Chez Arabidopsis thaliana, les gènes d’ARNr 5S sont transcrits par l’ARN polymérase III qui fait intervenir plusieurs facteurs de transcription dont TFIIIA qui reconnaît spécifiquement le promoteur interne de ces gènes et permet ainsi le recrutement de l’ensemble du complexe de transcription. Le gène TFIIIA est composé de sept exons dont le troisième, résultant de l’exonisation d’une molécule d’ARNr 5S, est épissé de façon alternative produisant ainsi deux transcrits. Le transcrit ES, qui ne contient pas cet exon, code pour la protéine TFIIIA pleine longueur et fonctionnelle. Le transcrit EI, dans lequel l’exon « 5S-like » est maintenu, est reconnu et dégradé par la voie NMD (Non-Mediated Decay). L’exon « 5S-like » a, en effet, la particularité de contenir un codon stop prémature qui en fait une cible de la voie NMD. Lors de l’étude de l’expression des transcrits ES et EI ainsi que celle de l’accumulation de la protéine TFIIIA au cours du développement, nous avons montré que le taux de la protéine TFIIIA fonctionnelle est soumis à plusieurs niveaux de régulation. En effet, la production de la protéine TFIIIA pleine longueur est le résultat de la synthèse du transcrit ES et de sa traduction mais également de l’efficacité du clivage protéolytique de la protéine. Lors de la maturation de la graine, l’accumulation croissante de protéine TFIIIA résulte d’une augmentation des quantités de transcrits ES couplée à une diminution du clivage protéolytique. Dans les premiers jours du développement, la protéine TFIIIA n’est détectée qu’après le 4ème jour, suite à la diminution du clivage. Sa présence est corrélée au remodelage de la chromatine de l’ADNr 5S. La combinaison de ces deux mécanismes permet ainsi la production de TFIIIA et de son produit de transcription l’ARNr 5S en fonction des besoins de la plante. / In Arabidopsis thaliana, 5S rRNA genes are transcribed by RNA polymerase III. TFIIIA, specifically required for transcription of these genes, binds to the internal control region of the 5S rRNA genes and allows the assembly of the full transcription complex pol III. The TFIIIA gene consists of seven exons, the third of which results in the exonisation of one 5S rRNA molecule. This exon “5S-like” is alternatively skipped or included to produce either of two transcript isoforms. The ES transcript encodes the fully functional TFIIIA protein. The EI transcript, which contains the exon “5S-like”, is a target of the NMD pathway (Non-Mediated Decay). Indeed, the exon “5S-like” contains a premature stop codon, which is recognized by this RNA decay pathway. During the study of the ES and EI transcripts expression and the TFIIIA protein accumulation throughout the plant development, we show that TFIIIA functional protein levels are under control of many regulation steps. Actually the production of the full-length TFIIIA protein results from production and translation of the ES transcript but also from the proteolytic cleavage efficiency of TFIIIA protein. During the seed maturation, the strong accumulation of TFIIIA results from an increase in ES amounts and a proteolytic cleavage decrease. After the fourth day post-germination, TFIIIA protein is detected because the proteolytic cleavage decreases. TFIIIA presence is correlated with 5S rDNA chromatin reorganization. The combination of these two mechanisms allows TFIIIA production and its transcription product 5S rRNA according to the plants needs.

