A alga marinha Gracilaria tenuistipitata var. liui Zhang et Xia (Gracilariales, Rhodophyta) tem grande importância econômica por ser a principal fonte para a produção de ficocolóides. Esta linhagem tem sido extensivamente cultivada em tanques ou diretamente no mar na China e Taiwan por exibir grande tolerância a variação de fatores ambientais. A maior fonte de nitrogênio no oceano está na forma de nitrato que é o principal fator limitante no crescimento das macroalgas. A assimilação de nitrogênio é um processo que ocorre em duas etapas catalisadas sequencialmente pelas enzimas nitrato redutase (NR) e nitrito redutase (NiR), respectivamente. A NR catalisa a redução de nitrato a nitrito usando NAD(P)H como fonte doadora de elétrons. O nitrito é reduzido a amônio (pela NiR) e é imediatamente assimilado em compostos orgânicos nitrogenados, como aminoácidos e bases nitrogenadas. A NR é considerada o primeiro passo regulador no processo de assimilação de nitrato. Neste trabalho descrevemos a oscilação circadiana da atividade da NR bem como do seu conteúdo protéico em G. tenuistipitata. A atividade da NR e os níveis da proteína NR apresentam um pico no meio do dia em algas mantidas sob regime de claro:escuro (12:12h). A atividade de NR é 30 vezes maior durante o período de claro em algas crescendo sob condições de luz:escuro, e o nível protéico de NR é cerca de 40 vezes maior em extratos do período de claro. A oscilação observada está sob controle do relógio biológico uma vez que a flutuação da atividade da NR persiste após 10 dias sob condições de claro constante. Nenhuma oscilação na atividade de NR foi encontrada quando a alga foi mantida sob escuro constante, demonstrando que a luz é um fator de grande importância no controle desta enzima. Experimentos de absorção de nitrato do meio realizados em culturas de algas crescendo sob condições de claro:escuro mostrou que a absorção de nitrato ocorre preferencialmente no período noturno, ocorrendo o pico máximo de absorção no meio da noite. Daí concluirmos que os processos de assimilação de nitrato e tomada do mesmo do meio estejam ocorrendo de maneira independente. A NR de G. tenuistipitata pertence a classe das NRs específicas para o NADH, com constante de Michaelis-Menten aparente de 95 µM. O KM aparente para o nitrato foi estimado em 197 µM, valor esse razoável com os descritos para outras algas. A focalização isoelétrica revelou que a NR é uma proteína básica com pI de 8,66. A NR desta espécie foi purificada em 4 etapas: cromatografia de troca iônica, precipitação com sulfato de amônio, filtração em gel e cromatografia de afinidade em resina Affigel-blue. A proteína não desnaturada apresenta massa molecular de aproximadamente 420 kDa, e 4 subunidades idênticas de 110 kDa, baseado em SDS-PAGE. A localização intracelular da NR de G. tenuistipitata foi examinada utilizando técnicas de imunofluorescência e \"immuno-gold\" e revelaram que a NR está associada com os cloroplastos. / The marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia) is economically important, being the main source for the production of phycocolloids. This strain is extensively cultivated in ponds in southern China and Taiwan and exhibits a wide tolerance to environmental factors. The major source of nitrogen in the ocean is in the form of nitrate being the main limiting macroalgal production. The nitrogen assimilation process occurs in a two-step reaction catalyzed by 2 enzymes working sequentially, nitrate reductase (NR) and nitrite reductase (NiR). NR catalyzes the reduction of nitrate to nitrite using NAD(P)H as electron donor. Nitrite is reduced to ammonium (by NiR) and it is immediately assimilated in organic nitrogen compounds, such as amino acids and bases. NR is considered the first and regulatory step in this assimilating process. We report the circadian oscillation of NR activity as well as NR protein content in G. tenuistipitata. Both NR activity and NR protein levels peaks at midday in algae maintained under light:dark cycle (12:12h). Toe NR activity is 30 fold higher in light period for alga growing under light-dark conditions, and the NR protein level is about 40 fold higher in extracts from the light period. The oscillation observed is under biological clock control since the fluctuation of NR activity persists after 10 days under constant light conditions. No oscillation in the NR activity is found when the algae were maintained under constant dark condition. The experiments on nitrate uptake carried out in algae growing under light:dark conditions had shown that the absorption peaks at the middle of the dark periods. So we can conclude that the process of nitrate assimilation and nitrate uptake is occurring in such an independent ways. NR from G. tenuistipitata is a NADH specific enzyme with Michaelis-Menten apparent constant of 95 µM. Toe apparent KM for nitrate was estimated to be 197 µM, which is reasonable with the values described to another algae. The isoeletric focusing revealed NR is a basic protein with pi of 8.66. The NR from was purified in four steps: ion exchange chromatography, ammonium sulfate precipitation, gel filtration chromatography, and Affigel-blue affinity column. Non-denaturated protein shows a molecular mass of about 420 kDa, and 4 identical subunits of 110 kDa, based on SDS-PAGE. Toe intracellular localization of NR in G. tenuistipitata was examined by applying both immunofluorescence and immunogold methods revealing NR associated with chloroplasts.

