No description available.

No description available.

BACKGROUND: Clostridium sordellii can cause severe infections in animals and humans, the latter associated with trauma, toxic shock and often-fatal gynaecological infections. Strains can produce two large clostridial cytotoxins (LCCs), TcsL and TcsH, related to those produced by Clostridium difficile, Clostridium novyi and Clostridium perfringens, but the genetic basis of toxin production remains uncharacterised. RESULTS: Phylogenetic analysis of the genome sequences of 44 strains isolated from human and animal infections in the UK, US and Australia placed the species into four clades. Although all strains originated from animal or clinical disease, only 5 strains contained LCC genes: 4 strains contain tcsL alone and one strain contains tcsL and tcsH. Four toxin-positive strains were found within one clade. Where present, tcsL and tcsH were localised in a pathogenicity locus, similar to but distinct from that present in C. difficile. In contrast to C. difficile, where the LCCs are chromosomally localised, the C. sordellii tcsL and tcsH genes are localised on plasmids. Our data suggest gain and loss of entire toxigenic plasmids in addition to horizontal transfer of the pathogenicity locus. A high quality, annotated sequence of ATCC9714 reveals many putative virulence factors including neuraminidase, phospholipase C and the cholesterol-dependent cytolysin sordellilysin that are highly conserved between all strains studied. CONCLUSIONS: Genome analysis of C. sordellii reveals that the LCCs, the major virulence factors, are localised on plasmids. Many strains do not contain the LCC genes; it is probable that in several of these cases the plasmid has been lost upon laboratory subculture. Our data are consistent with LCCs being the primary virulence factors in the majority of infections, but LCC-negative strains may precipitate certain categories of infection. A high quality genome sequence reveals putative virulence factors whose role in virulence can be investigated.

No description available.

The thesis addresses the potential application of ruthenium(II)-cobalt(III) heterodinuclear complexes as a new selective cancer treatment. The selectivity is to be achieved through the use of visible light to trigger activation of the drug. The majority of work conducted relates to the design and synthesis of the bridging ligand for the final ruthenium(II)-cobalt(III) heterodinuclear complex. In Chapter 2, a potential bridging ligand based on a functionalised terpyridine is described. The intention was to bind the ruthenium(II) metal centre to the terpyridine end of the bridging ligand and have a secondary binding domain available for coordination of the cobalt(III) metal centre. However, a reductive step in the synthetic pathway failed to produce the desired product and this potential bridging ligand had to be abandoned. In Chapter 3, two series of bridging ligands are described. The first of these series is based on Jurgen Sauer’s ‘LEGO’ system. In addition to describing the free synthesis of these ligands, their synthesis on a ruthenium(II) metal centre is described. The second series is based on disubstituted-1,2,4,5-tetrazines. These compounds are only able to be directly synthesised as the non-coordinated ligand. Coordination of these ligands to a single ruthenium(II) metal centre is then described. Ruthenium(II) complexes of both ligand series are then exposed to several transition metals and their ability to coordinate a second metal centre investigated. The formation of ruthenium(II)-cobalt(III) heterodinuclear complexes, using the ligand series detailed in Chapter 3, is described in Chapter 4. These complexes are formed by reacting the ruthenium(II) complex of the bridging ligand with either [Co(en)₂(OTf)₂](OTf) or [Co(tren)(OTf)₂](OTf). These heterodinuclear complexes exhibit photo-activated ligand release, which makes them candidates for development as a potential cancer treatment. The non-bridging ligands coordinated to the cobalt(III) metal centre in Chapter 4 were not cytotoxic. In order to make the system biologically active these ligands need to be changed. Chapter 5 describes how nitrogen mustards (a class of cytotoxic DNA alkylators) could be introduced as the non-bridging ligands. This involves the synthetic strategy of forming the cobalt(III) complex of the alcohol precursor of a nitrogen mustard. This precursor complex is then converted into the nitrogen mustard complex and coordinated to the ruthenium(II) bound bridging ligand. The synthetic strategies outlined in this thesis can be applied to a wide range of potential bridging ligands and could potentially lead to a large number of ruthenium(II)-cobalt(III) heterodinuclear complexes being synthesised. One journal article based on this research has been accepted for publication, in the Australian Journal of Chemistry. Three more articles are in preparation.