The saliva of hematophagous ectoparasites contains a cocktail of vasodilators, anticoagulants and immunosuppressors that maintain blood in a liquid state at the site of the lesion and evade the host's defense mechanisms in suppressing the immune response. Since ticks have evolved to utilize mammals as a source of food, our understanding of the tick material, especially the salivary glands will enhance the control of tick infestation and allow the exploitation of the tick's natural resources. SGE protease activity was determined by measuring the degradation of azocasein. Proteolytic activity was found in the pH range of 3 to 11 with the highest activity at pH 9 followed by pH 7. At pH 3-5 the activity was mainly due to aspartic proteases, whereas at pH 7-9 the activity was due to the action of metallo- and serine proteases. At pH 11, the activity was mainly ascribed to metallo- and aspartic proteinase activity The fibrinogenolytic activity was determined by incubating human fibrinogen in the presence of SGE and monitoring the fibrinogen degradation by SDS-PAGE. SGE degraded the Au-chain of fibrinogen within 2 hours of incubation and even after 24 hours incubation there was no hydrolysis of the Bβ and γ-chains of fibrinogen. Characterization of the fibrinogenolytic activity revealed that metalloprotease activity was present over pH range of 3-9 and at pH 3-5, the cysteine proteases were active. No serine protease activity was found under similar experimental conditions. CE-HPLC separation of the SGE revealed three regions of proteolytic activity. Further characterization of the activity containing fractions using protease inhibitors at various pH values showed that the activity associated with region A is mainly due to the presence of aspartic and cysteine proteases in the lower pH range (< 5). Region B was mainly due to the activity of the metallo- and serine proteases, while the activity in region C was mainly due to the metalloproteinases which were more active in the higher pH range (> 9). CE-HPLC separation of SGE resulted in three regions exhibiting fibrinogenolytic activity at pH 7-9. In region A all four enzyme classes were found while in regions B and C, serine, cysteine and metalloproteinases were found to be responsible for the activity. Region A was further purified on the HIC-column and activity eluted in several peaks which after individual application on SE-HPLC column had similar retention times. The pooled samples were analyzed for purity using C5 RP-HPLC and reducing tricine SDS-PAGE and three bands of relative molecular masses 15, 22 and 12 kDa, respectively were found. In an attempt to purify the proteins in region C, four individual CE-HPLC runs were combined and applied to a fibrinogen affinity column. Reducing SDS-PAGE analysis of bound material showed two bands of relative molecular masses of 31 and 39 kDa, respectively. CE-HPLC region C as well as the SGE control was found to disaggregate platelets aggregated by ADP, epinephrine, collagen as well as TRAP. No disaggregation was observed for the saline negative control. The disaggregation is most probably due to the hydrolysis of the fibrinogen cross-linking platelets by the metalloproteinase activity in region C. Understanding of the proteolytic activities present in the salivary gland and therefore identifying molecules crucial for tick feeding could aid in the development of experimental vaccines. Even though the fibrinogenolytic activity was not purified to homogeneity, this study has laid the groundwork for further experiments in this field. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

The aim of this study was to evaluate the effect of glycosylation on exogenous xylanase stability when incubated under proteolytic conditions. Xylanase produced by Trichoderma longibrachiatum, was purified using gel filtration chromatography, ammonium sulfate salt precipitation and dialysis. A partially purified xylanase with Mr of 20- and 10 kDa was identified and contained >65% of the original xylanase activity. Glycoproteins present in the xylanase were identified by thymol sulfuric acid staining or by the FITC-Iabeled lectin method, specific for glycoproteins. This naturally glycosylated xylanase was enzymatically deglycosylated with one of two endo-N-glycosidases: PNGase F or Endo H. Efficiency of deglycosylation was determined with electrophoresis by observing protein mobility shifts or by staining with FITC-Iabeled lectin. The effect of glycosylation on the stability of the exogenous xylanase was tested by incubating the glycosylated or deglycosylated xylanase with rumen fluid (Rf), Prevotella ruminicola culture supernatant (Pr) or a commercial protease from Bacillus subtilis (Bs) for 0, 3, 6, 9 and 24h at 37°C. Results indicated that glycosylated xylanase was significantly more stable (P<0.05) against proteolytic inactivation under the relatively low protease conditions of Rf and Pr (0.018 and 0.046 mg azocasein degraded/ml/h, respectively), but not under high proteolytic conditions of Bs (1.009 mg azocasein/mllh). Also, the glycosylation effect was observed earlier when incubated with the numerous proteases of Rf (3h), than with Pr (9h). These results indicate that glycosylation enhances xylanase stability and therefore is an important characteristic for exogenous enzyme supplements for ruminants. / Dissertation (MSc (Agric))--University of Pretoria, 2005. / Animal and Wildlife Sciences / unrestricted