Os artrópodes constituem as formas mais abundantes de vida animal no planeta. O sistema digestivo dos Arthropoda é uma das mais importantes superfícies de contato com o ambiente. Pouco se conhece a respeito do sistema digestivo dos aracnídeos. Identificamos as principais carboidrases digestivas em três Arachnida modelo: Tityus serrulatus, Amblyomma cajennense e Nephilengys cruentata. As α-fucosidases são glicosídeo hidrolases que catalisam a hidrólise de ligações glicosídicas entre uma fucose ligada a outro carboidrato ou outro tipo de molécula. Estas enzimas são fundamentais no processamento de glicoproteínas e glicolipídeos. Observamos altas atividades de α-fucosidases em todas as espécies estudadas e estas enzimas foram isoladas e caracterizadas. / Arthropods are the most abundant animal life form on the planet. The digestive system of Arthropoda is one of the most important areas of contact with the environment. The study of Arachnida digestive enzymes has been sporadic and remains a largely unexplored area. We identified the most important digestive carbohydrases in three Arachnida models: Tityus serrulatus, Amblyomma cajennense and Nephilengys cruentata. The α-fucosidases are glycoside hydrolases that catalyze the hydrolysis of glycosidic links between a fucose linked to other carbohydrate or another type of molecule. These enzymes are essential in the processing of glycoprotein and glycolipids. We showed that all models tested presented high activities of digestive α-fucosidases and these enzymes were isolated and characterized.

A paraoxonase sérica humana (PON) vem sendo amplamente estudada. Além da capacidade de PON1 em hidrolisar compostos organofosfatados, sabe-se, atualmente, que toda a família PON (composta por PON1, PON2 e PON3) promove a proteção de lípides, incluindo-se a lipoproteína de baixa densidade (LDL) contra a oxidação. O gene da PON1 sérica apresenta dois sítios polimórficos bem determinados: a troca Gln192Arg (Q/R) e Met55Leu, os quais estão associados com diferenças na atividade e concentrações séricas da enzima, respectivamente. Também o polimorfismo Cys311Ser parece contribuir sinergisticamente com o alelo PON1-192R quanto ao risco cardiovascular em algumas populações. Já foi demonstrado, por sua vez, que pacientes infectados pelo vírus HIV podem desenvolver dislipidemia e que tanto a atividade como a concentração de PON1 podem ser influenciadas por esta infecção. O objetivo deste estudo foi determinar as freqüências alélicas dos polimorfismos genéticos PON1-192QR, PON1-55LM, PON2-311SC e PON2-148AG, bem como avaliar a atividade de PON1 e a peroxidação lipídica no plasma de indivíduos portadores de HIV. Materiais e Métodos após aprovação pela comissão de ética e da aplicação do termo de consentimento pós-esclarecido, 350 (264 homens e 86 mulheres) pacientes infectados pelo HIV foram incluídos no estudo. Foi avaliado ainda um grupo de 32 (23 homens e 9 mulheres) indivíduos recentemente diagnosticados como portadores do vírus. Uma população saudável composta por 179 doadores de sangue, todos de sexo masculino, foi avaliada como controle. Após a extração do DNA, procedeu-se à genotipagem para os polimorfismos de PON1 e PON2 através de PCR-RFLP. A atividade paraoxonase de PON1 foi avaliada por espectrofotometria empregando-se paraoxon como substrato. O colesterol total, VLDL-colesterol, HDL-colesterol e triglicérides foram determinados por métodos padrão. A fração LDL-colesterol foi calculada pela fórmula de Friedwald. Resultados As freqüências