To investigate mechanisms responsible for the turnover of endogenous muscle protein, lysosomotropic proteinase inhibitors have been employed to elucidate the relative contributions of lysosomal and non-lysosomal degradation pathways functioning under varying nutritional states and for different classes of intracellular proteins. Proteolysis in cultured bovine aortic smooth muscle cells was measured as the percentage of ³H-phenylalanine released per hour from pre-labelled cellular proteins. To reduce background radioactivity, the intracellular ³H-phenylalanine pool was depleted by serial extraction at 37°C, effecting equilibration between the intracellular pool and the phenylalanine-free medium. Reutilization of labelled amino acids during subsequent incubation periods was minimized by the presence of excess non-labelled phenylalanine in the medium. ³H-phenylalanine was released at a constant rate of 1,5 % per hour for at least 4 h, from cells pre-labelled for 16 h ('long-lived' proteins). Leupeptin, an inhibitor of thiol proteinases including cathepsin 8, inhibited degradation by 12 %, whereas the general lysosomal inhibitors chloroquine and NH₄Cl inhibited degradation by 30 %, presumably the contribution by the lysosomal pathway. In the case of 'short-lived' proteins (pre-labelled for 1 hour), the initial degradation rate was 6,5% per hour, which rapidly declined, reaching the basal rate of 1,5 % after 4 h. Chloroquine and NH₄Cl reduced proteolysis by only 12-15% and leupeptin had no significant inhibition, consistent with the view that the majority of short-lived proteins a degraded by non-lysosomal pathways. Proteolysis rates of 'abnormal' proteins containing the arginine-analogue, canavanine, were found to be significantly elevated (80 %) over controls. Leupeptin had no significant inhibition, and chloroquine and NH₄Cl only reduced degradation by 12-16 %, showing that the rapid removal of 'abnormal' intracellular proteins proceeds mainly via extra-lysosomal mechanisms. Incubation of the cells under nutritional step-down conditions, increased the average degradation rate of long-lived proteins to 3% per hour, and chloroquine and NH₄Cl inhibited degradation by 55-60 %, indicating that the accelerated proteolytic condition is due to increased activity of the lysosomes. Nutritional deprivation did not increase the rate of degradation of short-lived proteins. The results were clarified by the parallel use of the well-characterized LDL degradation system in this cell type, known to occur almost exclusively via lysosomes. This allowed the effectiveness of lysosomotropic inhibitors to be tested. Chloroquine inhibited LDL degradation by over 90 % and NH₄Cl inhibited by 80-95 % in all cases. Other proteinase inhibitors such as chymostatin, pepstatin and the chloromethyl ketones were also tested, and of these chymostatin seemed to be the most valuable because of its additivity to the effect of chloroquine, indicating its selective inhibition of non-lysosomal degradative mechanisms. Incubations of smooth muscle cells under anoxic conditions or with metabolic inhibitors such as fluoride, azide and cyanide, resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of chloroquine, i.e. both lysosomal and non-lysosomal degradation pathways have some energy-dependence. The degradation of long-lived proteins appeared to be more sensitive to temperature than that of short-lived proteins, further indicating the activity of distinct proteolytic mechanisms for these two classes of intracellular proteins. Preliminary studies have indicated a role for Ca⁺⁺ in the regulation of proteolysis, since degradation rates were increased by elevated levels of Ca⁺⁺ in the extracellular medium. Inhibition of this increased proteolysis by leupeptin has indicated a role for a thiol proteinase, possibly Ca⁺⁺-activated neutral proteinase. In similar studies with cultured L8 skeletal muscle cells, an average proteolysis rate of 1,2 % per hour was found, which was increased by 50 % under nutritional step-down conditions. Once again, the lysosomal pathway was found to account for only about one-third of basal protein degradation but fully accounted for the increased proteolysis under nutrient deprivation. The degradation characteristics of intracellular smooth and skeletal muscle cell proteins was examined using double isotope labelling. It was found that large molecular weight proteins and glycoproteins tended to be degraded more rapidly than small proteins and non-glycoproteins. In smooth muscle cells, these correlations were markedly reduced or absent under the accelerated proteolysis associated with nutrient deprivation, possibly confirming the increased activity of the non-selective autophagic lysosomal pathway under these conditions. A similar loss of correlations was not so clearly seen for skeletal muscle cell proteins.

Christine May Briton-Jones. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 149-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thèse de doctorat : Parasitologie : Paris 12 : 2005. / Thèse uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 192 réf.

Thèse de doctorat : Sciences de la vie et de la santé : Tours : 2008. / Titre provenant de l'écran-titre.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Illinois State University, 1998. / Title from title page screen, viewed July 17, 2006. Dissertation Committee: Radheshyam K. Jayaswal (chair), Herman E. Brockman, Brian J. Wilkinson, Anthony J. Otsuka, Alan J. Katz. Includes bibliographical references and abstract. Also available in print.

