The finding that a post-log phase, cell-free media of a Porphyromonas gingivalis culture can stimulate collagenolytic activity in a keratinocyte culture system has led to work directed along two lines: to identify the factor(s) in the P.g. culture media responsible for this activity and to quantitatively assess the changes in the keratinocyte matrix metalloproteinases and inhibitors which are associated with collagen degradation. Through various purification schemes, it was found that the P.g. culture supernatant contained various molecular weight species whose relative abundance were concentration dependant. Polyclonal antibodies made to a 100 kD protease were found to inhibit stimulatory activity as well as all proteolytic activity of P.g. culture media. Immunopreciptation of 35S labeled P.g. cell extracts from early log phase culture using the polyclonal antibodies precipitated a 50 kD species protein suggesting that the 100 kD protease is a dimer. Biochemical analysis of the numerous proteolytic species in P.g. culture supernatant suggested that they are high molecular weight aggregates (>50 kD) of lower molecular weight species (>50 kD) which associate in a non-covalent, hydrophobic manner. Western blotting with monoclonal antibodies made to the various molecular weight species indicates that the high molecular weight proteolytic species are related through their cross-immunoreactivity. This work suggests that thiol-protease isoforms exist in Porphyromonas gingivalis culture. Future work includes the biochemical characterization of the protease with regard to kinetics and inhibition. Earlier analysis of the keratinocyte cell culture medium showed that the addition of the P.g. culture supernatant led to a relative increase in the amount of active collagenase present in the medium as compared to controls. Pre-incubation of purified human procollagenase with P.g. culture supernatant results in molecular weight reduction of the collagenase consistent with activation. Work is planned to address the effects in keratinocyte culture produced by the addition of the P.g. culture supernatant and includes quantitative analysis of the keratinocyte medium by western blotting and enzyme analysis.
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