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.

Adenosine deaminase has been shown to consist of three molecular weight forms, A, B, and C. In higher mammals, the A and C forms are dominant while in lower mammals, the B and C forms are dominant. In this work, the B and C forms were isolated from the frog liver and several kinetic parameters were determined.Ammonium sulfate salt fractionation, starting at 40 percent and increased by 5 percent increments to 80 percent was used to separate the two forms. The B form adenosine deaminase was predominantly found in the 50 to 60 percent precipitate fractions while the C form was predominant in precipitate fractions containing more than 60 percent ammonium sulfate. The rechromatographed B and C forms were subjected to isoelectric focusing and thin layer electrophoresis. The B form separated into three activity bands while the C form separated into two activity bands`. Michaelis constant values were determined to be 4.61 X 10-5M and 2.00 X 10-5M for the B and C forms with adenosine as a substrate, respectively. The relative substrate specificity ratio showed that the B form was very specific for adenosine.In conclusion, the B form adenosine deaminase was found to be dominant in the frog liver. The Michaelis constant, relative substrate specificity ratio, thin layer electrophoresis and isoelectric focusing distinguished between the adenosine deaminase B and C forms. The technique of ammonium sulfate fractionation gave excellent separation between the B and C forms of adenosine deaminase.Ball State UniversityMuncie, IN 47306

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xii, 195 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Made available in DSpace on 2016-01-26T12:51:34Z (GMT). No. of bitstreams: 1 danielaprudenteteixeiranunes_dissert.pdf: 2007548 bytes, checksum: 1b9b96cf5d50556af914f8157e7d26ec (MD5) Previous issue date: 2010-12-16 / Adenosine deaminase (ADA), an enzyme coded by ADA gene (20q13.11) acts in adenosine metabolism and it is involved in the modulation of the immune response. ADA gene G22A polymorphism originates two co-dominants alleles ADA*01 and ADA*02 and influences the level of ADA enzyme in the organism. Apparently it has a fundamental role in gestational maintenance. The ADA*02 allele has been associated as protector effect against recurrent spontaneous abortion (RSA) in European Caucasian women. Aim: To investigate if ADA gene G22A polymorphism is associated with occurrence of RSA in Brazilian women. Methods: After obtaining the written consent 311 women were selected to compose two groups: G1 with previous history of RSA (n=129) and G2 without previous history of RSA (n=182). Genomic DNA was isolated from peripheral blood using commercial kits. The PCR-RFLP method was used to identify ADA gene G22A polymorphism. p>0005 was considered statistically significant. Results: The frequencies of ADA*01;*01, ADA*01;*02 and ADA*02;*02 genotypes were similar in both groups (G1 and G2) with no statistically significance differences observed (p = 0,7170; x2 = 0,6653; GL = 2). ADA*01 and ADA*02 alleles frequencies were 95,6% and 4,4% in G1 group and 94,9% and 5,1% in G2 group, respectively (p = 0,8433; OR = 1,179; CI 95%: 0,5340 2.601). Conclusion: The results suggest that ADA alleles ADA*01 and ADA*02 are not associated with RSA. It xvi is possible that the reduction of ADA levels resulting from the presence of at least one ADA*02 allele do not have a role against abortion in Brazilian women. / Polimorfismo G22A do gene ADA e abortamento espontâneo recorrente: ausência de