A Bothrops atrox é uma serpente de amplia distribuição na Sul América e é responsável por um número importante de mortes de pessoas, principalmente na Amazônia. As alterações na composição do veneno desta espécie têm sido associados a fatores como a ontogenia, distribuição geográfica e alimentação. Assim, este projeto visa comparar e identificar a partir da diferença entre os sexos, as características bioquímicas e biológicas do veneno de irmãos de B. atrox, sob condições ambientais controladas, contribuindo no conhecimento das mudanças nas características do veneno da espécie e pudendo auxiliar no aprimoramento da produção de antissoros mais efetivos. Os venenos foram coletados de 5 fêmeas e 4 machos irmãos de B. atrox, nascidas em cativeiro. Os venenos foram analisados quanto individualmente como o pool de cada grupo. As análises consistiram em dosagem de proteína através de BCA, eletroforese mono e bidimensional, cromatografia liquida, espectrometria de massas, atividades caseinolítica, fosfolipásica A2, L-aminoácido oxidase, zimografias contendo gelatina e caseína como substrato, dose mínima coagulante sobre o plasma e fibrinogênio, dose leta 50% e dose mínima hemorrágica. A análise individual dos venenos mostrou que os machos apresentaram maior concentração de proteínas e atividade fosfolipásica A2. No quanto aos pools de veneno, o das fêmeas apresentou maior letalidade e capacidade coagulante sobre plasma e fibrinogênio e o dos machos apresentaram maior capacidade hemorrágica e atividade L-aminoácido oxidase. O perfil espectrométrico mostrou que o pool de veneno das fêmeas, teve um 29% a mais na quantidade de proteínas identificadas em relação aos machos. Em conclusão, a ação do veneno das fêmeas estaria relacionado a uma maior capacidade para gerar dano sistêmico na presa, entanto que os venenos dos machos poderiam ocasionar um maior dano local. Além, a variabilidade nas atividades biológicas dos venenos confirma que além dos fatores ambientais existem outros que poderiam influir na plasticidade da composição dos venenos. / Bothrops atrox snake is widespread in South America and causing a large number of human deaths, mainly in the Amazon. Changes in the composition of the venom of this species have been linked to factors such as ontogeny, geographical distribution and feeding. Thus, this study aims to compare and identify from the sex difference, the biochemical and biological characteristics of venom of B. atrox siblings, under controlled environmental conditions, contributing to the knowledge of changes in the characteristics of the venom of the species and can assist in improving the production of more effective antisera. Venoms were collected from 5 females and 4 males of B. atrox siblings, born in captivity. The venoms were analyzed both, individually and as a pool of each group. The assays consisted in protein quantification using BCA, one and two-dimensional electrophorese, liquid chromatography, mass spectrometry, caseinolytic, phospholipase A2, and L-amino acid oxidase activities, zimography containing gelatin and casein as substrate, minimum coagulant dose upon plasma and fibrinogen, lethal dose 50 % and minimum hemorrhagic dose. Individual analysis of venoms showed that males had higher proteins concentration and phospholipase A2 activity. Concerning the venoms pool, the female showed higher lethality and coagulant capacity upon plasma and fibrinogen and the male had higher L-amino acid oxidase activity and hemorrhagic capacity. Spectrometric profile showed that the venom pool of female snakes had a 29 % increase in the number of proteins identified in comparison to males. In conclusion, the action of the female venom would be related to a higher capacity to generate systemic damage in the prey and male venoms could lead to higher local damage. In addition, variability in the biological activities of venoms confirms that there are other factors that could would be influencing the plasticity of the composition of venoms, in addition to environmental.

Presented diploma thesis is focused on testing of an appropriate form of encapsulated enzymes intended for application in cosmetic and pharmaceutical industry. For encapsulation, proteolytic enzymes bromelain, papain and collagenase were used. These enzymes were encapsulated into alginate and chitosan microparticles prepared by an encapsulator and packed into liposomes. Encapsulation effectiveness was evaluated by analysis of total proteins. Particles stability was evaluated in model and real conditions by photometrical analysis of released proteins. Proteolytic activity of released enzymes in model and real conditions were observed too. Alginate and chitosan microparticles prepared by the encapsulator were found as an appropriate form of encapsulated enzymes designed to wound healing. Encapsulation effectiveness of these particles and stability in model conditions were good in comparison with liposomes. Hydrogel and water-oil emulsion were used for analysis of particles stability at real conditions. Hydrogel was found as a good option for preservation of particles as well as proteolytic enzyme activity. Emulsion made particles less stable and proteolytic activity of enzymes decreased rapidly. Encapsulation enables long-term stabilization of biologically active compounds as well as possibility of targeted transport and controlled releasing. Presented diploma thesis suggests possibilities of application encapsulated enzymes in designing more effective formulations for wound healing.

