In this research, a model system for studying the effects of the toxic metabolite, methylglyoxal, was created using Chinese Hamster Ovary (CHO) cells which produce tissue plasminogen activator (t-PA). The human gene for glyoxalase I was subcloned into an inducible mammalian expression vector. This vector was then used to create three stable CHO integrants, two control and one putative glyoxalase I producing cell lines. The CHO clones were characterized for the production of glyoxalase I using both SDS-PAGE gels and glyoxalase activity assays. In addition, the cell lines were evaluated to determine the levels of free methylglyoxal produced. The putative glyoxalase producer showed higher levels of glyoxalase I activity than the parent cell line and produced a unique protein band at the correct molecular weight. They also had a significantly lower level of free methylglyoxal than either of the two control cell lines. These cells can now be used as a tool to determine the specific effect of methylglyoxal on the yield and quality of tissue plasminogen activator produced. / Graduation date: 2000

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves.

published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis supervisor: David R. Sheff. Includes bibliographic references (p. 84-93).

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Pathology, 1985. / Some ill. mounted. Includes bibliographical references (leaves 145-160).

Some ill. mounted / Bibliography: leaves 145-160 / vii, 160, [58] leaves : ill. (some col.), maps ; 31 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, 1985

Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i_b), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i_a) antigen, had little to no biological activity. When fractions containing i_a and i_b were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i_a or i_b, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i_a and i_b, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i_a and i_b were mixed. Monospecific antisera against purified i_a and i_b neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i_a and i_b proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i_a protein is heat stable (85° C/15 min) while i_b lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i_a, but not i_b, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor. / Ph. D.

Orientador: Caio Antonio Carbonari / Coorientador: Edivaldo Domingues Velini / Banca: Fernando Tadeu de Carvalho / Banca: Marcus Barifouse Matallo / Banca: Cleber Daniel de Goes Maciel / Banca: Elza Alves Corrêa / Resumo: O glufosinate é derivado do fosfinotricina, uma toxina microbiana natural isolada a partir de duas espécies de fungos Streptomyces. Atua inibindo a atividade da enzima glutamina sintetase, que é necessária para a produção do aminoácido glutamina e para a desintoxicação da amônia pela planta. Objetivou-se neste trabalho avaliar a intoxicação e alterações fisiológicas e bioquímicas causadas pelo glufosinate em plantas de soja. O experimento foi conduzido em casa-de-vegetação, no Núcleo de Pesquisas Avançadas em Matologia (Nupam), pertencente à Faculdade de Ciências Agronômicas - FCA/UNESP. Foi utilizado o cultivar de soja Nidera NS 7100 (RR) plantada em vasos de cinco de litros de solo. Foram realizados dois experimentos, sendo o primeiro experimento instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio a 2,5 L p.c. ha-1 e glufosinate de potássio 2,5 L p.c. ha-1), três períodos de coleta de folhas dois, quatro e seis dias após aplicação e um tratamento testemunha sem aplicação. O segundo experimento foi instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio e glufosinate de potássio), três doses de cada produto (0,625 L p.c. ha-1; 1,25 L p.c. ha-1; 2,5 L p.c. ha-1) e um tratamento testemunha sem aplicação. As variáveis analisadas em ambos os experimentos foram, amônia, compostos pertencentes à rota metabólica de ação do glufosinate (glutamato, glutamina, serina, ácido aminolevulênico, glufosinate), análise visual de fitotoxicicidade e ETR (taxa de transporte de elétrons). Para determinação de amônia foram desenvolvidas metodologias de extração para posterior quantificação em espectrofotômetro. Para determinação dos compostos, foram realizados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The glufosinate is derived from phosphinothricin, a natural microbial toxin isolated from Streptomyces of two species of fungus. Acts inhibits the activity of glutamine synthetase, which is necessary for the production of the amino acid glutamine and for the detoxification of ammonia by the plant. The objective of this study was to evaluate the toxicity and physiological and biochemical changes caused by glufosinate in soybean plants. The experiment was carried out in green-house, in Faculty of Agronomic Sciences at São Paulo State University. We used soybean the cultivar NS 7100 Nidera planted in vases containing 5 liters of soil. Two experiments were carried out, the first experiment set with the following treatments: two formulations (glufosinate ammonium 2.5 L c.p. ha-1 and glufosinate potassium 4 2.5 L c.p. ha-1), three periods of leaf collection two, for and six days after application and a control without application. The second experiment was carried out with the following treatments: two formulations (glufosinate ammonium and glufosinate potassium), three doses of each product (0.625 L c.p. ha-1, 1.25 c.p. ha-1 L, 2.5 L c.p. ha -1) and a control without application. The variables analyzed in both experiments were ammonia, compounds belonging to the metabolic pathway of action of glufosinate (glutamate, glutamine, serine, aminolevulenic acid, glufosinate), visual analysis of plant injury and ETR (electron transport rate). To determine the ammonia were developed extraction methodologies for a quantification in spectrophotometric and for determination of compounds, extraction tests were performed to choose the most appropriate methodology, and development of analytical methods in LC-MS/MS (liquid chromatography and mass spectrometry). Plant injury evaluations were based on visual criteria and ETR measurements are given... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:30:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-16Bitstream added on 2014-06-13T19:00:30Z : No. of bitstreams: 1 barberis_lrm_dr_botfca.pdf: 840865 bytes, checksum: 2158433b6e8b218bb15ad11592a60db9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O glufosinate é derivado do fosfinotricina, uma toxina microbiana natural isolada a partir de duas espécies de fungos Streptomyces. Atua inibindo a atividade da enzima glutamina sintetase, que é necessária para a produção do aminoácido glutamina e para a desintoxicação da amônia pela planta. Objetivou-se neste trabalho avaliar a intoxicação e alterações fisiológicas e bioquímicas causadas pelo glufosinate em plantas de soja. O experimento foi conduzido em casa-de-vegetação, no Núcleo de Pesquisas Avançadas em Matologia (Nupam), pertencente à Faculdade de Ciências Agronômicas – FCA/UNESP. Foi utilizado o cultivar de soja Nidera NS 7100 (RR) plantada em vasos de cinco de litros de solo. Foram realizados dois experimentos, sendo o primeiro experimento instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio a 2,5 L p.c. ha-1 e glufosinate de potássio 2,5 L p.c. ha-1), três períodos de coleta de folhas dois, quatro e seis dias após aplicação e um tratamento testemunha sem aplicação. O segundo experimento foi instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio e glufosinate de potássio), três doses de cada produto (0,625 L p.c. ha-1; 1,25 L p.c. ha-1; 2,5 L p.c. ha-1) e um tratamento testemunha sem aplicação. As variáveis analisadas em ambos os experimentos foram, amônia, compostos pertencentes à rota metabólica de ação do glufosinate (glutamato, glutamina, serina, ácido aminolevulênico, glufosinate), análise visual de fitotoxicicidade e ETR (taxa de transporte de elétrons). Para determinação de amônia foram desenvolvidas metodologias de extração para posterior quantificação em espectrofotômetro. Para determinação dos compostos, foram realizados... / The glufosinate is derived from phosphinothricin, a natural microbial toxin isolated from Streptomyces of two species of fungus. Acts inhibits the activity of glutamine synthetase, which is necessary for the production of the amino acid glutamine and for the detoxification of ammonia by the plant. The objective of this study was to evaluate the toxicity and physiological and biochemical changes caused by glufosinate in soybean plants. The experiment was carried out in green-house, in Faculty of Agronomic Sciences at São Paulo State University. We used soybean the cultivar NS 7100 Nidera planted in vases containing 5 liters of soil. Two experiments were carried out, the first experiment set with the following treatments: two formulations (glufosinate ammonium 2.5 L c.p. ha-1 and glufosinate potassium 4 2.5 L c.p. ha-1), three periods of leaf collection two, for and six days after application and a control without application. The second experiment was carried out with the following treatments: two formulations (glufosinate ammonium and glufosinate potassium), three doses of each product (0.625 L c.p. ha-1, 1.25 c.p. ha-1 L, 2.5 L c.p. ha -1) and a control without application. The variables analyzed in both experiments were ammonia, compounds belonging to the metabolic pathway of action of glufosinate (glutamate, glutamine, serine, aminolevulenic acid, glufosinate), visual analysis of plant injury and ETR (electron transport rate). To determine the ammonia were developed extraction methodologies for a quantification in spectrophotometric and for determination of compounds, extraction tests were performed to choose the most appropriate methodology, and development of analytical methods in LC-MS/MS (liquid chromatography and mass spectrometry). Plant injury evaluations were based on visual criteria and ETR measurements are given... (Complete abstract click electronic access below)

