The hydrolysis of adenosine, 2-deoxyadenosine and 6-chloropurine riboside by adenosine deaminase preparations from human spleen has been investigated, and Km and Vm values have been determined. The effect of the substrate on the reaction velocity was followed over a 250-fold range Results showed no deviation from Michaelis-Menten kinetics. The effect of pH on Vm, Vm/Km, and the apparent activation energy was examined. Inactivation of the enzyme by p-chloromercuribenzoate suggests the involvement of one or more SH groups. Competitive inhibition by inosine and the fact that ammonia is not, an inhibitor support a reaction mechanism involving a ternary complex. The apparent activation energy of the a parameter was smaller or less sensitive to temperature than the ß parameter.Ball State UniversityMuncie, IN 47306

Upstream oil and gas production has caused soil salinity problems across western Canada. In this work we investigated the use of ACC (1-aminocyclopropane-1-carboxylate) deaminase-producing plant growth-promoting rhizobacteria (PGPR) and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices to enhance the efficiency and feasibility of phytoremediation of saline soils. This work involved laboratory and field research for three sites in south east Saskatchewan, Canada. The three research sites were Cannington Manor South (CMS), Cannington Manor North (CMN) and Alameda (AL). CMS and AL were highly saline, while the CMN site had moderate salinity. Indigenous PGPR were isolated from these sites and tested in greenhouse experiments using authentic salt-contaminated soils taken from the research sites. Increased plant biomass by PGPR and/or AMF was observed. This growth promotion effect varied with plant species, soil salinity and soil fertility. The combination treatment of two previously isolated PGPR Pseudomonas putida UW3 and UW4 (noted as UW3+4) from farm soil in Ontario consistently promoted shoot growth of both barley and oats grown in saline soils by approximately 100%. The indigenous PGPR Pseudomonas corrugata (CMH3) and Acinetobacter haemolyticus (CMH2) also promoted plant growth on par with UW3+4. In addition, in one experiment where alfalfa was tested, UW3+4, CMH2 and CMH3 treatments not only enhanced shoot biomass but also increased root nodulation. For AMF effects, G. intraradices enhanced biomass of oats and barley. Furthermore, the AMF+CMH3 was effective in promoting growth of Topgun ryegrass, while AMF+CMH2 was beneficial for Inferno tall fescue growth in salt impacted soils. The concentration of NaCl in the plants grown in salt-impacted soils ranged from 24 – 83 g/kg. There was no evidence of an increase in NaCl concentrations of plant tissue by PGPR and/or AMF treatments. In addition, to determine the importance of nutrient addition to research sites, liquid fertilizer was applied to 2-week old plants. Results demonstrated that fertilizer effectively increased biomass, and more importantly the biomass of PGPR treated plants supplied with fertilizer was approximately 20% higher than that of plants treated with fertilizer alone. Therefore, research sites were then amended with compost before planting of the 2007 field trial. Plant growth promotion by UW3+4 and CMH3 was tested in the summer of 2007 in the field. Prior to planting, soils were sampled from each site for soil salinity analysis. Barley, oats, tall fescue and ryegrass treated with and without PGPR were sown in plots. The plant coverage condition, NaCl concentrations and biomass of plant shoots were assessed to evaluate the PGPR effect. The results showed that PGPR promoted shoot dry weight by 30% - 175%. The NaCl concentrations of barley, oats and tall fescue averaged 53 g/kg, 66 g/kg and 35 g/kg, respectively. There was no evidence of an increase in NaCl concentrations of plant tissue by PGPR in the field. The salt removal of the CMN site was the highest among three sites due to the large amount of shoot biomass produced. The amount of salt accumulated in the shoots on the CMN site is estimated to be 1580 kg per hectare per year when both barley and ryegrass are planted together as a mix and treated with PGPR. Based on the field data, the estimated time required to remove 50% salt in the top 50 cm soil is seven years with PGPR treatments, while it takes fifteen years to do so without PGPR. In conclusion, PGPR-promoted phytoremediation was proven to be a feasible and effective remediation technique for soils with moderate salinity.