O carrapato bovino, Rhipicephalus (Boophilus) microplus, é um artrópode hematófago, responsável por importantes perdas na pecuária. Durante o desenvolvimento do embrionário do carrapato a cisteíno endopeptidase degradadora de vitelina (VTDCE) participa do processo de hidrólise de vitelina (Vt), a principal fonte de energia e aminoácidos durante esse período. Devido à sua importância na fisiologia do parasita, a VTDCE é um alvo potencial para o desenvolvimento de vacinas anti-carrapatos. Estudos anteriores demonstraram que a VTDCE nativa, purificada de ovos confere proteção contra R. microplus, desencadeada pela resposta imune dos bovinos após a administração deste antígeno. Entretanto, o extenso e dispendioso processo de purificação desta proteína, acrescido do baixo rendimento e fonte escassa de material (ovos de carrapato) inviabiliza a utilização da proteína nativa para fins comerciais. Tais dificuldades podem ser ultrapassadas por meio do uso de uma proteína recombinante. O presente trabalho tem por objetivo a expressão, purificação e caracterização da VTDCE recombinante (rVTDCE). Parte da sequência de aminoácidos da VTDCE nativa foi obtida por sequenciamento de edman e pela análise por especrometria de massas de petídeos obtidos pelo tratamento da proteína com tripsina e. As sequências obtidas assim obtidas foram utilizadas para buscar a sequência da ORF da VTDCE em um banco de cDNA. Por meio de PCR, a ORF da enzima foi clonada em vetor de clonagem e posteriormente de expressão. A rVTDCE expressa em Escherichia coli foi purificada por cromatografia de afinidade e utilizada para determinar algumas de suas propriedades, como atividade enzimática e antimicrobiana. A atividade enzimática foi avaliada através de ensaios fluorimétricos, onde a rVTDCE mostrou características similares à VTDCE nativa. Ensaios antimicrobianos foram realizados para avaliar a possível atividade sugerida após análise da sequencia da ORF da VTDCE. A análise molecular mostrou que a sequência da VTDCE apresenta semelhança com peptídeos antimicrobianos, fato comprovado através de ensaios antimicrobianos. Efetivamente, a VTDCE apresentou atividade antimicrobiana tanto na sua forma nativa como na forma desnaturada desprovida de atividade peptidásica, demonstrando que ambas as atividades são independentes. Foram feitos também ensaios imunológicos, onde anticorpos policlonais anti-rVTDCE foram produzidos em coelhos e bovinos. Por Western blot foi observado que os anticorpos policlonais produzidos contra VTDCE recombinante reconheceram a VTDCE nativa, e o inverso também, indicando que a forma recombinante pode ser utilizada para experimentos de vacinação. / The cattle tick, Rhipicephalus (Boophilus) microplus is a hematophagous arthropod, responsible for significant losses in livestock. During the tick embryo the vitellin-degrading cysteine endopeptidase (VTDCE) participates in the vitellin (Vt) hydrolysis, the main energy source and amino acids during this period. Due to the importance of this enzyme in parasite physiology the VTDCE is a potential target for development of anti-tick vaccine. In previous studies, the immunization with native VTDCE, purified from tick egg, conferred protection against R. microplus. However, the extensive and expensive purification process, plus the low achievement precludes the use of the native protein for commercial purposes. This difficulty can be overcome by the expression of a recombinant protein in heterologous organism. This work aims the expression, purification and characterization of recombinant VTDCE (rVTDCE). Part of the native VTDCE amino acid sequence was determined by Edman sequencing. Native protein was also trypsinized and peptides sequenced by mass spectrometry. The sequences obtained from mass spectrometry and Edman sequencing were used to search the ORF sequence of VTDCE in a tick cDNA library. Through PCR enzyme ORF was cloned into a cloning vector and further into an expression vector. The rVTDCE expressed in Escherichia coli was purified by affinity chromatography and used to determine some of its properties such as antimicrobial and enzyme activity. The enzymatic activity was measured using fluorimetric assays, where rVTDCE had similar characteristics to VTDCE. Antimicrobial assays were performed to evaluate the possible activity suggested after sequence analysis. Molecular analysis showed that the sequence of VTDCE shows similarity with antimicrobial peptides, which was proven through antimicrobial assays. Effectively, the VTDCE presented antimicrobial activity even when the protein didn’t present enzymatic activity, demonstrating that both activities are independent. Immunological assays were also performed. Polyclonal anti-rVTDCE serum were produced in rabbits and cattle. Western blot showed that the polyclonal antibodies raised against recombinant VTDCE recognized the native form, indicating that the recombinant form may be used for vaccination experiments.

