Recently, the use of multipotent stem cells has been studied as for regeneration medicine of missing craniofacial bone. Most craniofacial structures are derived from cranial neural crest cells. We investigated the use of craniofacial bone induction from mouse embryonic stem (ES) cells differentiated into neural crest cells via neuroectoderm. First, we cultured mouse ES cells in serum-free neuroinduction medium for sphere formation and confirmed they were precursors of neuronal and neural crest cells by immunostaining using specific antibodies for Nestin and AP2α, respectively. The sphere contained Nestin and AP2α positive cells, indicating that a portion of the ES cells differentiated into neural crest cells through neural precoursor cells during sphere formation. These findings indicated the possibility that neural crest cells can be generated from mouse ES cells through spheres cultured in serum-free medium.

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Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. Because T and NK cells infiltrate inflammatory gingival tissues during periodontal disease, a local immunoresponse is believed to be involved. Interleukin 17 is a proinflammatory cytokine secreted by activated T-lymphocytes. IL-17 plays essential roles in regulating inflammation and host defense. While the IL-17 cytokine is expressed only by T-cells, its receptor is expressed in many cells such as neutrophils and macrophages. However, the IL-17 function to NK cells remains incompletely understood. We examined the cell proliferation ability by IL-17 stimulation on NK92 cells. There was no effect of IL-17 alone on NK92 cell proliferation activity, nor was there coordinate action of IL-2 and IL-17. We also investigated the effect of IL-17 on NK92 cell cytotoxicity against K562 cells. NK92 cells stimulated by IL-17 suppressed the cytotoxicity against K562 cells. These results suggest that IL-17 stimulation may mediate an inhibitory signal on NK92 cells.

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A previous study by Ishida et al. disclosed that the ethanol extract from Sciadopitys vertillata was active not only against periodontopathic bacteria Porphyromonas gingivalis and Prevotella intermedia/nigrescens, but also against facultative cocci and rods including biofilmforming bacteria that possess meshwork-like structures around cells. In this study we attempted to separate antimicrobial fractions from the ethanol extract by various organic solvents such as hexane, diisopropyl ether, ethyl acetate and butanol and to characterize the antimicrobial spectra of these fractions. The diisopropyl ether layer contained 74.1% of the total antimicrobial activity, followed by the hexane layer (19.8%) and ethyl acetate layer (6.2%), respectively. However, there were no activity in both butanol and water layers. After gel filtration of hexane layer, active fractions were found between the first and second peak (Hb). In the diisopropyl ether layer, antimicrobial activities were found in the first peak (Ea), the second peak (Eb), and the low molecular weight fractions (Ec). The antimicrobial spectra of the fractions Hb, Ea, Eb and EC were each different. These results suggest that the ethanol extract from Sciadopitys vertillata contains different antimicrobial molecules with respect to polarity, molecular weight and antimicrobial spectra.

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We previously demonstrated that the expression of DnaK, one of the major heat shock proteins (HSPs), was up-regulated in biofilm-forming Prevotella intermedia strain 17 (strain 17). The entire encoding sequence for the strain 17 DnaK protein was cloned into pET and expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS. The DnaK was expressed in a soluble form at a high level in the E. coli cytoplasm. This recombinant DnaK protein was used as an immunogen to obtain rabbit antibodies against the wild-type DnaK of this organism. The alignment of 10 amino-acid residues from an N-terminus of this recombinant protein was identical to that of the wild-type DnaK protein. The rabbit polyclonal antibodies recognized 70 kD protein whose molecular weight corresponds to that of the wild-type DnaK in the sonicate of strain 17 cells as well as in the culture supernatant. These results suggest that the recombinant P. intermedia DnaK and the antibodies obtained in this study can be used to study the function of this HSP in the biofilm formation of this organism.

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Recent studies have revealed that Actinomyces spp. plays an important role as an initial colonizer in dental plaque formation. However, only a few systems for genetic engineering have been available in Actinomyces spp. In this study, we determined whole sequences of pJRD215, the wide host-range conjugative vector, and evaluated its transformation efficiency in several Actinomyces spp. pJRD215 was transformed with high efficiency to Actinomyces oris strain MG-1, and with low efficiency to Actinomyces viscosus strain ATCC 27044, Actinomyces naeslundii strain ATCC 27039 and Actinomyces naeslundii strain ATCC 51655. We then cloned either a thiostrepton resistant gene or a trimethoprim resistant gene into the downstream of the kanamycin resistant gene in pJRD215. The transformants could express functional thiostrepton resistance or trimethoprim resistance, respectively. We were able to demonstrate the availability of pJRD215 as an efficient expression vector in Actinomyces spp.

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