Upstream oil and gas production has caused soil salinity problems across western Canada. In this work we investigated the use of ACC (1-aminocyclopropane-1-carboxylate) deaminase-producing plant growth-promoting rhizobacteria (PGPR) and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices to enhance the efficiency and feasibility of phytoremediation of saline soils. This work involved laboratory and field research for three sites in south east Saskatchewan, Canada. The three research sites were Cannington Manor South (CMS), Cannington Manor North (CMN) and Alameda (AL). CMS and AL were highly saline, while the CMN site had moderate salinity. Indigenous PGPR were isolated from these sites and tested in greenhouse experiments using authentic salt-contaminated soils taken from the research sites. Increased plant biomass by PGPR and/or AMF was observed. This growth promotion effect varied with plant species, soil salinity and soil fertility. The combination treatment of two previously isolated PGPR Pseudomonas putida UW3 and UW4 (noted as UW3+4) from farm soil in Ontario consistently promoted shoot growth of both barley and oats grown in saline soils by approximately 100%. The indigenous PGPR Pseudomonas corrugata (CMH3) and Acinetobacter haemolyticus (CMH2) also promoted plant growth on par with UW3+4. In addition, in one experiment where alfalfa was tested, UW3+4, CMH2 and CMH3 treatments not only enhanced shoot biomass but also increased root nodulation. For AMF effects, G. intraradices enhanced biomass of oats and barley. Furthermore, the AMF+CMH3 was effective in promoting growth of Topgun ryegrass, while AMF+CMH2 was beneficial for Inferno tall fescue growth in salt impacted soils. The concentration of NaCl in the plants grown in salt-impacted soils ranged from 24 – 83 g/kg. There was no evidence of an increase in NaCl concentrations of plant tissue by PGPR and/or AMF treatments. In addition, to determine the importance of nutrient addition to research sites, liquid fertilizer was applied to 2-week old plants. Results demonstrated that fertilizer effectively increased biomass, and more importantly the biomass of PGPR treated plants supplied with fertilizer was approximately 20% higher than that of plants treated with fertilizer alone. Therefore, research sites were then amended with compost before planting of the 2007 field trial. Plant growth promotion by UW3+4 and CMH3 was tested in the summer of 2007 in the field. Prior to planting, soils were sampled from each site for soil salinity analysis. Barley, oats, tall fescue and ryegrass treated with and without PGPR were sown in plots. The plant coverage condition, NaCl concentrations and biomass of plant shoots were assessed to evaluate the PGPR effect. The results showed that PGPR promoted shoot dry weight by 30% - 175%. The NaCl concentrations of barley, oats and tall fescue averaged 53 g/kg, 66 g/kg and 35 g/kg, respectively. There was no evidence of an increase in NaCl concentrations of plant tissue by PGPR in the field. The salt removal of the CMN site was the highest among three sites due to the large amount of shoot biomass produced. The amount of salt accumulated in the shoots on the CMN site is estimated to be 1580 kg per hectare per year when both barley and ryegrass are planted together as a mix and treated with PGPR. Based on the field data, the estimated time required to remove 50% salt in the top 50 cm soil is seven years with PGPR treatments, while it takes fifteen years to do so without PGPR. In conclusion, PGPR-promoted phytoremediation was proven to be a feasible and effective remediation technique for soils with moderate salinity.

