Pantothenate-p-nitroanilide has been synthesized for use as a substrate in a continuous spectrophotometric assay of pantetheinase activity monitoring absorbance at 41 0 nm. Pantothenate-p-nitroanilide is a crystalline compound with a molecular weight of 338.0 and a melting point of 146-149°C. Use of this substrate in the described assay is suitable for enzyme activity determination in high protein content media such as blood serum. Serum pantetheinase activity was determined for rats of varying pantothenate nutriture. Rats with mildly (but significantly, p

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Orientador: Sergio de Toledo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T06:56:35Z (GMT). No. of bitstreams: 1 Pinto_FernandoRodrigues_D.pdf: 254395 bytes, checksum: e7aa584ae3a971974f0129d6af2c160e (MD5) Previous issue date: 2005 / Resumo:A doença periodontal tem como fator etiológico primário o acúmulo de biofilme dental que pode ser favorecido por uma grande variedade de fatores retentivos locais, como por exemplo, próteses parciais removíveis, que podem alterar a progressão e o risco da doença. O objetivo deste trabalho foi avaliar o efeito do uso de próteses parciais removíveis (PPRs) sobre as condições clínicas periodontais e alterações qualitativas do biofilme dos dentes suportes por um período de dois anos. Foram selecionados 13 pacientes com média de idade de 55,8 (±14,8) anos, de acordo com os seguintes critérios: parcialmente dentados e que não utilizaram PPRs nos últimos 18 meses previamente ao início do estudo, sem envolvimento sistêmico e não fumantes. Os seguintes parâmetros clínicos periodontais foram avaliados: Índice de Placa de Silness & Löe (1964) ¿ IPL, o Índice Gengival de Löe (1967) ¿ IG, profundidade de sondagem ¿ PS, recessão gengival - RG e Nível de inserção clínico ¿ NIC; medidos através de uma sonda periodontal eletrônica de pressão controlada (Florida Probeâ, Gainesville, FL, USA). A atividade de enzimas tipo tripsina no biofilme dental dos dentes suportes foi analisada por um teste enzimático (teste BAPNA). Os dados foram coletados em quatro períodos experimentais: T0 = antes da reabilitação protética, T6 = após 6 meses de uso, T18 ¿ 18 meses e T24 após 24 meses de uso continuado das próteses. Os resultados obtidos em todos os períodos (T0, T6, T18 e T24) foram respectivamente: valores medianos para IPL (1, 1, 1 e1) e IG (0, 0, 1 e 1), valores médios e desvio padrão para PS (2,3mm ±0,3; 2,28mm ±0,4; 2,17mm ±0,2 e 2,26mm ±0,3), RG (0,78mm ±0,7; 0,87mm ±0,7; 0,87mm ±0,7 e 0,95mm ±0,8), NIC (3,08mm ±0,8; 3,15mm ±0,9; 3,04mm ±0,9 e 3,21mm ±0,9) e teste BAPNA (5,479 nmol/mg de biofilme X min ±2,2; 6,22nmol/mg de biofilme X min ±2,9; 8,199 nmol/mg de biofilme X min ±2,7 e 13,852 nmol/mg de biofilme X min ±11,7). A análise estatística demonstrou diferença para o índice gengival entre os períodos T24 e T18 em relação ao T0 e do T24 para T6 (p<0,05) e também para o teste BAPNA entre os períodos T24 e T18 em relação ao T0 e do T24 para T6 (p<0,05) indicando aumento na atividade enzimática. Concluiu-se que a simples presença das próteses parciais removíveis, mesmo em pacientes com adequado controle de placa, foi capaz de alterar um parâmetro indicativo de inflamação gengival e a composição qualitativa do biofilme destes pacientes durante um período de dois anos de uso das próteses / Abstract: The dental biofilm is the primary etiological factor of the periodontal disease and its accumulation can be influenced by a great variety of local factors, such as removable partial dentures. The aim of this study was to evaluate the possible influence of the presence of removable partial dentures (RPDs) on the periodontal clinical parameters and in the dental biofilm of the connectors¿ teeth. Thirteen partially edentulous volunteers with a mean age of 55.8 (±14.8), without RPDs, no systemic diseases, no smokers were selected. The following clinical parameters were evaluated: Plaque Index (Silness & Löe, 1964) PI, Gingival Index (Löe, 1967) GI, Probing Depth - PD, Gingival Recession ¿ GR and Clinical Attachment Level ¿ CAL; PD, GR and CAL were obtained with an electronic computerized probe (Florida Probeâ, Gainesville, Fla, USA). The tripsin-like activity on the biofilm of the connectors¿ teeth was evaluated by an enzymatic test (BAPNA). The data were collected in four experimental periods: T0 = before the rehabilitation, T6 = six months after rehabilitation, T18 = eighteen months and T24 = 24 months of continuous use of RPDs. The clinical measurements in T0, T6, T18 and T24 were respectively: median value for PI (1, 1, 1, 1) and GI (0, 0, 1, 1); mean value and standard deviation for PD (2.3mm ±0.3; 2.28mm ±0.4; 2.17mm ±0.2 e 2.26mm ±0.3), GR (0.78mm ±0.7; 0.87mm ±0.7; 0.87mm ±0.7 e 0.95mm ±0.8), CAL (3.08mm ±0.8; 3.15mm ±0.9; 3.04mm ±0.9 e 3.21mm ±0.9). For the trypsin-like activity in T0, T6, T18 and T24 the mean value and standard deviation were T0 ¿ 5.479nmol/mg of biofilm X min ±2.2, T6 ¿ 6.22nmol/mg of biofilm X min ±2.9, T18 ¿ 8.199 nmol/mg of biofilm X min ±2.7 and T24 ¿ 13.852 nmol/mg of biofilm X min ±11.7. Statistical analysis showed differences for GI for T18 and T24 compared to T0 and T24 compared to T6 (p<0.05) and the same was observed for BAPNA harboring differences for T24 and T18 compared to T0 and between T24 and T6. It was concluded that, even in patients with good plaque control, the presence of Removable Partial Dentures does altered the health status of periodontal tissues and the trypsin-like activity of dental biofilm over a period of two years of continuous use of RPDs / Doutorado / Periodontia / Doutor em Clínica Odontológica