Orientador: Eduardo Maffud Cilli / Banca: Patrícia Soares Santiago / Banca: Ederlan de Souza Ferreira / Resumo: Devido ao avanço científico e tecnológico, notável sucesso tem sido alcançado na identificação de novos compostos antitumorais. Entretanto, problemas associados à baixa estabilidade e seletividade tem limitado o sucesso da grande maioria destes compostos. Dentre as estratégias para aumentar a estabilidade de moléculas bioativas, a bioconjugação em domínios de ligação a albumina (DLA) tem se mostrado promissor para ampliar o tempo de vida médio de moléculas susceptíveis à degradação proteolítica. Neste trabalho, a estrutura e a atividade de um composto contendo um peptídeo citotóxico, um DLA e um sítio de clivagem específico foram avaliadas. Motivado pela perspectiva do peptídeo melitina apresentar atividade antitumoral, o nosso grupo de pesquisa avaliou a síntese e atividade biológica deste composto com o peptídeo RQKRSLGG-WQRPSSW. O peptídeo obtido foi testado contra tipos de celulas tumorais e não tumorais (linhagem MCF-7 e HaCaT, respectivamente), mostrando-se potente, porém tóxico. Nos estudos de dicroísmo circular, os peptídeos não apresentaram estrutura secundária em solução aquosa. Em presença de miméticos de membrana, os peptídeos adquiriram uma estrutura em α-hélice exceto o peptídeo sítio de clivagem-DLA. Estudos de vazamento de carboxifluoresceína em LUVs (POPC:POPS), através da técnica de espectroscopia de fluorescência, mostraram que o peptídeo completo tem capacidade de permeabilização similar ao da melitina e que é dependente da concentração. Os resultados de f... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the scientific and technological advanced permitted remarkable successful in the field of identification of new antitumour compounds. However, problems associated with low stability and selectivity have been a restriction in the successf ul of majority of this compounds. Inside the strategic for increase the stability of bioactive molecules, bioconjugation with albumin - binding domain (ABD) have been promising for increasing t he lifetime of molecules susceptible for the proteolytic degradat ion. Herein, structure and activity of a compound containing a cytotoxic peptide, ABD, and cleavage site were evaluated. The ABD (WQRPSSW) can give more stability for molecules, while the cle avage site RQKRSLGG can give more selectivity to the peptide. Thi s sequence is degraded for the protease Kallikrein 4 (KLK4), which occurs in elevated quantity in the tumours cells, releasing the cytotoxic compound, especially in the microenvironments of t hese cells, promoting the higher selectivity. Inspired for the pe rspective of Melittin peptide holds a promising antitumour activity, our research group evaluated the synthesis and biological activity with this peptide containing the sequence RQKRSLGG - WQRP SSW. The peptide was evaluated against diversity of tumours and n o tumours cells (MCF - 7 and HaCaT respectively), presenting effective activity but toxic. In circular dichroism studies the peptides did not show second structure in aqueous solution. In the p resence of membrane mimet... (Complete abstract click electronic access below) / Mestre

NADPH:cytochrome P450 reductase (CPR) is a 78 kDa flavoprotein, which is together with cytochrome P450 component of monooxygenase system bound in the membrane of the endoplasmic reticulum. Monooxygenase system is involved in the metabolism of a wide range of organic substances, including drugs or various pollutants present in the environment (polycyclic aromatic hydrocarbons, aromatic amines, etc.). CPR works as a transporter of reducing equivalents from NADPH to the cytochromes P450. For proper interaction with cytochromes P450, intact N-terminal hydrophobic domain anchoring protein in the membrane is needed. Removing this domain, e.g. during trypsin proteolysis, gives rise a soluble CPR (72 kDa) and cause loss of catalytic activity towards cytochrome P450. During heterologous expression in E. coli proteolytically sensitive site of CPR (Lys56 - Ile57) is cleaved by intracellular trypsin-like proteases, that may negatively affect the yields of native 78 kDa protein. This thesis describes the heterologous expression, purification and characterization of two forms of rat CPR. WtCPR is a protein naturally occurring in rats (Wistar strain), while mCPR contains one amino acid substitution (K56Q) in the site of proteolytic degradation. The result of that substitution is proteolytically stable CPR,...