Previous studies of serum adenosine deaminase have neglected consideration of the two molecular forms of this enzyme that exist in human tissues. The purpose of this study was to survey the distribution of these forms in normal and pathological human sera. Both molecular forms were present in normal serum, though the small form predominated. This form also predominates in lymphocytes, erythrocytes, and in tissues with high specific activity of this enzyme. The ratio of the two forms is different for plasma and serum and can change with sample storage. The activity of the small form varied over a wider range than the activity of the large form in normal serum. Many pathological samples showed an altered distribution of the two forms. This study demonstrates the potential usefulness of serum forms of adenosine deaminase for distinguishing some pathological conditions.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / IntroduÃÃo: A leishmaniose visceral (LV) à uma doenÃa causada por protozoÃrios do gÃnero Leishmania e transmitida por insetos flebotomÃneos. O Brasil à uma das mais importantes Ãreas endÃmicas para a doenÃa, sendo necessÃrio o acompanhamento da ocorrÃncia dos casos atravÃs de uma vigilÃncia epidemiolÃgica ativa. A adenosina desaminase (ADA) à uma enzima que catalisa a desaminaÃÃo da adenosina produzindo inosina e amÃnia. Em humanos, està presente como duas isoenzimas e trÃs isoformas (ADA1, ADA1-CD26 e ADA2), sendo fundamental para o funcionamento do sistema imunolÃgico, uma vez que a deficiÃncia genÃtica da ADA1 provoca uma imunodeficiÃncia severa e combinada (SCID), bem como o vÃrus HIV à capaz de promover uma diminuiÃÃo na contagem dos linfÃcitos T via interaÃÃo com a ADA1-CD26. Objetivos: Descrever o perfil epidemiolÃgico da LV no Brasil (de 2001 a 2011) e no Cearà (de 2007 a 2011), bem como a atividade de ADA em pacientes acometidos pela LV. Metodologia: Realizou-se um estudo observacional descritivo da LV no Brasil e no estado do Cearà utilizando os dados secundÃrios disponibilizados pelo SINAN/MS, sendo categorizadas as Ãreas de transmissÃo; faixa etÃria, sexo e escolaridade dos casos; incidÃncia, prevalÃncia e evoluÃÃo da doenÃa; co-infecÃÃo HIV-LV e casos em gestantes. A ADA e suas isoenzimas foram determinadas em plasma de pacientes acometidos por LV utilizando o mÃtodo de Giusti, EHNA e por eletroforese. Os valores de cutoff para a ADA foram determinados utilizando a curva ROC. Resultados: A LV à endÃmica no Brasil, presente em 26 dos 27 estados, com uma mÃdia anual de ~3.600 casos, incidÃncia de ~1,79 casos/100.000hab e prevalÃncia de ~1,96 casos/100.000hab. No CearÃ, està presente em ~88% dos municÃpios, com mÃdia anual de ~600 casos, incidÃncia de 6,1casos/100.000hab e prevalÃncia de 7,1casos/100.000hab. A ADA està significativamente aumentada na LV, com uma atividade mÃdia de 106,8Â4,5U/L (vs controle 21,1Â0,6U/L). Este aumento ocorre com ambas isoenzimas, contudo a ADA2 à a principal isoenzima presente em pacientes acometidos por LV, com um valor de cutoff de 47,6 U/L para ADA total e 29,6U/L para a ADA2, utilizando adenosina. ConclusÃo: A LV à uma doenÃa endÃmica presente em todos os estados brasileiros, a exceÃÃo do Acre. Os estados do CearÃ, MaranhÃo e Minas Gerais apresentaram a maior quantidade de casos e o estado de Tocantins a maior incidÃncia e prevalÃncia. O Cearà à uma Ãrea endÃmica para a LV e a cidade de Fortaleza à o municÃpio que registrou a maior quantidade de casos no paÃs. A ADA pode ser utilizada como um marcador da resposta inflamatÃria na LV, e a determinaÃÃo da isoenzima ADA2 pode ser utilizada para avaliar a ativaÃÃo ou participaÃÃo de monÃcitos e macrÃfagos no processo infeccioso. / Introduction: Visceral leishmaniasis (VL) is a disease caused by protozoa of the genus Leishmania and transmitted by sandflies. Brazil is one of the most important endemic areas for the disease, being necessary monitoring of the occurrence of the disease through active epidemiological surveillance. Adenosine deaminase (ADA) is an enzyme that catalyzes the deamination of adenosine to produce inosine and ammonia. In humans, it is present as two isoenzymes and three isoforms (ADA1, ADA2 and ADA1-CD26) and