alélicas para os polimorfimos de PON1 nos pacientes foram: 36,43% para o alelo PON1-192R, 57,86% para PON1-55L, 65,57% para PON2-311S e 76,43% para o alelo PON2-148A. No grupo de indivíduos recentemente diagnosticados como portadores de HIV estas freqüências foram 37,50%, 51,56%, 81,25% e 68,75, respectivamente. No grupo composto pelos saudáveis, a freqüência alélica de PON1-192R foi 43,02%, a de PON1-55L foi 68,99%, a do alelo PON2-311S foi 67,60% e, por fim, a freqüência do alelo PON2-148A foi 75,14%. Conclusões As distribuições alélicas dos polimorfimos em PON1 e PON2 foram similares dentre os portadores de HIV e os controles. A relação entre os genótipos PON1-192QR e/ou PON1-55LM e a atividade de PON1 em pacientes não diferiu daquela observada nos controles. Observou-se ainda, aumento do colesterol, dos triglicérides e da peroxidação lipídica no plasma dos infectados pelo vírus e, nos pacientes, uma maior atividade enzimática dentre os possuidores de contagem de linfócitos CD4+ acima de 500 células por mm3. / Human serum paraoxonase (PON) has been the subject of a number of studies. Beside the capacity of PON1 in hydrolyzing organophosphate compounds, it is known now that the entire PON family (which comprises PON1, PON2 and PON3) protects lipids, including low-density lipoprotein (LDL), from oxidation. Serum PON1 gene presents two well-determined genetic polymorphic sites: a Gln192 Arg (Q/R) and Met55 Leu, which are associated with differences in enzymatic activity and serum concentrations, respectively. Moreover, PON2 Cys311 Ser polymorphism seems to contribute synergistically with PON-192R allele to cardiovascular risk in some populations. It has been shown that HIV infected patients may develop dyslipidemia and that PON1 activity and concentration may be influenced by this infection. The aim of this study was to determine allelic frequencies of PON1-192QR, PON1-55LM, PON2-311SC and PON2-148AG genetic polymorphisms, evaluate PON1 activity and lipid peroxidation in plasma of HIV patients. Methods and Subjects after ethical committee approval and written consent, 350 (264 men and 86 women) HIV infected patients were included in the study. It was also evaluated a group of 32 recently diagnosed HIV individuals (23 men and 9 women). As controls, a healthy population formed by 179 men, blood donors, was studied. After DNA extraction PON1 and PON2 genotyping were performed by PCR-RFLP. Paraoxonase activity of PON1 was evaluated spectrofotometrically using paraoxon as substrate. Serum cholesterol, VLDL-cholesterol, HDL-cholesterol and triglycerides were analyzed by standard methods. LDL-cholesterol was calculated by Friedewald formula. Results: Allelic frequencies for PON1 polymorphisms in patients were: 36,43% for PON1-192R, 57,86% for PON1-55L, 65,57% for PON2-311S and 76,43% for PON2-148A. In recently diagnosed individuals these frequencies were 37,50%, 51,56%, 81,25% and 68,75% respectively. In controls, PON1-192R allelic frequency was 43,02%, PON1-55L was 68,99%, PON2-311S was 67,60% and PON2-148A was 75,14%. Conclusion: Allelic distributions of PON1 and PON2 polymorphisms were similar in HIV patients and controls. The relationship between PON1-192 QR and/or PON1-55LM genotypes and enzyme activity in patients were not different from controls. It was also observed an elevation of cholesterol, tryglicerides and lipid peroxidation levels in plasma of infected patients and, in this group, a higher activity in those which CD4+ cells counting was more than 500 cell/mm3.