associação Introdução: A adenosina deaminase (ADA), uma enzima codificada pelo gene ADA (20q13.11), atua no metabolismo da adenosina e modula a resposta imune. O polimorfismo G22A deste gene origina os alelos co-dominantes ADA*01 e ADA*02 e influencia o nível de expressão da enzima ADA, que possui papel fundamental na manutenção da gestação. O alelo ADA*02 tem sido associado a um efeito protetor contra o abortamento espontâneo recorrente (AER) em mulheres caucasianas européias. Objetivo: Investigar se o polimorfismo G22A do gene ADA se associa à ocorrência de AER em brasileiras. Métodos: Após obtenção do Termo de Consentimento Livre e Esclarecido (Parecer CEP FAMERP 308/2008), 311 mulheres foram selecionadas para compor dois grupos: G1 com histórico de AER (N=129) e G2 sem histórico de AER (N=182). O DNA genômico foi extraído a partir de sangue periférico com o uso kit comercial. O polimorfismo G22A do gene ADA foi identificado com o uso do método PCR-RFLP. O valor p>0,005 foi considerado significante. Resultados: As frequências dos genótipos ADA*01;*01, ADA*01;*02 e ADA*02;*02 foram semelhantes entre os grupos e não apresentaram diferenças estatisticamente significantes (p = 0,7170; χ2 = 0,6653; GL = 2). As frequências dos alelos ADA*01 e ADA*02 em G1 foram iguais a 95,6% e 4,4%; em G2, 94,9% e 5,1%, respectivamente (p=0,8433; OR=1,179; IC 95%: 0,5340-2.601). Conclusões: Os resultados sugerem que xiv os alelos ADA*01 e ADA*02 do gene ADA não estão associados ao AER. É possível que a redução nos níveis da ADA resultantes do alelo ADA*02 não apresente um efeito protetor contra o AER em brasileiras.

The tilapias are a group of African and Middle Eastern cichlid fish that are widely cultured in developed and developing countries. With many different species and sub-species, and extensive use of interspecies hybrids, identification of tilapia species is of importance in aquaculture and in wild populations where introductions occur. This research set out to distinguish between tilapia species and sub-species by retrieving species-specific nuclear DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions of the ADA gene; and (ii) next-generation sequencing, both standard RADseq and double-digest RADseq (ddRADseq). The mitochondrial DNA (mtDNA) marker cytochrome c oxidase subunit I (COI) was used to verify tilapia species status. ADA gene sequence analysis was partially successful, generating SNP markers that distinguished some species pairs. Most species could also be discriminated using the COI sequence. Reference based analysis (RBA: using only markers found in the O. niloticus genome sequence) of standard RADseq data identified 1,613 SNPs in 1,002 shared RAD loci among seven species. De novo based analysis (DBA: based on the entire data set) identified 1,358 SNPs in 825 loci and RBA detected 938 SNPs in 571 shared RAD loci from ddRADseq among 10 species. Phylogenetic trees based on shared SNP markers indicated similar patterns to most prior phylogenies based on other characteristics. The standard RADseq detected 677 species-specific SNP markers from the entire data set (seven species), while the ddRADseq retrieved 38 (among ten species). Furthermore, 37 such SNP markers were identified from ddRADseq data from a subset of four economically important species which are often involved in hybridization in aquaculture, and larger numbers of SNP markers distinguished between species pairs in this group. In summary, these SNPs are a valuable resource in further investigating hybridization and introgression in a range of captive and wild stocks of tilapias.

