RESUMO
Characterization of the molecular response under caries lesions requires a robust and reliable transcript isolation system, and analysis of data indicated that collection of extracted teeth in either liquid nitrogen/RNA-stabilizing solution facilitated this. Subsequent transcriptional analysis indicated higher general activity in carious pulps, while characterization of inflammatory mediators, including cytokines and S100 proteins, highlighted increasing expression levels associated with both microbial front progression and elevated cellular immune response. Analysis of the pleiotropic hormone adrenomedullin (ADM) indicated that transcript and protein levels are increased in pulpal tissue during caries, and that protein levels sequestered in dentin due to primary dentinogenesis are comparable with those of TGF-ß1. Expression analysis of a leucine-rich-repeat-containing protein (LRRC15/Lib) indicated that this highly conserved molecule was up-regulated during caries, is transcriptionally regulated by pro-inflammatory stimuli, and is relatively abundant in mineralized tissues.
Assuntos
Cárie Dentária/genética , Polpa Dentária/metabolismo , Mediadores da Inflamação/metabolismo , Regeneração/genética , Adolescente , Adrenomedulina/análise , Adrenomedulina/genética , Adulto , Citocinas/análise , Citocinas/genética , Cárie Dentária/imunologia , Cárie Dentária/microbiologia , Polpa Dentária/imunologia , Polpa Dentária/microbiologia , Dentina/metabolismo , Progressão da Doença , Matriz Extracelular/química , Matriz Extracelular/genética , Humanos , Imunidade Celular/genética , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Sequências Repetitivas de Aminoácidos/genética , Proteínas S100/análise , Proteínas S100/genética , Transcrição Gênica/genética , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/genética , Adulto JovemRESUMO
Quantitative structure-activity relationships were determined for the diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), OA diol-ester and dinophysistoxin-4 (DTX-4), using a sensitive bioassay procedure with the diatom Thalassiosira weissflogii. OA diol-ester was found to be nearly as toxic as OA. This result contradicted the accepted idea that only the free acid toxins, such as DTX-1 and OA, are potent phosphatase inhibitors. Postassay analyses using liquid chromatography-mass spectrometry (LC-MS) of cultures incubated with OA diol-ester showed that the ester had partially decomposed to OA, which explained some but not all of the observed toxicity. The formation of OA during the bioassay raised the possibility that cells exposed to inactive DSP toxin esters could metabolically activate them. This was examined in an additional experiment which showed that the hydrolysis of both DTX-4 and OA diol-ester was spontaneous and apparently not mediated by the presence of T. weissflogii cells. However, cells of T. weissflogii challenged with OA diol-ester rapidly metabolized most of the toxin to a more water-soluble product. From interpretation of mass spectral data obtained using ion-spray LC-MS, the metabolite was identified as an oxygenated diol-ester of OA, implying that it was the product of a monooxygenase-detoxification pathway. It is postulated that OA diol-ester, as a lipid-soluble, uncharged molecule with a propensity to hydrolyse to OA, may facilitate the transfer of OA across cell walls and membranes.
Assuntos
Diatomáceas/efeitos dos fármacos , Dinoflagellida , Toxinas Marinhas/toxicidade , Ácido Okadáico/análogos & derivados , Ácido Okadáico/toxicidade , Piranos/toxicidade , AnimaisRESUMO
The diarrhetic shellfish poisoning toxin-producing dinoflagellate, Prorocentrum lima, isolated from Nova Scotian waters, contained both okadaic acid (OA) and dinophysistoxin-1 (DTX-1) throughout its growth cycle in culture; maximum concentrations of toxins and highest OA/DTX-1 ratios occurred during the stationary phase. Cells of P. lima survived 0 degrees C for 5 weeks and recovered when brought to a higher temperature. During the cold period, some cell damage probably occurred with concomitant losses of toxins to the medium. Nitrogen concentration in the medium was used to limit growth or stress the cells physiologically, and when growth was limited, increases in toxin associated with the cells were recorded. The relative amounts of okadaic acid were always greater than dinophysistoxin-1, but the significance of these ratios remains to be determined.
Assuntos
Dinoflagellida/metabolismo , Éteres Cíclicos/metabolismo , Toxinas Marinhas/metabolismo , Nitrogênio/química , Piranos/metabolismo , Animais , Contagem de Células , Cromatografia Líquida de Alta Pressão , Temperatura Baixa , Meios de Cultura , Dinoflagellida/citologia , Dinoflagellida/crescimento & desenvolvimento , Nitrogênio/metabolismo , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Padrões de Referência , Frutos do Mar , Espectrometria de FluorescênciaRESUMO
Four fluorescent brighteners (Fluorescent Brightener 28, Fluostain 1, Fluostain II and Cellufluor) were examined with respect to their binding affinity, toxicity (their ability to stunt growth), and teratogenic effects on the red alga Antithamnion kylinii. Maximum binding occurred with FB-28 and F-II but these stains showed the greatest inhibition of growth when plants were exposed to concentrations of 0.01% for 30 min. Filaments incubated in low stain concentrations (0.0005%) showed cell abnormalities with all stain types, with FB-28 producing the most extreme deformations of both intercalary and apical cells. The experiments suggest that extensive experimentation is required to develop protocols for vital cell wall stains that minimize toxicity and maximize binding.