Fu Lai Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-110). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Lists of Figures --- p.x / Lists of Tables --- p.xiii / List of Abbreviations --- p.xiv / Amino acid abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2 / Chapter 1.1.1 --- Lysosomal storage disease --- p.2 / Chapter 1.1.2 --- Treatments of MPS 1 --- p.4 / Chapter 1.2 --- Plant cells as bioreactors --- p.5 / Chapter 1.3 --- The Plant secretary pathway --- p.7 / Chapter 1.3.1 --- Transport of soluble proteins --- p.9 / Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10 / Chapter 1.4 --- Differences between plant and human proteins --- p.11 / Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12 / Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13 / Chapter 1.7 --- Project objectives --- p.14 / Chapter 1.8 --- Long term significance --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials --- p.18 / Chapter 2.2.1 --- Chemical --- p.18 / Chapter 2.2.2 --- Plant materials --- p.18 / Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18 / Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19 / Chapter 2.2.5 --- Primers --- p.20 / Chapter 2.3 --- Methods --- p.22 / Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22 / Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23 / Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23 / Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25 / Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25 / Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25 / Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29 / Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29 / Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31 / Chapter 2.3.2 --- Affinity-purified antibodies --- p.33 / Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33 / Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34 / Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39 / Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39 / Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39 / Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40 / Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40 / Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40 / Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40 / Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41 / Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42 / Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42 / Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42 / Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43 / Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43 / Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44 / Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Generation of IDUA antibodies --- p.48 / Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48 / Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51 / Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51 / Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55 / Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58 / Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66 / Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67 / Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70 / Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72 / Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72 / Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76 / Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81 / Chapter Chapter 4 --- Discussion --- p.82 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.89 / Chapter 5.1 --- Summary --- p.90 / Chapter 5.2 --- Future perspectives --- p.92 / Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Objectives --- p.94 / Chapter 6.3 --- Materials and Methods --- p.95 / Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95 / Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95 / Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95 / Chapter 6.4 --- Results --- p.97 / Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97 / Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98 / Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104 / Chapter 6.5 --- Summary and Future Perspectives --- p.105 / Chapter 6.5.1 --- Summary --- p.105 / Chapter 6.5.2 --- Future Perspectives --- p.105 / References --- p.106

Seto Tai Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 122-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.v / Abstract (in Chinese) --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xvi / List of Figures --- p.xv / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tobacco seed as bioreactor --- p.4 / Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4 / Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5 / Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8 / Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10 / Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10 / Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11 / Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12 / Chapter 1.3 --- The plant secretory pathway --- p.13 / Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16 / Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17 / Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17 / Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19 / Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19 / Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20 / Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21 / Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21 / Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21 / Chapter 1.4.2 --- Physiological role --- p.22 / Chapter 1.4.3 --- Molecular property --- p.24 / Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24 / Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24 / Chapter 1.4.5 --- Biochemical property --- p.25 / Chapter 1.4.6 --- Clinical application --- p.27 / Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27 / Chapter 1.4.6.2 --- Clinical trial --- p.28 / Chapter 1.4.6.3 --- Economic value --- p.29 / Chapter 1.4.7 --- Expression system --- p.29 / Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30 / Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30 / Chapter 1.5 --- Project objective and long-term significance --- p.30 / Chapter 1.5.1 --- Project objective --- p.30 / Chapter 1.5.2 --- Long-term significance --- p.31 / Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Chemical --- p.33 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35 / Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35 / Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39 / Chapter 2.3.1.3 --- Immunization of rabbit --- p.39 / Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40 / Chapter 2.3.2.1 --- Dot-blot analysis --- p.40 / Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42 / Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42 / Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42 / Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43 / Chapter 2.5 --- Discussion --- p.51 / Chapter 2.6 --- Conclusion --- p.51 / Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54 / Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54 / Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54 / Chapter 3.2 --- Materials --- p.55 / Chapter 3.2.1 --- Chemical --- p.55 / Chapter 3.2.2 --- Primers --- p.55 / Chapter 3.2.3 --- Bacterial strain --- p.58 / Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58 / Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61 / Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61 / Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61 / Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62 / Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62 / Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62 / Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66 / Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66 / Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66 / Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73 / Chapter 3.3.2.1 --- Tobacco plant --- p.73 / Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73 / Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74 / Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75 / Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75 / Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75 / Chapter 3.3.3.2 --- Southern blot analysis --- p.76 / Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77 / Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78 / Chapter 3.3.3.5 --- Western blot analysis --- p.79 / Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81 / Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81 / Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81 / Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82 / Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82 / Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82 / Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83 / Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83 / Chapter 3.3.5.5 --- Image collection --- p.84 / Chapter 3.4 --- Results --- p.85 / Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85 / Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86 / Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88 / Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91 / Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93 / Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93 / Chapter 3.4.7 --- Western blot analysis --- p.96 / Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96 / Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98 / Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98 / Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102 / Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.117 / References --- p.122 / Appendix 1 --- p.139 / Appendix II (List of Abbreviations) --- p.141