O carrapato bovino, Rhipicephalus (Boophilus) microplus, é um artrópode hematófago, responsável por importantes perdas na pecuária. Durante o desenvolvimento do embrionário do carrapato a cisteíno endopeptidase degradadora de vitelina (VTDCE) participa do processo de hidrólise de vitelina (Vt), a principal fonte de energia e aminoácidos durante esse período. Devido à sua importância na fisiologia do parasita, a VTDCE é um alvo potencial para o desenvolvimento de vacinas anti-carrapatos. Estudos anteriores demonstraram que a VTDCE nativa, purificada de ovos confere proteção contra R. microplus, desencadeada pela resposta imune dos bovinos após a administração deste antígeno. Entretanto, o extenso e dispendioso processo de purificação desta proteína, acrescido do baixo rendimento e fonte escassa de material (ovos de carrapato) inviabiliza a utilização da proteína nativa para fins comerciais. Tais dificuldades podem ser ultrapassadas por meio do uso de uma proteína recombinante. O presente trabalho tem por objetivo a expressão, purificação e caracterização da VTDCE recombinante (rVTDCE). Parte da sequência de aminoácidos da VTDCE nativa foi obtida por sequenciamento de edman e pela análise por especrometria de massas de petídeos obtidos pelo tratamento da proteína com tripsina e. As sequências obtidas assim obtidas foram utilizadas para buscar a sequência da ORF da VTDCE em um banco de cDNA. Por meio de PCR, a ORF da enzima foi clonada em vetor de clonagem e posteriormente de expressão. A rVTDCE expressa em Escherichia coli foi purificada por cromatografia de afinidade e utilizada para determinar algumas de suas propriedades, como atividade enzimática e antimicrobiana. A atividade enzimática foi avaliada através de ensaios fluorimétricos, onde a rVTDCE mostrou características similares à VTDCE nativa. Ensaios antimicrobianos foram realizados para avaliar a possível atividade sugerida após análise da sequencia da ORF da VTDCE. A análise molecular mostrou que a sequência da VTDCE apresenta semelhança com peptídeos antimicrobianos, fato comprovado através de ensaios antimicrobianos. Efetivamente, a VTDCE apresentou atividade antimicrobiana tanto na sua forma nativa como na forma desnaturada desprovida de atividade peptidásica, demonstrando que ambas as atividades são independentes. Foram feitos também ensaios imunológicos, onde anticorpos policlonais anti-rVTDCE foram produzidos em coelhos e bovinos. Por Western blot foi observado que os anticorpos policlonais produzidos contra VTDCE recombinante reconheceram a VTDCE nativa, e o inverso também, indicando que a forma recombinante pode ser utilizada para experimentos de vacinação. / The cattle tick, Rhipicephalus (Boophilus) microplus is a hematophagous arthropod, responsible for significant losses in livestock. During the tick embryo the vitellin-degrading cysteine endopeptidase (VTDCE) participates in the vitellin (Vt) hydrolysis, the main energy source and amino acids during this period. Due to the importance of this enzyme in parasite physiology the VTDCE is a potential target for development of anti-tick vaccine. In previous studies, the immunization with native VTDCE, purified from tick egg, conferred protection against R. microplus. However, the extensive and expensive purification process, plus the low achievement precludes the use of the native protein for commercial purposes. This difficulty can be overcome by the expression of a recombinant protein in heterologous organism. This work aims the expression, purification and characterization of recombinant VTDCE (rVTDCE). Part of the native VTDCE amino acid sequence was determined by Edman sequencing. Native protein was also trypsinized and peptides sequenced by mass spectrometry. The sequences obtained from mass spectrometry and Edman sequencing were used to search the ORF sequence of VTDCE in a tick cDNA library. Through PCR enzyme ORF was cloned into a cloning vector and further into an expression vector. The rVTDCE expressed in Escherichia coli was purified by affinity chromatography and used to determine some of its properties such as antimicrobial and enzyme activity. The enzymatic activity was measured using fluorimetric assays, where rVTDCE had similar characteristics to VTDCE. Antimicrobial assays were performed to evaluate the possible activity suggested after sequence analysis. Molecular analysis showed that the sequence of VTDCE shows similarity with antimicrobial peptides, which was proven through antimicrobial assays. Effectively, the VTDCE presented antimicrobial activity even when the protein didn’t present enzymatic activity, demonstrating that both activities are independent. Immunological assays were also performed. Polyclonal anti-rVTDCE serum were produced in rabbits and cattle. Western blot showed that the polyclonal antibodies raised against recombinant VTDCE recognized the native form, indicating that the recombinant form may be used for vaccination experiments.