Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 77-84).

Marburg, Univ., Diss., 2009

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Orientador: Ladaslav Sodek / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-23T07:48:30Z (GMT). No. of bitstreams: 1 Goncalves_KatiaViviane_M.pdf: 2523264 bytes, checksum: 6d12338edf721f6a804d16a963fad313 (MD5) Previous issue date: 1997 / Resumo: Foi implementado um sistema in vitro para o crescimento de cotilédones imaturos de Crotalaria juncea L. Com a finalidade de estudar o metabolismo do nitrogênio, cotilédones com 39 dias após a antese cresceram por 8 dias em meio líquido de cultivo contendo diferentes fontes nitrogenadas. As atividades das enzimas asparaginase, glutamina sintetase, glutamato sintase e glutamato desidrogenase foram maiores quando asparagina foi utilizada como fonte de nitrogênio. O uso de glutamina proporcionou os melhores níveis de crescimento e deposição de proteínas de reserva. Asparagipa, entre as fontes testadas, foi a segunda melhor para a promoção do crescimento dos cotilédones. A menor eficiência da asparagina não pode ser explicada por deficiência na atividade de glutamina sintetase, limitação de asparaginase ou insuficiência de glutamato para a reação de glutamina sintetase / Abstract: An in vitro system for the growth of Crotalaria juncea L. was implemented. In order to study the metabolism of nitrogen, cotyledons collected 39 days after anthesis were grown for 8 days in a liquid culture medium containing different nitrogen sources. It was observed that the activities of the enzimes asparaginase, glutamine synthetase, glutamate syntase and glutamate dehydrogenase were greater when asparagine was used as a nitrogen source. Among the tested sources, glutamine provided the greatest growth rates and deposition of storage proteins, followed by asparagine. It was concluded that the lower efficiency obtained with asparagines can not be explained by a deficiency of the glutamine sinthetase activity, asparaginase limitation or glutamate insufficiency in the glutamine sinthetase reaction / Mestrado / Mestre em Ciências Biológicas

Heterologous enzymes have been investigated for a variety of therapeutic applications, including the treatment of a number of cancers that are sensitive to the systemic depletion of specific amino acids. One such example is acute lymphoblastic leukemia (ALL) for which enzyme-mediated L-Asparagine (L-Asn) depletion by the Escherichia coli L-Asparaginase II (EcAII) has been proven critical for treatment. However, the repeated or prolonged therapeutic administration of such enzymes is restricted by their immunogenicity, which frequently results in the generation of anti-enzyme antibodies that may in turn mediate a variety of adverse hypersensitivity reactions and neutralization of the enzymes themselves. Thus, while the therapeutic efficacy of asparaginase is well established, a significant number of patients still develop adverse immune responses to the enzyme. Here, we have developed and explored novel strategies towards engineering an asparaginase with reduced immunogenicity for ALL therapy. First, we identified and investigated human enzymes that putatively shared functional similarity to asparaginase with the long-term aim of engineering such enzymes to acquire biochemical and pharmacological properties requisite for eventual therapeutic application. In one study, we described the bacterial expression and characterization of the human asparaginase-like protein 1 (hASRGL1). We presented evidence that hASRGL1 exhibited an activity profile consistent with enzymes previously designated as [Beta]-aspartyl peptidases, which had only been previously identified in plants and bacteria. Similar to non-mammalian [Beta]-aspartyl peptidases, hASRGL1 was revealed to be an N-terminal nucleophile (Ntn) hydrolase whereby Thr168 serves as the essential Ntn for both intramolecular processing and catalysis. In a second study, we described the optimized bacterial expression and biochemical characterization of the human N-terminal asparagine amidohydrolase 1 (hNTAN1). We demonstrated that hNTAN1 catalysis is dependent upon direct involvement of a thiol group, and subsequently identified Cys75 as an essential residue that may act as the catalytic nucleophile. Further, we presented the first description of hNTAN1 kinetics, secondary structure composition, and thermal stability. Second, we devised and validated a novel therapeutic deimmunization approach by combinatorial T-cell epitope removal using neutral drift. We showed that combinatorial saturation mutagenesis coupled with a robust neutral drift screen enabled the isolation of engineered EcAII variants that contained multiple amino acid substitutions yet exhibited catalytic efficiencies nearly indistinguishable to that of the parent enzyme. Three regions of EcAII were computationally identified as putative T-cell epitopes and then subjected to saturation mutagenesis at 4 positions (per region) believed to be critical for MHC-II binding. The resulting libraries were then sequentially subjected to a neutral drift FACS screen in order to isolate EcAII mutants that retained wild-type function. Pools of neutral drift variants were then computationally evaluated for MHC-II binding and those that displayed scores indicative of compromised binding were purified and biochemically characterized. Finally, T-cell activation assays and antibody titers in HLA-transgenic mice were used to evaluate T-cell epitope removal and immunogenicity, respectively. Ultimately, we revealed that mice immunized with an EcAII neutral variant containing 8 amino acid substitutions -- 3 of which were non-phylogenetically conserved -- within computationally predicted T-cell epitopes, displayed a significant 10-fold reduction in serum anti-EcAII IgG titer relative to mice similarly immunized with the parent enzyme. / text