This project was focused on development of extraction and purification protocol for novel cytotoxic compound isolated from soil cyanobacterium Nostoc muscorum which would allow to determine its molecular structure via NMR (nuclear magnetic resonance) and MS (mass spectrometry) techniques. Purified cytotoxin was subjected to several biochemical and microscopical experiments in order to assess its toxicological parameters, determine the mechanism of action and evaluate potential application in biotechnology or pharmacy.

We have previously reported a high prevalence of H. pylori DNA in saliva. In this study, the cytotoxin genotypes of H. pylori strains from both stomach and saliva were compared in 31 patients with gastritis and peptic ulcer. The cagA, vacA m1, vacA m2, and vacA s1 genotypes were analyzed by PCR. The 417 bp PCR products from three patients were also subjected to DNA sequencing analysis. There was 95% agreement between stomach H. pylori isolates and their corresponding saliva DNA in at least one cytotoxin genotype; 86% agreement with two cytotoxin genotypes; 59% agreement with three cytotoxin genotypes; and 27% agreement with all four cytotoxin genotypes studied. DNA sequencing from three patients showed 78.0%, 64.0%, and 66.9% homology of H. pylori from both sources, respectively. The data suggest that more than one H. pylori strain may exist in the stomach and saliva in the same patient.

Staphylococcus aureus employs an array of virulence factors to aid in its pathogenesis. A subset of cytotoxins termed bi-component leucocidins have been characterized as important determinants in the host-pathogen interaction in S. aureus infections. While they have been studied over a century, bi-component leucocidins’ complex mechanisms in pathogenesis have not been fully elucidated. Secreted as monomers, with the exception of LukAB/GH, many combinations of the S- (HlgA, HlgC, LukE, LukS, LukA/H) and F- (HlgB, LukD, LukF, LukB/G) components have demonstrated cell lysis via pore formation and a magnified proinflammatory response at sublytic concentrations. While studies have described various host cellular receptors and therapeutic strategies to evade leucocidin binding, a common receptor for all the leucocidins has yet to be classified. Challenges in data extrapolation have occurred due to non-native protein expression methods and species specificity of the leucocidin components. In turn, developing successful therapeutic strategies has proven problematic with a need for multimodal therapy. Thus, our studies aimed to clarify the bi-component leucocidins’ cytotoxic effects on multiple subsets of leukocytes using a native protein expression system and to identify a novel human leukocyte receptor common to all leucocidins. Overall, combinations with HlgA and LukE demonstrated the highest degrees of cytotoxicity against PMNs and PBMCs. Coronin 1A was discovered as a common receptor for all cognate pairs of bi-component leucocidins, except LukAB/GH. In conclusion, our results have expanded the knowledge of the cellular targets for leucocidin cytotoxicity and have described a common leucocidin receptor as a potential therapeutic target against the bi-component leucocidins.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-04T16:19:04Z No. of bitstreams: 1 2011 - B?rbara Amorim Silva.pdf: 5390134 bytes, checksum: 32ec2143250d4b2b5d5a8de98ea88d9d (MD5) / Made available in DSpace on 2016-10-04T16:19:04Z (GMT). No. of bitstreams: 1 2011 - B?rbara Amorim Silva.pdf: 5390134 bytes, checksum: 32ec2143250d4b2b5d5a8de98ea88d9d (MD5) Previous issue date: 2011-02-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / The castor bean (Ricinus communis L) is a plant family Euphorbiaceae. Considered an oil of high economic value because this has a clearly defined market for the oil extracted from its seeds. The cake, which is a residue of this extraction, stands out for its high protein content. Among the proteins found in the cake stands ricin, a cytotoxin, which not allows its use as an alternative protein source for animal feed. In this study was used the extrusion-cooking technology, combined application of calcium hydroxide Ca(OH)2 in order to inactivate the protein fraction containing ricin. The extrusion process of cake castor oil was carried out using a Brabender DS20 cannon