No epitélio do intestino médio de S. frugiperda encontra-se 90% da atividade de trealase solúvel. A trealase solúvel foi purificada até a homogeneidade por uma série de passos cromatográficos. A enzima possui um sítio hidrofóbico adjacente ao sítio ativo. Mudanças conformacionais aparentemente ocorrem quando metil-a-glicosídeo liga-se ao sítio ativo. A trealase solúvel é inibida competitivamente por amigdalina (Ki=0,21 mM), prunasina (Ki=0,92 mM), mandelonitrila (Ki = 1,14 mM), metil-α-glicosídeo (Ki=89 mM), metil-α-manosídeo (Ki=6,2 mM )e salicina (Ki= 19 mM). Florizina é um inibidor acompetitivo hiperbólico da trealase solúvel (Ki=0,087 mM, α =β =0,35) e seu aglicone floretina é um inibidor não competitivo (Ki=0,029 mM). Tris e mandelonitrila ligam-se a regiões diferentes da enzima enquanto mandelonitrila e floretina não podem ligar-se concomitantemente à enzima. Os pKs da enzima livre (pKe) e do complexo enzima substrato (pKes) foram determinados a partir de valores de Km e Vmáx/Km obtidos em vários pHs. Os valores encontrados foram: pKe1=4,47; pKe2=8,0l ; pKes1=4,83; pKes2=7,59. A trealase solúvel não perde a atividade quando incubada com reagentes que modificam grupos sufidrila, thiol e fenol. Com modificador de grupo imidazol, a enzima perde 60% da atividade somente na presença de metil-α-glucosídeo (inibidor competitivo da trealase). Essa modificação é protegida por trealose, indicando a presença de uma Histidina não essencial para catálise. Modificadores de grupo carboxila e guanidino inativam a enzima, com pKs de, respectivamente, 4,87 e 7,84. a similaridade desses pKs com os determinados cineticamente sugere que os resíduos envolvidos em catálise são uma arginina e um asparto ou glutamato. β-glicosídeos tóxicos produzidos por plantas e seus aglicones inibem trealoses presentes em diferentes órgãos de várias ordens de insetos. Essa inibição foi menor em insetos que se alimentam somente de vegetais, provavelmente devido a uma adaptação desses organismos. / The epithelium of S. frugiperda midgut has a trehalase activity that is mainly soluble (90%). The soluble trehalase was purified by several chromatographic steps. The enzyme has an hydrophobic site near the active site. Conformational changes apparently occur in the enzyme when methyl-α-glucoside binds to the active site. Trehalase has a Km=0.37 mM and is a competitively inhibited by methyl-α-glucoside (Ki=89 mM); methyl-α-mannosideo (Ki=6.2 mM); amygdalin (Ki=0.21); prunasin (Ki=0.92 mM); mandelonitrile (Ki=l.14 mM); Tris (Ki=0.55 mM) and salicin (Ki=l9 mM). Phlorizin is an hyperbolic acompetitive inhibitor (α=β=0.35; Ki=0.0087 mM), whereas its aglycon, phloretin, is a non-competitive inhibitor (Ki=0.029 mM). Tris and mandelonitrile bind at the same region in the active site. On the other hand, mandelonitrile and phloretin cannot be bound to the enzyme at the same time. Enzyme pKs (pKe) and enzyme substrate pKs (pKes) were determined from Km and Vmax/Km values obtained at different pHs. The values are: pKe1=4.47; pKe2=8.0l ; pKes1=4.83; pKes2=7.59. Trehalase is not inactivated when incubated with compounds that react with thiol, imidazole or phenol groups. Trealase lose 60% of its activity in the presence of methyl-α-glucoside (acompetitive inhibitor) plus a compound that reacts with imidazole groups. This inactivation is decreased by trehalose, indicating that there is a non-essential histidine in the active site substances that react with carboxyl guanidine groups inactivate the enzyme. The modified groups have pH of respectively, 4.87 and 7.84. The resemblance of these pKs with the one determined from Km and Vmax values suggest that the prototropic groups of the enzyme are in residues of arginine and aspartic acid or glutamic acid. Toxic β-glucosides from plants and their aglycons inhibit trealase from different organs of insects from several orders. This inhibition is lower in herbivorous insects, possibly due to their adaptation in ingesting vegetal tissues.

No epitélio do intestino médio de S. frugiperda encontra-se 90% da atividade de trealase solúvel. A trealase solúvel foi purificada até a homogeneidade por uma série de passos cromatográficos. A enzima possui um sítio hidrofóbico adjacente ao sítio ativo. Mudanças conformacionais aparentemente ocorrem quando metil-a-glicosídeo liga-se ao sítio ativo. A trealase solúvel é inibida competitivamente por amigdalina (Ki=0,21 mM), prunasina (Ki=0,92 mM), mandelonitrila (Ki = 1,14 mM), metil-α-glicosídeo (Ki=89 mM), metil-α-manosídeo (Ki=6,2 mM )e salicina (Ki= 19 mM). Florizina é um inibidor acompetitivo hiperbólico da trealase solúvel (Ki=0,087 mM, α =β =0,35) e seu aglicone floretina é um inibidor não competitivo (Ki=0,029 mM). Tris e mandelonitrila ligam-se a regiões diferentes da enzima enquanto mandelonitrila e floretina não podem ligar-se concomitantemente à enzima. Os pKs da enzima livre (pKe) e do complexo enzima substrato (pKes) foram determinados a partir de valores de Km e Vmáx/Km obtidos em vários pHs. Os valores encontrados foram: pKe1=4,47; pKe2=8,0l ; pKes1=4,83; pKes2=7,59. A trealase solúvel não perde a atividade quando incubada com reagentes que modificam grupos sufidrila, thiol e fenol. Com modificador de grupo imidazol, a enzima perde 60% da atividade somente na presença de metil-α-glucosídeo (inibidor competitivo da trealase). Essa modificação é protegida por trealose, indicando a presença de uma Histidina não essencial para catálise. Modificadores de grupo