Orientador: Ladaslav Sodek / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-01T13:36:16Z (GMT). No. of bitstreams: 1 Lima_JulianaDomingues_D.pdf: 5527721 bytes, checksum: 83d0069fbdc08e5ae3fada2738225514 (MD5) Previous issue date: 2002 / Resumo: Foram conduzidos experimentos com plantas de soja e outras leguminosas em condições de casa-de-vegetação. Plantas noduladas e não-noduladas apresentaram aumento significativo do nível de ácido aspártico e redução da asparagina transportada na seiva do xilema sob condições que limitam a assimilação do nitrogênio. Em soja, a resposta foi revertida quando as condições ótimas para assimilação do nitrogênio foram restauradas. Plantas não-noduladas, crescidas em vasos contendo vermiculita, usando nitrato como fonte de nitrogênio, apresentaram este fenômeno quando transferidas para hidroponia em solução deficiente em nitrogênio. Subseqüentemente, quando nitrato foi novamente fornecido o efeito foi revertido. Plantas noduladas crescidas em vermiculita recebendo solução deficiente em nitrogênio e totalmente dependente da fixação do N2 como fonte de nitrogênio apresentaram a mesma resposta quando transferi das para hidroponia com aeração. Neste caso, após a transferência para hidroponia os níveis de ureídeos na seiva do xilema decresceram significativamente sugerindo, com outras evidências, que a fixação do nitrogênio foi prejudicada nessas condições. Com o retomo destas plantas para vermiculita, ambos a razão ASPI ASN e o nível de ureídeos retomaram aos valores iniciais. Durante o crescimento e a nodulação de plantas de soja cultivadas em vermiculita em solução nutritiva deficiente em nitrogênio, o fenômeno foi observado durante curto período que corresponde à fase de "fome de N". Esta fase é caracterizada pelo transitório amarelecimento das folhas e ocorre quando as reservas são exauridas e a nodulação não está suficientemente desenvolvida para manter a planta com nitrogênio fixado. Seguindo os tratamentos descritos acima, a análise dos aminoácidos livres do floema, raízes e nódulos também revelou aumento da razão ASP/ASN mas, muito menos pronunciado do que o encontrado na seiva do xilema. A atividade da asparagina sintetase foi investigada no sistema radicular para tentar elucidar os fatores envolvidos no fenômeno. A atividade da enzima nos nódulos sofreu uma queda nos tratamentos que elevaram a razão ASPI ASN como a transferência das plantas noduladas para hidroponia e a queda foi recuperada com o retomo das plantas para vermiculita quando a razão ASP/ASN atingiu o nível normal. Entretanto, houve insucesso na dosagem da atividade da asparagina sintetase nas raízes. Evidentemente, em termos qualitativos, o transporte de nitrogênio na seiva do xilema parece ser um indicador bastante sensível do status de nitrogênio da planta / Abstract: Experiments with soybean plants and other legumes were conducted under greenhouse conditions. Both nodulated and non-nodulated plants were found to present substantially increased aspartic acid levels and lower asparagine in the xylem bleeding sap under conditions that limit nitrogen assimilation. In soybean, the response was reversed when optimum conditions for nitrogen assimilation were restored. Non-nodulated plants, grown in pots with vermiculite using nitrate as sole source of nitrogen, presented this phenomenon when transferred to N-deficient water-culture. Subsequent return to nitrate reversed the effect. Nodulated plants grown in vermiculite with N-deficient nutrient solution and totally dependent on N2-fixation as a source of nitrogen presented the same response when transferred to aerated water -culture. In this case xylem ureide levels also fell substantially suggesting, along with other evidence, that nitrogen fixation was impaired under these conditions. On return of plants to vermiculite, both the Asp/Asn ratios and ureide levels returned to initial values. During growth and nodulation of soybean plants cultivated in vermiculite with N-deficient nutrient solution, the phenomenon was observed over a short period corresponding to the "N hunger" phase. This phase is characterised by transient yellowing of the leaves and occurs when reserves are depleted and nodulation is not yet sufficiently developed to maintain the plant with fixed nitrogen. Following the above-described treatments, an analysis of free amino acids in the phloem, roots and nodules also revealed some increase in the Asp/ Asn ratios but these were much less pronounced than those found for the xylem. Asparagine synthetase activity was investigated in the root system in an attempt to elucidate the factors underlying the phenomenon. Activity in the nodules was found to fall during treatments which elevated the Asp/Asn ratio such as transfer of nodulated plants to water-culture and the fall in activity reversed on return of the plants to vermiculite when ratios returned to normal. However, measurement of asparagine synthetase activity in the roots was unsuccessful. Evidently, in qualitative terms, nitrogen transport in the xylem appears to be very sensitive to the nitrogen status of the plant / Doutorado / Doutor em Biologia Vegetal