short. Was used the response surface methodology to investigate the effect of the interaction of parameters in the study. Was used a central composite design in order to evaluate the moisture effects (14%, 16%, 18%, 20%, and 21%), temperature (116?C, 130?C, 150?C, 170?C and 180?C) and concentration of Ca(OH)2.. As a comparative sample was used castor beans which were crushed to extract oil and cake which used as a sample without heat treatment, as standard. For the evaluation of physical-chemical, it was utilized methods of AOAC. The quantification of proteins was realized using the method of Bradford and the protein profile was electrophoresis-SDS-PAGE. Was used to mark the Biorad electrophoresis the methodology observed using the of preparation of the gels described by LaemmLi (1970). The results indicated that most of the different treatments performed totally changed the structure of ricin. Indizatily the possibility of total hydr?lise of this macromolecule. The application of extracts of castor oil in the gel electrophoresis allowed the observation of a clear chain A (RTA) and 38 kDa molecular weight B (RTB) molecular weight 36kDa. For the crude protein extract of bran molar mass chains A and B were approximately 37 kDa and 35 kDa respectively. The levels of protein extracted from the bran decreased 89% after extrusion. The results indicated that all treatments changed entirely the structure of ricin with the possibility of their having been a total hydrolysis of the polymer. According to tests performed and observed results, we conclude that the thermoplastic extrusion process is a suitable technology to decrease the activity of ricin / A mamoneira (Ricinus communis L.) ? uma planta da fam?lia Euforbi?cea que vem sendo muito estudada como fonte alternativa na produ??o de biodisel. O farelo, res?duo gerado ap?s a extra??o do ?leo da semente utilizando solvente, apresenta alto teor de prote?nas. Dentre as prote?nas encontradas na torta destaca-se a ricina, uma citotoxina, que inviabiliza sua utiliza??o como fonte prot?ica alternativa para alimenta??o animal. No presente estudo empregamos a extrus?o termopl?stica, associada ? aplica??o de hidr?xido de c?lcio Ca (OH)2 com o objetivo de inativar a fra??o prot?ica que contem a ricina. O processo de extrus?o do farelo de mamona foi realizado utilizando uma extrusora BRABENDER DS20 de canh?o curto. Utilizou-se a metodologia de superf?cie de resposta para verificar o efeito da intera??o dos par?metros estabelecidos no estudo. Foi utilizado um delineamento composto central rotacional a fim de avaliar os efeitos dos n?veis de umidade (14%, 16%, 20% e 21%), temperatura (116?C, 130?C, 150?C, 170?C e 180?C) e concentra??o Ca(OH)2.. Como amostra comparativa foi utilizada sementes de mamona da cultivar Paragua?? as quais foram esmagadas em prensa expeller at? extrair o ?leo e cuja torta foi utilizada como padr?o por apresentar maior integridade das cadeias polipept?dicas. Para a avalia??o das caracter?sticas f?sico-qu?micas, foram utilizados m?todos da AOAC, para quantifica??o de prote?nas foi utilizado o m?todo de Bradford e para avalia??o do perfil prot?ico foi utilizada a eletroforese- SDS-PAGE, atrav?s do sistema de eletroforese da marca Biorad com a metodologia de prepara??o dos g?is descrita por Laemmli (1970). Os m?todos de purifica??o usando a di?lise e precipita??o com sulfato de am?nio favoreceu para um bom rendimento quanto ao teor de prote?na, principalmente a ricina. A aplica??o dos extratos da torta de mamona no gel de eletroforese permitiu a observa??o n?tida das cadeias A (RTA) massa molar 38 kDa e B (RTB) massa molar 36kDa. Para o extrato prot?ico do farelo bruto a massa molar das cadeias A e B ficaram em torno 37 kDa e 35 kDa respectivamente. Os n?veis de prote?na extra?da do farelo diminu?ram 89% ap?s a extrus?o. Os resultados indicaram que todos os tratamentos realizados modificaram totalmente a estrutura da ricina, com possibilidade de ter havido hidr?lise total desta macromol?cula. De acordo com os ensaios realizados e resultados observados, conclui-se que o processo de extrus?o termopl?stica ? uma tecnologia adequada para diminuir a atividade da ricina. Palavras-chave: Citotoxina, Eletroforese

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363363 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009