is pivotal to immune system function, since ADA1âs genetic deficiency causes a severe combined immunodeficiency (SCID), as well as HIV is able to promote a decrease in the count of T lymphocytes via interaction with ADA1-CD26. Objectives: To describe the epidemiology of VL in Brazil (2001-2011) and Cearà (2007-2011); and the ADA activity in patients suffering from VL. Methods: We conducted an observational descriptive study of VL in Brazil and in the state of Cearà using secondary data provided by SINAN/MS, being categorized the transmission areas, age, sex and education of cases, incidence, prevalence and evolution of disease, co-infection HIV-VL and cases among pregnant women. The ADA and its isoenzymes were determined in plasma of patients suffering from LV using the method of Giusti, EHNA and electrophoresis. The cutoff values for the ADA were determined using the ROC curve. Results: VL is endemic in Brazil and is present in 26 of 27 states, with an annual average of ~ 3,600 cases, incidence of ~1.79 cases/100.000 inhabitants and prevalence of ~1.96 cases/100.000 inhabitants. In CearÃ, it is present in ~88 % of the municipalities, with an annual average of ~ 600 cases, incidence of 6.1 cases/100.000 inhabitants and prevalence of 7.1 cases/100.000 inhabitants. The ADA activity is significantly increased in LV, with an average activity of 106.8  4.5 U / L (vs. control 21.1  0.6 U / L). This increase occurs with both isoenzymes, but the ADA2 is the major isoenzyme present in patients suffering from LV, with a cutoff value of 47.6 U / L for total ADA activity and 29.6 U/L for ADA2 activity, using adenosine. Conclusion: LV is an endemic disease present in all Brazilian states, with the exception of Acre. The states of CearÃ, MaranhÃo and Minas Gerais had the highest number of cases and the state of Tocantins a higher incidence and prevalence. Cearà is an endemic area for VL and the city of Fortaleza is the municipality that the highest number of cases in the country. ADA can be used as a marker of the inflammatory response in VL, and the determination of isoenzyme ADA2 can be used to evaluate the activation or participation of monocytes and macrophages in the infectious process.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objectives: Diagnosis of tuberculous meningitis (TM) is a challenge in countries with a high burden of the disease and constrained resources and clinical prediction rules (CPRs) could be of assistance. We aimed at developing a CPR for diagnosis of TM in a Latin American setting with high tuberculosis incidence and a concentrated HIV epidemic. Methods: We enrolled adult patients with clinical suspicion of TM attending two hospitals in Lima, Peru. We obtained information on potential anamnestic, clinical and laboratory predictive findings that are easy to collect and promptly available. We independently diagnosed TM according to a composite reference standard that included a series of microbiological tests. We performed bivariate analysis and constructed a logistic regression model to select the predictive findings associated with TM. With the selected predictors included in the model, we developed a score-based CPR. We assessed its internal validity and diagnostic performance. Results: Of 155 analysed patients, 59 (38%) had TM. The CPR we derived includes three predictors: cough for 14 days or more, 10–500 cells in CSF and adenosine deaminase ≥ 6 U/l in CSF. It classifies patients into high-, moderate- or low-score groups and has an overall area under the ROC curve of 0.87. 59% of patients were assigned to either the high- or the low-score group, permitting prompt decision-making. In patients in the high-score group, it attains a positive likelihood ratio for TM of 10.6 and in patients with low scores, a negative likelihood ratio of 0.10. Bootstrap analysis indicated high internal validity. Conclusion: This CPR could support decision-making in patients with clinical suspicion of TM. External validation and further assessment of its clinical impact are necessary before application in other settings. / Revisión por pares

No description available.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 139-158).