Thesis (M. Tech. Environmental health) -- Central University of technology, Free State, 2011 / In nature microorganisms do not exist alone, but in association with one another. These kinds of associations can also be found in food industries, where cells of the same or different species can attach to pipes (biofilm formation) and a variety of surfaces in food processing environments and in food product such as yoghurt which can contain both yeast and bacteria originating from the starter culture as well as fruit. To control food spoilage organisms and food-borne pathogens preventative measures such as good manufacturing processes, the use of sanitizers and preservatives as well as hazard analysis critical control points (HACCP) are crucial in food industries. Sanitation of the working surface, floors, pipes, containers and equipment is a stepwise application of a detergent, acid or alkali rinse, a disinfectant treatment followed by final rinsing. If rinsing of the sanitizer is not done properly it may end up in the product in sub-lethal doses. In this study the influence of Liquid Hypochlorite (LH) and Liquid Iodophore (LI) sanitizers on organism growth and toxicity was evaluated. The organisms investigated included Escherichia coli 0113, Escherichia coli 026 and Zygosaccharomyces bailii Y-1535 in yeast malt broth, which was supplemented with LH and LI at sub-lethal concentrations 0.05% LH, 0.2% LH and 0.075% LI. Subsequently, bacterial and yeast growth responses as pure cultures and in combination (E. coli + Z. bailii) were measured as colony forming units and optical density values. Incorporation of the sanitizers in the growth media resulted in different levels of growth inhibition. Z. bailii proved more robust and the growth rate was not influence significantly by the addition of sanitizers or communal growth with either E. coli strains. The growth rate of both E. coli strains decreased where grown in combination with Z. bailii as well as in the presence of sanitizers, with the most influence exerted by LH. Changes in endotoxicity following the growth of the test samples (stressed cells) and the control (unstressed) were measured by the limulus amoebocyte lysate (LAL) and porcine IL-6 ELISA methods. Where E. coli strains were cultured together with Z. bailii the toxicity of tire mixture showed a decrease over time when measured with the limulus amoebocyte assay method. Interestingly the communal growth of the E. coli strains and Z bailii produced different toxicity profiles when the IL-6 porcine method was used, hi both cases, where E. coli strains were cultured together with Z. bailii the toxicity of the mixture showed an increase over tune when measured by this assay. Other than a similar toxicity profile for E. coli 0113 grown in pure culture, the comparison between results obtained using the LAL or porcine IL-6 methods yielded no correlation in determined toxicity. It was established that LH and LI sanitizers as well as communal growth had an influence in the toxicity of LPS/EPS and the method used to determine such toxicity should be carefully considered.