carboxila e guanidino inativam a enzima, com pKs de, respectivamente, 4,87 e 7,84. a similaridade desses pKs com os determinados cineticamente sugere que os resíduos envolvidos em catálise são uma arginina e um asparto ou glutamato. β-glicosídeos tóxicos produzidos por plantas e seus aglicones inibem trealoses presentes em diferentes órgãos de várias ordens de insetos. Essa inibição foi menor em insetos que se alimentam somente de vegetais, provavelmente devido a uma adaptação desses organismos. / The epithelium of S. frugiperda midgut has a trehalase activity that is mainly soluble (90%). The soluble trehalase was purified by several chromatographic steps. The enzyme has an hydrophobic site near the active site. Conformational changes apparently occur in the enzyme when methyl-α-glucoside binds to the active site. Trehalase has a Km=0.37 mM and is a competitively inhibited by methyl-α-glucoside (Ki=89 mM); methyl-α-mannosideo (Ki=6.2 mM); amygdalin (Ki=0.21); prunasin (Ki=0.92 mM); mandelonitrile (Ki=l.14 mM); Tris (Ki=0.55 mM) and salicin (Ki=l9 mM). Phlorizin is an hyperbolic acompetitive inhibitor (α=β=0.35; Ki=0.0087 mM), whereas its aglycon, phloretin, is a non-competitive inhibitor (Ki=0.029 mM). Tris and mandelonitrile bind at the same region in the active site. On the other hand, mandelonitrile and phloretin cannot be bound to the enzyme at the same time. Enzyme pKs (pKe) and enzyme substrate pKs (pKes) were determined from Km and Vmax/Km values obtained at different pHs. The values are: pKe1=4.47; pKe2=8.0l ; pKes1=4.83; pKes2=7.59. Trehalase is not inactivated when incubated with compounds that react with thiol, imidazole or phenol groups. Trealase lose 60% of its activity in the presence of methyl-α-glucoside (acompetitive inhibitor) plus a compound that reacts with imidazole groups. This inactivation is decreased by trehalose, indicating that there is a non-essential histidine in the active site substances that react with carboxyl guanidine groups inactivate the enzyme. The modified groups have pH of respectively, 4.87 and 7.84. The resemblance of these pKs with the one determined from Km and Vmax values suggest that the prototropic groups of the enzyme are in residues of arginine and aspartic acid or glutamic acid. Toxic β-glucosides from plants and their aglycons inhibit trealase from different organs of insects from several orders. This inhibition is lower in herbivorous insects, possibly due to their adaptation in ingesting vegetal tissues.

Diaminopropionate ammonia lyase (DAPAL) which belongs to the  class of PLP enzymes is reported only from prokaryotes. It is involved in the removal of two amino groups from its substrate, diaminopropionate, to form ammonia and pyruvate. DAPAL from Escherichia coli (eDAPAL) and Salmonella typhimurium (sDAPAL) was cloned, over expressed and purified using either affinity chromatography or conventional procedures. It was observed that eDAPAL (90 units / mg) was comparatively less active than sDAPAL (200 units / mg). Also the enzymes with the N-terminal His tag were found to be many fold less active than the enzymes without tag. DAPAL had a characteristic absorption maximum at 414nm due to the Schiff`s linkage between PLP and the € - amino group of the active site lysine residue. The apoenzyme was prepared by reaction with L-cysteine, and the resulting thiazolidine complex was easily dialyzed. On reconstitution with PLP, complete regain of absorption spectrum and 60% activity was seen. All the three enzymes (apo-, holo and reconstituted), when subjected to gel filtration chromatography were found to be homodimers of 88 kDa. The active site lysine 78 was mutated to glutamine, and the enzyme was purified to homogeneity. In the mutant enzyme PLP continued to be bound at the active site, but in a different orientation with an absorbance maximum at 406nm. The K78Q enzyme had negligible activity as compared to the wild type enzyme confirming the role of K78 in catalysis. Only a few of the enzymes of the  class have been investigated for their unfolding pathways. Urea induced unfolding studies on sDAPAL revealed that at lower concentrations of urea there was a loss in activity due to the disruption of Schiff's linkage. No gross conformational changes were observed at these concentrations of urea as seen from fluorescence and gel filtration experiments. Increase in concentration of urea led to unfolding of the protein thereby causing a shift in fluorescence maximum from 340nm to 357 nm due to the exposure of the buried tryptophans to the less hydrophobic environment. A considerable amount of aggregation was seen at intermediate urea concentrations, which was possibly the reason for the inability of the protein to refold completely. Based on the results, a concerted mechanism for dissociation and unfolding was proposed for sDAPAL. Aminooxy compounds, which are mechanism-based inhibitors for PLP enzymes have been used as drugs against various disorders for the last few decades. In order to probe the mechanism and efficiency with which these compounds inhibit sDAPAL, cycloserine (D and L), methoxyamine (MA) and