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Orientador: Marcos Antônio de Oliveira / Resumo: L-asparaginases (ASNases) bacterianas são importantes biofármacos utilizados no tratamento de leucemia linfoide aguda (LLA), uma vez que este tipo tumoral é dependente da disponibilidade de asparagina (Asn) extracelular. As ASNases bacterianas são capazes de hidrolisar eficientemente Asn em ácido aspártico (Asp) e amônia (NH3), diminuindo a disponibilidade de Asn para células tumorais e induzindo apoptose. Comercialmente, indústrias farmacêuticas internacionais produzem ASNases de Escherichia coli e Erwinia chrysanthemi, entretanto, ASNase de nenhuma origem é produzida pelas indústrias farmacêuticas brasileiras. Adicionalmente, o tratamento com estas enzimas produz efeitos colaterais, entre eles imunogênicos, que estão relacionados com a alta massa molecular da enzima (140kDa) e neurológicos, atribuídos a atividade secundária de glutaminase (GLNase). Neste contexto, fontes alternativas destas enzimas e também a auto-suficiência em suas produções são importantes para mitigar os efeitos colaterais e evitar falhas no tratamento devido a flutuações internacionais de sua fabricação. Neste trabalho, realizamos a caracterização de uma ASNase de levedura, denominada de ASNaseM que compartilha elevada homologia (maior que 30% de identidade e 40% de similaridade) com as enzimas bacterianas utilizadas no tratamento da LLA e que possui todos os aminoácidos envolvidos na catálise conservados, sugerindo uma fonte alternativa potencial para o tratamento da LLA. Experimentos de cromatografia... (Resumo completo, clicar acesso eletrônico abaixo) / Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chro / Doutor

Growth of Escherichia coli A-l under aerobic conditions in an enriched medium with a total amount of 0.2 per cent glucose was biphasic and asparaginase II activity was detected after depletion of ammonia from the growth medium in the second phase of growth. Glucose was exhausted two hours before ammonia and three hours before asparaginase II activity was detected. The concentration of 3',5'-cyclic adenosine monophosphate was found to fluctuate when the dissolved oxygen in the medium reached a low level, when glucose and ammonia were exhausted, and when the cells entered the second stationary phase of growth. Culture tube studies of the growth of E_j_ coli A-l in three per cent nutrient broth with varied concentrations of ammonium chloride and potassium nitrate gave lower specific activity of asparaginase II when this was compared to that seen in three per cent nutrient broth alone. The addition of glucose to the same medium before asparaginase II activity was detected resulted in the production of acid by E. coli A-l with cessation of growth; however, addition after L-asparaginase synthesis had started did not affect the specific activity of the enzyme. The addition of ammonium chloride suppressed L-asparaginase synthesis, but addition after enzyme synthesis started had no affect. These findings suggest that asparaginase II is produced by E. coli A-l in response to low concentrations of ammonia and that exogenously supplied nitrogen compounds may play a major role in the regulation of this enzyme. It is suggested that E. coli A-l produced L-asparaginase in order to obtain ammonia for the synthesis of glutamine from glutamate. The synthesis of glutamine from glutamate is the first step of a highly branched pathway which ultimately leads to the synthesis of many of the important macromolecules of the cell.