An affinity column for cathepsin D was prepared making use of the strong affinity of pepstatin for cathepsin D. Pepstatin is an N-acylated pentapeptide from Actinomycetes with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy- 6-methyl heptanoic acid. A relatively rapid and efficient method for cathepsin D purification has been developed; Steps include homogenization, ammonium sulfate fractionation, and chromatography on pepstatin-Sepharose column. The final preparation has a specific activity of 38 units/mg. and shows a single protein band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate corresponding to a subunit molecular weight of 42,000. Polyacrylamide gel electrophoresis studies did not reveal any impurities. The proteolytic activity of isolated cathepsin D on bovine myofibrils and myosin was examined at pH 3.80, 37 °C. The heavy chains of myosin, as well as other smaller regulatory proteins of the myofibrils were degraded. Actin was degraded less rapidly than myosin heavy chain. Degradation became more extensive when the substrate-enzyme incubation time was increased.

The Mozzarella cheese market is growing rapidly. Major concerns with cheese meltability and color have arisen in the fast food industry. Pre starter culture was used in this study to improve the physical properties of Mozzarella cheese. Three tests (stretch test, melt test, and browning test) were modified to evaluate the quality of cheese. A stretch test using the Brookfield helipath viscometer to stretch the cheese sample at 60°C was successful in distinguishing cheeses from different make procedures and from different proteolytic strains. A melt test using a glass tube to hold the cheese flow at 110°C for 60 min was used to determine meltability of cheese. A chroma meter was used to measure color change after the cheese sample was subjected to boiling water for 60 min. The b* value was used to indicate the color change. Cheese made with Pre strains of Lactobacillus bulgaricus stretched less but showed longer melting flow than that from Prt+ strains. Cheese made with Pre strains was lighter in color than cheese from Prt+ strains. An inverse relationship existed between stretchability and meltability. When mixed cultures of L. bulgaricus and Streptococcus thermophil us were used, the symbiotic interaction in acid production of Prt+ strains was more effective than mixed cultures of Prt- strains. Stretchability of...

String cheese, a Mozzarella cheese, has the unique ability to string in fibrous strands when pulled apart. Graders judge string cheese by its stringy texture; samples with copious amounts of string are awarded high ratings. But just as the texture of natural cheeses softens with time, the stringy texture of string cheese can diminish with age too. Age related softening in cheese is due primarily to an important biochemical event known as proteolysis, which is attributed to inherent milk proteinases, residual coagulant activity, and enzymes from the lysis of starter culture microorganisms. It is hypothesized that a post manufacture heat treatment of string cheese could inactivate these proteolytic enzymes and slow or eliminate proteolysis during storage. Therefore, the main objective of this study was to determine the effects of a post manufacture thermal dip treatment on proteolytic activity in packaged, commercial string cheese. Proteolysis was examined qualitatively by Urea-PAGE electrophoresis, quantitatively by measuring percentage of water-soluble nitrogen (%WSN), and by using a scoring method to analyze stringy texture during refrigerated storage. Fresh, commercial string cheese was sourced on two separate occasions and treated six days after manufacture. Treatment consisted of dipping the packaged cheese sticks in water baths at 55°C, 75°C, and 95°C for 30 and 60 seconds. String cheese that did not undergo treatment served as the control. Treated and control cheeses were stored at 4°C until sampling for Urea-PAGE, WSN extraction, and texture analysis on days 1, 11, 22, 29, 49, 91, and 172 after treatment. The degree of β-CN breakdown was not observed to be different between all treatment levels throughout the storage period. This was not expected since Mozzarella cheese exposed to a higher temperature should have more plasmin activity than that of cheese exposed to a lower temperature. There was a trend of slightly more intact αs1-CN in the most severely treated string cheese (95°C for 60s) when compared to the control at the final time point of the study. This suggests the possibility of successful inactivation of residual coagulant, intracellular enzymes, or other proteolytic enzymes in the string cheese at this treatment. However, only storage time had a significant effect on %WSN (p The research completed in this study provides insight of the proteolytic effects from a thermal treatment process applied post string cheese manufacture. Though relationships between the treatments to the extent of secondary proteolysis and stringy texture were not significant, it was still found that there was more intact αs1-CN due to one of the treatments. These results suggest that it is possible that the use of other heat treatment parameters, longer storage period, or a combination of the two could show a significant relationship between thermal treatment and proteolysis. These results also suggest that further work to improve shelf life of string cheese or other cheese varieties through the concept of a post manufacture heat treatment may be promising.