INTERACTIONS OF HUMAN ORAL CELLS WITH ORAL BACTERIAL

poster abstractIntroduction: Streptococcus mutans is the main etiological cause of den-tal caries, and it has been shown that individuals who smoke have increased dental caries. S. mutans is known to bind to or interact with MG63 osteo-blasts. However, very little is known about the effects of tobacco directly on these bacteria on their ability to affect human pulp MG63 osteoblasts. We are hypothesizing that tobacco upregulates the expression of pro-inflammatory cytokines and MMPs to increase the pathogenic potential of S. mutans. The objective of this research project is to investigate the effects that nicotine, cigarette smoke condensate (CSC), and dissolvable smokeless tobacco (DST)-extract treated bacterial cells have on humanMG63 osteo-blasts, in respect to their release of pro-inflammatory and anti-inflammatory cytokines, as well as MMP expression. In addition, the effects of the S. mutans cells will be examined for the ability to affect MG63 osteoblast growth. The long-term goal is to develop treatment modalities to reduce the effects of smoking on dental caries. Materials and Methods: S. mutans UA159 was incubated in Tryptic Soy Broth (TSB), with the following concentrations: 2 mg/mL nicotine, 0.125 mg/mL CSC, 100 uL/3 mL DST-extract, and a 0 mg/mL control group. The cultures were grown in the presence of the tobacco products for 8 h at 37oC in 5% CO2, and centrifuged to isolate cells and supernatants. The cells were washed and heat-killed for 1 h at 60oC. Human MG63 osteoblasts were iso-lated from extracted teeth, and cell passages 3-8 will be used. The tobacco-treated S. mutans cells and supernatants will be incubated with the osteo-blasts in culture plates for 72 h and cytokine expression evaluated by re-verse transcriptase polymerase chain reaction. Results: The protein concentration of each tobacco-treated sample was found. The undiluted concentrations of the nicotine- and CSC-treated cells were slightly lower and the DST-treated cells was slightly higher than the control cells. The undiluted nicotine (p<0.05) and DST-treated supernatants were higher than the control, while the CSC supernatant protein concentra-tion was lower. From our previous studies, it was found that nicotine in-creases bacteriocin production of S. mutans, so we might hypothesize that nicotine induces bacteriocin secretion, thus increasing dental caries

Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.

Contains fulltext : 47379.pdf (publisher's version ) (Open Access)The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To analyze the role of MMPs and TIMPs in tissue remodeling under normal and pathological conditions, it is important to have reliable detection methods. This review will focus on zymographical techniques for the analysis of MMPs and TIMPs. MMPs can be analyzed with several zymographical techniques, but substrate zymography is the most commonly used. This technique identifies MMPs by the degradation of their preferential substrate and by their molecular weight. Several substrates that can be used for zymography are described. Reverse zymography, which detects TIMPs by their ability to inhibit MMPs, is also discussed. Finally, in situ zymography is described, which is used to localize MMPs in tissue sections. Common problems encountered during sample preparation, zymography itself and the data analysis are discussed. Hints are given to improve the sensitivity and accuracy of zymographical methods. In conclusion, zymography is a valuable tool for research purposes and for the development of new diagnostic techniques and therapies for pathological conditions such as rheumatoid and osteoarthritis, and tumor progression

Insights into MMP-TIMP interactions

Bode W, Fernandez-Catalan C, Grams F, et al. Insights into MMP-TIMP interactions. In: INHIBITION OF MATRIX METALLOPROTEINASES: THERAPEUTIC APPLICATIONS. Annals of the New York Academy of Sciences. Vol 878. NEW YORK ACAD SCIENCES; 1999: 73-91.The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis, Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions, Within the last few years, several three-dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs, Very recently, structural information also became available for some TIMP structures and MMP-TIMP complexes, and these new data elucidated important structural features that govern the enzyme-inhibitor interaction