The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides or “conotoxins”. Conotoxins are bioactive peptides found in the venom duct of Conus spp. Like other neuropeptides, conotoxins are expressed as propeptides that undergo posttranslational proteolytic processing. Peptides derived from propeptides are typically cleaved at a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys or Arg-Lys) by proteases found in secretory vesicles. However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes. As many of the substrate-specific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible. In order to understand more about the substrate specificity of conotoxin substrate-specific proteases a characterisation study of one such endoprotease isolated from the venom duct of Conus textile was undertaken. The C. textile mollusc was chosen as a good source from which to isolate the endoprotease for two reasons; firstly, these cone shells are found in great abundance on the Great Barrier Reef (Queensland, Australia) and are readily obtainable and secondly, a number of conotoxin precursors and their cleavage products have been previously identified in the venom duct. In order to purify the endoprotease an activity-guided fractionation protocol that included a para-nitroanilide (p-NA) substrate assay was developed. The p-NA substrate mimicked the cleavage site of the conotoxin TxVIA, a member of the C. textile O-superfamily of toxins. The protocol included a number of chromatographic techniques including ion exchange, size-exclusion and reverse-phased HPLC and resulted in isolation of an active protease, termed Tex31, to >95% purity. The purification of microgram quantities of Tex31 made it possible to characterise the proteolytic nature of Tex31 and to further characterise the O-superfamily conopeptide propeptide cleavage site specificity. Specificity experiments showed Tex31 requires a minimum of four residues including a leucine in the P4 position (LNKR↓) for efficient substrate processing. The complete sequence of Tex31 was determined from cDNA. A BLAST search revealed Tex31 to have high amino acid sequence similarity to the CAP (abbreviated from CRISP (Cysteine-rich secretory protein), Antigen 5 and PR-1 (pathogenesis-related protein)) superfamily and most closely related to the CRISP family of mammalian and venom proteins that, like Tex31, have a cysteine-rich C-terminal domain. The CAP superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in processes as diverse as human brain tumour growth and plant pathogenesis. This is the first report of a biological role for the N-terminal domain of CAP proteins. A homology model of Tex31 constructed from two PR-1 proteins, Antigen 5 and P14a, revealed the highly conserved and likely catalytic residues, His78, Ser99 and Glu115. These three amino acids fall within a structurally conserved N-terminal domain found in all CAP proteins. It is possible that other CAP proteins are also substrate-specific proteases. With no homology to any known proteases, Tex31 may belong to a new class of protease. The sequence alignment of five Tex31-like proteins cloned from C. marmoreus, C. litteratus, C. arentus, C. planboris, and C. omaria show very high sequence similarity to Tex31 (~80%), but only one weakly conserved serine residue was identified when the conserved residues of the new Tex31-like protein sequences were aligned with members of the CAP superfamily. Future work to identify members of catalytic diad or triad, e.g. by site-directed mutagenesis, will rely on the expression of active recombinant Tex31. In this study neither Escherichia coli nor Pichia pastoris expression systems yielded active recombinant Tex31 protein, possibly due to the number of cysteine residues hindering the expression of correctly folded active Tex31. This study has shown Tex31 to be highly sequence specific in its cleavage site and it is likely that this high substrate specificity has confounded previous attempts to identify the proteolytic nature of other CAP proteins. With the proteolytic nature of one member of the CAP protein family confirmed, it is hoped this important discovery may lead the way to discovering the role of other CAP family members.