrotain, was applied to Ryegrass (Lolium perenne L.) grown in standard plant trays maintained at soil moisture contents of 75–80% field capacity, at rates equivalent to 25 or 50 kg Nha⁻¹. These treatments were compared with other common forms of N fertilisers (ammonium nitrate, ammonium sulphate and sodium nitrate). In a separate pot experiment, granular ¹⁵N urea (10 atom %) with or without Agrotain, was applied at 25 kg Nh⁻¹ to track N use-efficiency and the fate of ¹⁵N-labelled fertiliser. In both experiments, Agrotain-treated urea improved bioavailability (defined as the fraction of total soil N that can interact with a biological target in the plant or that can be taken up by plant) of added N and resulted in significantly higher herbage DM yield and N uptake than urea alone or other forms of N fertilisers. Results from the ¹⁵N experiment support the suggestion that a delay in urea hydrolysis by Agrotain provided an opportunity for direct plant uptake of an increased proportion of the applied urea-N than in the case of urea alone. In chapter 3, two more glasshouse-based experiments were conducted to investigate if urea applied in fine particle application (FPA), with or without Agrotain, had any effect on fertiliser-N uptake efficiency (defined as the difference in N uptake between the fertiliser treatment and the control as a percentage of the amount of N applied) under optimum soil moisture (75-80% field capacity) and temperature (25 °C) conditions, in comparison with other common forms of N fertilisers applied, either in FPA or in granular form. In a separate pot experiment, ¹⁵N urea (10 atom %), with or without Agrotain, was applied to either shoots or leaves only or to the soil surface (avoiding the shoots and leaves) to determine urea hydrolysis, herbage DM and ¹⁵N uptake. In both experiments, herbage DM yield and N uptake were significantly greater in the FPA treatments than in those receiving granular application. Agrotain-treated urea FPA resulted in significantly higher N response efficiency (difference between the dry matter produced by the various fertiliser treatments and the control, divided by the amount of N applied) than urea FPA alone or other forms of N fertilisers. Results from the ¹⁵N experiment support the idea that Agrotain treatment improves the N response of urea applied in FPA form due to a delay in hydrolysis of urea, thus providing herbage an extended opportunity to absorb added urea directly through leaves, cuticles and roots. A further glasshouse-based study was conducted to investigate the effect of Agrotain and irrigation on urea hydrolysis and its movement in a Typic Haplustepts silt loam soil (Chapter 4). A total of 72 repacked soil cores (140 mm inner diameter and 100 mm deep) were used - half (36) of these cores were adjusted to soil moisture contents of 80% field capacity (FC) and the remaining 36 cores to 50% FC. Granular urea, with or without Agrotain, was applied at a rate equivalent to 100 kg N ha⁻¹. Twelve pots were destructively sampled at each day after 1, 2, 3, 4, 7, and 10 days of treatment application to determine urea hydrolysis and its lateral and vertical movement in different soil layers. Agrotain-treated urea delayed urea hydrolysis compared with urea alone during the first 7 days of its application. This delay in urea hydrolysis by Agrotain enabled added urea to disperse and move away from the surface soil layer to the sub-surface soil layer both vertically and laterally. In contrast, most urea in the absence of Agrotain hydrolysed within 2 days of its application. Irrigation after 1 day resulted in further urea movement from the surface soil layer (0-10 mm) to the sub-soil layer (30-50 mm) in Agrotain-treated urea. These results suggest that Agrotain delayed urea hydrolysis and allowed more time for rainfall or irrigation to move the added urea from the surface layer to sub-soil layers where it is likely to make good contact with plant roots. This distribution of urea in the rooting zone (0-200 mm) has the potential to enhance N use efficiency and minimise N losses via ammonia (NH₃) volatilisation from surface-applied urea. Finally, a field study using lysimeters (300 mm inner diameter and 400 mm deep), and small field plots (1 m² in area) was established using a silt loam Typic Haplustepts soil (Soil Survey Staff 1998) to investigate the effect of FPA and granular applications of urea, with or without Agrotain, on N losses and N use efficiency (Chapter 5). The five treatments were: control (no N) and ¹⁵N-labelled urea (10 atom %), with or without Agrotain, applied to lysimeters or mini plots (un-labelled urea), either in granular form to the soil surface or in FPA form (through a spray) at a rate equivalent to 100 kg N ha⁻¹. Gaseous emissions of NH₃ and N₂O, NO₃⁻ leaching, herbage production, N response efficiency, total N uptake and total recovery of applied ¹⁵N in the plant and soil were determined up to 63 days. Urea-alone and urea with Agrotain, applied in FPA form, was more effective than its granular form and reduced N2O emissions by 5-12% and NO3- leaching losses by 31-55%. Urea-alone applied in FPA form had no significant effect in reducing NH₃ losses compared with granular form. However, urea with Agrotain applied in FPA form reduced NH₃ emissions by 69% compared with the equivalent granular treatment. Urea-alone and with Agrotain applied in FPA form increased herbage dry matter production by 27% and 38%, and N response efficiency compared with the equivalent granular urea application, respectively. Urea applied in FPA form resulted in significantly higher ¹⁵N recovery in the shoots compared with granular treatments – this was improved further when urea in FPA form was applied with Agrotain. Thus, treating urea with Agrotain in FPA under field conditions has the potential to delay its hydrolysis, minimise N losses and improve N use efficiency and herbage production. The lower dry matter production and N-response efficiency to urea applied in FPA form in Chapter 3 are probably because of additional factors such as lower application rates (25 kg N ha⁻¹ ) or lack of interception of urea by the leaves. Applying urea in FPA form is a good management strategy and I conclude that combining FPA urea with Agrotain has the potential to increase N use efficiency and herbage production further.