by Mo-Lung Chen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 70-75). / Abstract also in Chinese. / ABBREVIATIONS --- p.iv / LIST OF TABLES --- p.v / LIST OF FIGURES --- p.vii / ACKNOWLEDGEMENT --- p.ix / ABSTRACT --- p.xi / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- BACKGROUND --- p.4 / Chapter 2.1 --- Production of pleural fluid --- p.4 / Chapter 2.2 --- Pathophysiology of pleural effusion --- p.5 / Chapter 2.3 --- Separating exudate from transudate --- p.8 / Chapter 2.4 --- Receiver operating characteristic curve --- p.9 / Chapter CHAPTER 3. --- ADENOSINE DEAMINASE --- p.12 / Chapter 3.1 --- Background --- p.12 / Chapter 3.2 --- Differentiation of tuberculous and non-tuberculous pleural effusion --- p.12 / Chapter CHAPTER 4. --- MATERIALS AND METHODS --- p.17 / Chapter 4.1 --- Patients --- p.17 / Chapter 4.2 --- Collection and handling of specimens --- p.17 / Chapter 4.3 --- Diagnostic criteria --- p.18 / Chapter 4.4 --- Methods --- p.19 / Chapter 4.4.1 --- Routine chemistries --- p.19 / Chapter 4.4.2 --- Protein zone electrophoresis --- p.19 / Chapter 4.4.3 --- Adenosine deaminase --- p.19 / Chapter 4.4.3.1 --- Instrumentation --- p.22 / Chapter 4.4.3.2 --- Optimization of reaction time --- p.24 / Chapter 4.4.4 --- Analytical performance --- p.24 / Chapter 4.4.4.1 --- Imprecision --- p.24 / Chapter 4.4.4.2 --- Recovery --- p.26 / Chapter 4.4.4.3 --- Lowest detection limit --- p.26 / Chapter 4.4.4.4 --- Linearity --- p.26 / Chapter 4.4.4.5 --- Interference by ammonia --- p.26 / Chapter 4.4.4.6 --- Interference by turbidity --- p.28 / Chapter 4.4.4.7 --- Interference by haemoglobin --- p.28 / Chapter 4.4.4.8 --- Interference by bilirubin --- p.29 / Chapter 4.4.4.9 --- Storage stability of ADA at -80°C --- p.29 / Chapter 4.4.5 --- Statistical analysis --- p.30 / Chapter CHAPTER 5. --- RESULTS OF OPTIMIZATION AND EVALUATION EXPERIMENTS --- p.31 / Chapter 5.1 --- Optimization of reaction time --- p.31 / Chapter 5.2 --- Analytical performance --- p.31 / Chapter 5.2.1 --- Imprecision --- p.31 / Chapter 5.2.1.1 --- Within-run --- p.31 / Chapter 5.2.1.2 --- Between-run --- p.31 / Chapter 5.2.2 --- Recovery --- p.31 / Chapter 5.2.3 --- Lowest detection limit --- p.34 / Chapter 5.2.4 --- Linearity --- p.34 / Chapter 5.2.5 --- Interference by / Chapter 5.2.5.1 --- ammonia --- p.34 / Chapter 5.2.5.2 --- turbidity --- p.34 / Chapter 5.2.5.3 --- haemoglobin --- p.37 / Chapter 5.2.5.4 --- bilirubin --- p.37 / Chapter 5.2.6 --- Storage stability of ADA at -80°C --- p.37 / Chapter CHAPTER 6. --- TRANSUDATIVE AND EXUDATIVE PLEURAL EFFUSION --- p.39 / Chapter 6.1 --- Results of routine chemistries --- p.39 / Chapter 6.2 --- Decision thresholds by ROC curve --- p.39 / Chapter 6.3 --- Discussion --- p.39 / Chapter 6.4 --- Results of protein zone electrophoresis --- p.49 / Chapter 6.5 --- Discussion --- p.51 / Chapter 6.6 --- Comparison of protein zone electrophoresis and Light's criteria --- p.55 / Chapter 6.7 --- Discussion --- p.55 / Chapter CHAPTER 7. --- TUBERCULOUS AND NON-TUBERCULOUS EXUDATIVE PLEURAL EFFUSION --- p.59 / Chapter 7.1 --- Results of adenosine deaminase assay --- p.59 / Chapter 7.2 --- Combinations of analysis --- p.59 / Chapter 7.3 --- Decision thresholds by ROC curve --- p.64 / Chapter 7.4 --- Discussion --- p.64 / Chapter CHAPTER8. --- GENERAL DISCUSSION --- p.69 / REFERENCES --- p.70