aminooxyacetic acid (AAA) were chosen for the inhibition studies. The inhibition rates were measured by monitoring decrease in absorbance at 414nm, increase in the range of 320-330nm due to the product formation and loss of activity upon incubation with the inhibitor. It was seen that both the enantiomers of cycloserine were equally effective in disrupting the Schiff’s linkage with the second order rate constants of 15.8 and 36 M -1 sec –1 respectively. Spectral measurements showed two isosbestic points in the case of DCS and one in the case of LCS. Product of this inhibition reaction was identified to be a heat and acid stable compound namely a hydroxyisooxazole derivative of PMP. It was similar in nature to that reported from GABA aminotransferase. These results showed that unlike in the case of alanine racemase, sDAPAL could be inhibited equally well by both the enantiomers. The inhibition studies with the other two inhibitors namely AAA and MA, showed AAA to be more efficient at disrupting the Schiff’s linkage and causing inactivation of the enzyme. The visible absorbance spectrum showed a single isosbestic point in both the cases, indicative of a single step involved in the formation of the final product. The elution profile of the product of the enzymatic as well as non-enzymatic reactions on a C-18 HPLC column was similar and the product was identified to be an oxime. These inhibitors reacted with sDAPAL many fold better than the other PLP dependent enzymes and therefore these compounds can serve as potential drugs for sDAPAL.

Disruptions in folate metabolism are known to increase the risk for neural tube defects (NTD) and this is preventable by folic acid supplementation. However, the relationship between folate metabolism and cardiac development remains unclear. The interaction between other folate pathway nutrients, choline and riboflavin, and folate metabolism was studied in a murine model of methylenetetrahydrofolate reductase (MTHFR) deficiency. Maternal choline deficiency, riboflavin deficiency and MTHFR deficiency adversely affected embryonic or heart development. The promoters of MTHFR were also examined for interactions with GATA-4, TBX5, MEF2A and NKX-2.5, known transcription factors of cardiac development. Upstream promoter activity was increased in the presence of GATA-4 and this interaction was further enhanced upon the addition of MEF2A. TBX5 appeared to decrease upstream promoter activity. GATA-4 modestly increased downstream promoter activity. These results highlight the importance of adequate nutrient intake during pregnancy and provide a link between folate metabolism and cardiac development.

This study forms part of an investigation into the possible relevance of the lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this study, the main technique adopted was the Tokuyasu "cryo" method, in which the tissues were fixed, frozen and sectioned and labelled using the relevant antibodies, which were detected with protein A gold probes. In order to implement the Tokuyasu technique, it was necessary to rebuild a knife maker, for the production of adequately sharp glass knives, and to modify a sputter-coater into a glow-discharger, for rendering carbon-coated grids hydrophilic, to promote adhesion of hydrated sections. This study was directed towards human tissues and peptide antibodies were investigated as a means of avoiding isolation of proteins from scarce human tissue, and as a means of obtaining antibodies that will target specific regions of proteins of interest. Peptide antibodies were also considered promising for studies of proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer therapy. Various prediction programmes were investigated for their effectiveness in predicting whether a given peptide sequence will elicit antibodies that will react with the native protein. Successful prediction would increase the success rate of peptide antibody production and thus lower the cost. Leucocytes were studied as a model of an invasive cell, since they are more readily available than tumour cells and serve the purpose during the development of methods. In the course of these studies, an optimal protocol for the fixation of PMNs was developed, involving lateral fixation of cut sections, that should be useful for future studies on these cells. Elastase and cathepsins D and G were found on the surface of activated PMNs and could thus play a role in the invasive properties of these cells. Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed Neo-T counterparts revealed that upon transformation, lysosomes shift from a perinuclear position, to a more peripheral position. None of the cathepsins studied was found on the cell surface of either the normal or ras-transfected cells, suggesting that surface distribution of these enzymes may not be a requirement for invasiveness. These studies suggest that immunocytochemical investigation of cells, in the process of invading through a barrier membrane, might be profitable in elucidating the role of proteinases in invasive cancer. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.