No description available.

Thesis (Ph.D.)--Murdoch University, 2008. / Thesis submitted to the Faculty of Sustainability Environmental and Life Sciences. Includes bibliographical references (leaves 237-251)

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on May 12, 2009) Includes bibliographical references.

Thesis (Ph.D.)--Michigan State University. Cell and Molecular Biology Program, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.

Humboldt-Universiẗat, Diss., 2001--Berlin.

A cultura algodoeira tem significativa participação na economia mundial. O algodão (Gossypium hirsutum) é predado por uma grande variedade de insetos, entre eles o percevejo manchador Dysdercus peruvianus (Hemiptera: Pyrrhocoridae). O hábito alimentar deste inseto provoca danos nas sementes e fibras depreciando-as para a utilização comercial. Como alternativa para o aumento da resistência das plantas ao ataque de insetos predadores, estratégias biotecnológicas vêm sendo consideradas. A Canavalia ensiformis é fonte de proteínas com atividade inseticida como a urease, a canatoxina e a concanavalina-A. Tanto a canatoxina como a urease exercem efeitos tóxicos em ninfas deste percevejo, causando mortalidade e atraso no desenvolvimento dos insetos sobreviventes. De maneira análoga a outros modelos de insetos já estudados, a toxicidade em D. peruvianus dependeria da ativação proteolítica das proteínas por enzimas digestivas (catepsinas) do inseto e formação de um peptídeo tóxico. Neste trabalho, estudos de hidrólise in vitro na presença de inibidores seguidos por SDS-page e Western-blot confirmaram a ativação proteolítica da urease por enzimas do tipo catepsina-B. O homogeneizado de intestinos de ninfas hidrolisa também substratos típicos para catepsinas, como hemoglobina e Abz-AIAFFSRQ-EDDnp, e a atividade proteolítica é bloqueada por pepstatina-A e E-64, inibidores específicos para aspártico e cisteíno proteinases, respectivamente. Duas cisteíno proteinases (DpQ-NR e DpQ-E) de intestinos de ninfas de D. peruvianus foram parcialmente purificadas e caracterizadas. Ambas apresentaram pH ótimo de atividade proteolítica entre 4,5 e 6,5, na presença de DTT e foram totalmente inibidas por E-64. No entanto o perfil de inibição evidencia a presença de outras enzimas nas frações. Ensaios de especificidade da fração DpQ-NR revelaram a preferência da enzima por substratos contendo resíduo hidrofóbico em P2, enquanto a enzima DpQ-E tem preferência por substratos contendo aminoácidos básicos em P1. Assim, o trabalho desenvolvido sugere a presença de proteinases múltiplas no sistema digestivo de D. peruvianus e o possível envolvimento destas peptidases na ativação proteolítica da urease. / Cotton (Gossypium hirsutum), an important agricultural commodity, is attacked by a number of pests, such as the cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) which may cause severe losses in cotton plantations. Bug feeding on the cotton seeds and stains the cotton fibers resulting in damage to the seed, besides being a vector for phytopathogenic bacteria and fungi. Biotechnology strategies focusing on plant defensive molecules are being considered as an alternative to increase host plant resistance against this pest. The jack bean Canavalia ensiformis is the source of proteins displaying insecticidal activity such as urease, canatoxin and concanavalin-A. Canatoxin and urease were shown to cause lethality and severe detrimental effects in surviving insects, delaying the development stages of D. peruvianus. As in other insect models studied previously, this would depend on the proteolytic activation of the protein by insect cathepsin-like digestive enzymes to produced toxic peptides. The in vitro hydrolysis of urease in the presence of inhibitors, followed by SDS-page and Wester-blot confirm the proteolytic activation by insect cathepsin B-like enzymes. A nymphal gut extract hydrolysed typical cathepsin substrates, such as hemoglobin and Abz-AIAFFSRQ-EDDnp, and hydrolysis was blocked by pepstatin-A and E-64, specific inhibitors of aspartic and cysteine proteinases. Two cysteine peptidases (DpQ-NR and DpQ-E) of D. peruvianus nymphs were partially purified and characterized. Both enzymes showed maximum activities at pH 4.5-5.0 and 5.5-6.5 in the presence of dithiotreitol and were totally inhibited by E-64. However, the inhibition profile points out to the presence of other types of proteinases in both fractions. DpQ-NR preferentially hydrolysed substrate with hydrophobic amino acids in P2, while DpQ-E had preference for substrate with basic residues in P1. Therefore, our work suggests the presence of multiple proteinases with distinct biochemical properties in the digestive system of D. peruvianus and the potencial involvement of these peptidases on the proteolytic activation of urease.