Phenotypic analyses of frz and dif double mutants of Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.

Function of gingival fibroblasts and periodontal ligament cells in the presence of methyl mercaptan.

OBJECTIVE: This study examined the effects of methyl mercaptan, a product of the bacterial putrefaction of protein in periodontal pockets, on the function of cells in culture. METHOD AND MATERIALS: Human gingival fibroblasts and periodontal ligament cells were exposed to a constant, continuous flow of methyl mercaptan in vitro. Control and test cultures were then examined for changes in intracellular pH, an event often associated with alterations in cellular function. Intracellular pH was determined by single-cell image analysis of cells loaded with a fluorescent, pH-sensitive dye. Periodontal ligament cells were also tested for changes in synthesis of total protein and fibronectin. RESULTS: Test cells exhibited a consistent decrease in intracellular pH following exposure to methyl mercaptan. Measurements of total protein production showed that test periodontal ligament cell cultures produced approximately 30% less protein than control cultures (P < 0.05). Western-blot analysis of fibronectin in medium demonstrated that abnormal monomeric fibronectins were a major protein in test, but not in control, cell cultures. CONCLUSION: Exposure to methyl mercaptan induced alterations in intracellular events that paralleled changes in extracellular matrix proteins. The observed changes in extracellular matrix proteins support the hypothesis that methyl mercaptan contributes to the progression of periodontal disease.

The response of periodontal ligament cells to fibronectin.

Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.

Exposure of periodontal ligament cells to methyl mercaptan reduces intracellular pH and inhibits cell migration.

Volatile sulfur compounds such as hydrogen sulfide and methyl mercaptan have been associated with adult periodontitis as well as with healing surgical wounds. To examine the effects of these compounds on the periodontium, we assayed periodontal ligament (PDL) cells for changes in intracellular pH, total protein, and cell migration following chronic exposure to CH3SH. Intracellular pH was quantitated by fluorescence measurements of cells loaded with BCECF, a pH-sensitive dye. Data show that 48-hour exposure to mercaptan lowered resting intracellular pH but did not consistently alter activity of the Na/H exchanger. This effect was seen in PDL cells from three different patients. Lowered pH was accompanied by decreases in both total protein and mature alpha 1 and alpha 2 chains of type I collagen. Since reductions in intracellular pH and total protein have been associated with inhibition of cell motility, migration was quantitated by sequential computer imaging, which measured the increase in size of plated cell circles at different times of migration. Incubation of PDL cells in pH 7.4 and 6.6 buffers reversibly altered intracellular pH. Migration was reversibly inhibited in pH 6.8 buffer. Exposure to CH3SH reduced intracellular pH in pH 7.4 buffer and in three independent assays inhibited enlargement of cell circles in pH 7.4 medium. These effects were therefore not related to alterations of extracellular pH, which remained at 7.4. The results support the hypothesis that gases such as methyl mercaptan may play a role in both surgical wound healing and periodontal disease by adversely affecting cell function and suggest that alterations in intracellular pH may be part of the mechanism for these changes.