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A adenosina desaminase (ADA â E.C.3.5.4.4.) à uma enzima fundamental no catabolismo das purinas. Ela catalisa a desaminaÃÃo da adenosina ou 2âdeoxi-adenosina produzindo amÃnia e inosina ou 2â-deoxi-inosina, respectivamente. Sua atividade à expressa por 2 isoenzimas presentes em 3 isoformas. A ADA1 (36kDa) ou ADA1 ligada ao CD26 (280kDa) sÃo amplamente distribuÃdas nos tecidos. Sua aÃÃo à particularmente importante porque altos nÃveis de 2âdeoxi-adenosina sÃo tÃxicos para as cÃlulas do sistema imunolÃgico. A ADA2 (100kDa) à normalmente encontrada no soro e sintetizada somente pelo sistema monocÃtico-macrofÃgico. A importÃncia biolÃgica da ADA2 ainda nÃo està totalmente estabelecida, principalmente devido as suas caracterÃsticas cinÃticas. O presente trabalho teve como objetivo discriminar as isoenzimas da adenosina desaminase humana atravÃs de eletroforese em gel de agarose e pelo modelo proposto por Vale e Almeida (1998), bem como realizar um estudo descritivo retrospectivo sobre o perfil dos exames de ADA no Estado do CearÃ. As amostras de lÃquido ascÃtico, pleural e pericÃrdico foram submetidas à eletroforese em agarose a 1% a 80 V por 7 horas. O gel foi fatiado e cada fatia foi incubada em adenosina (22 ou 0,55mM) por 20 horas para a detecÃÃo da amÃnia liberada pela reaÃÃo enzimÃtica. Os resultados encontrados a partir da eletroforese foram comparados com os resultados achados pelo modelo de Vale e Almeida (1998). O lÃquido pleural à o fluido que à mais frequentemente solicitado para a determinaÃÃo da ADA, seguido pelos lÃquidos ascÃtico, cefalorraquidiano, pericÃrdico e soro. Observamos que os valores de atividade enzimÃtica sÃo influenciados pelo tipo de lÃquido corporal onde a enzima se encontra, podendo estar relacionada Ãs barreiras corporais, tais como a barreira hematoencefÃlica. A partir dos resultados obtidos, podemos concluir que o modelo matemÃtico proposto pode ser usado em laboratÃrios clÃnicos para discriminar as isoenzimas da ADA. / Adenosine deaminase (ADA â E.C.3.5.4.4.) is a fundamental enzyme in the catabolism of the purines. It catalyzes the deamination of adenosine or 2âdeoxy-adenosine producing ammonium and inosine or 2â-deoxyinosine, respectively. Its activity is expressed by two isoenzymes presented in three isoforms. ADA1 (36 kDa) and ADA1 bound to CD26 (280kDa) are widely distributed in the body tissues. Their action is particularly important because high levels of 2âdeoxy-adenosine are toxic for the immune system cells. ADA2 (100kDa) is normally found in serum and is synthesized only in monocyte-macrophage system. The biological importance of ADA2 is not yet fully clear, especially for its kinetics characteristics. The objective of the present work was to discriminate the isoenzymes of human adenosine deaminase using agarose electrophoresis and by mathematical model proposed by Vale and Almeida (1998). In addition, we performed a study of the profile of ADA tests in State of Ceara (Brazil). Samples of of ascites, pleural and pericardial effusion were submitted to electrophoresis in 1% agarose at 80V for 7 hours. The gel was sliced and each slice was incubated in adenosine (22 or 0,55mM) for 20 hours to detect the ammonium released by enzymatic reaction. The results found from electrophoresis were compatible with the model proposed by Vale and Almeida (1998). The pleural fluid is the most frequently requested for the determination of ADA, followed by ascitic fluid, cerebrospinal fluid, pericardial fluid and serum. We observed that the value of enzymatic activity is influenced by corporal fluid type where the enzyme is localized. These data can be associated with the corporal barrier, like brain barrier. We concluded that the proposed mathematical model could be used in clinical laboratories to discriminate ADA isoenzymes to improve the diagnostic method.