18­α­glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Application of Different Wavelengths of LED Lights in Antimicrobial Photodynamic Therapy for the Treatment of Periodontal Disease.

Therapeutic light has been increasingly used in clinical dentistry for surgical ablation, disinfection, bio-stimulation, reduction in inflammation, and promotion of wound healing. Photodynamic therapy (PDT), a type of phototherapy, has been used to selectively destroy tumor cells. Antimicrobial PDT (a-PDT) is used to inactivate causative bacteria in infectious oral diseases, such as periodontitis. Several studies have reported that this minimally invasive technique has favorable therapeutic outcomes with a low probability of adverse effects. PDT is based on the photochemical reaction between light, a photosensitizer, and oxygen, which affects its efficacy. Low-power lasers have been predominantly used in phototherapy for periodontal treatments, while light-emitting diodes (LEDs) have received considerable attention as a novel light source in recent years. LEDs can emit broad wavelengths of light, from infrared to ultraviolet, and the lower directivity of LED light appears to be suitable for plaque control over large and complex surfaces. In addition, LED devices are small, lightweight, and less expensive than lasers. Although limited evidence exists on LED-based a-PDT for periodontitis, a-PDT using red or blue LED light could be effective in attenuating bacteria associated with periodontal diseases. LEDs have the potential to provide a new direction for light therapy in periodontics.

Effects of various light-emitting diode wavelengths on periodontopathic bacteria and gingival fibroblasts: An in vitro study.

BACKGROUND: In recent years, light has been used for bacterial control of periodontal diseases. This in vitro study evaluated the effects of light-emitting diode (LED) irradiation at different wavelengths on both Porphyromonas gingivalis and human gingival fibroblasts (HGF-1). METHODS: P. gingivalis suspension was irradiated with LEDs of 365, 405, 450, 470, 565, and 625 nm at 50, 100, 150, and 200 mW/cm2 for 3 min (radiant exposure: 9, 18, 27, 36 J/cm2, respectively). Treated samples were anaerobically cultured on agar plates, and the number of colony-forming units (CFUs) was determined. Reactive oxygen species (ROS) levels were measured after LED irradiation. The viability and damage of HGF-1 were measured through WST-8 and lactate dehydrogenase assays, respectively. Gene expression in P. gingivalis was evaluated through quantitative polymerase chain reaction. RESULTS: The greatest reduction in P. gingivalis CFUs was observed on irradiation at 365 nm with 150 mW/cm2 for 3 min (27 J/cm2), followed by 450 and 470 nm under the same conditions. While 365-nm irradiation significantly decreased the viability of HGF-1 cells, the cytotoxic effects of 450- and 470-nm irradiation were comparatively low and not significant. Further, 450-nm irradiation indicated increased ROS production and downregulated the genes related to gingipain and fimbriae. The 565- and 625-nm wavelength groups exhibited no antibacterial effects; rather, they significantly activated HGF-1 proliferation. CONCLUSIONS: The 450- and 470-nm blue LEDs showed high antibacterial activity with low cytotoxicity to host cells, suggesting promising bacterial control in periodontal therapy. Additionally, blue LEDs may attenuate the pathogenesis of P. gingivalis.

The effect of antimicrobial photodynamic therapy using yellow-green LED and rose bengal on Porphyromonas gingivalis.

INTRODUCTION: This study aimed to investigate the effects of a new antimicrobial photodynamic therapy (aPDT) system using yellow-green light-emitting diode (YGL) and rose bengal (RB) on Porphyromonas gingivalis (Pg) in vitro. MATERIALS AND METHODS: Pg suspension mixed with RB was irradiated with YGL (565 nm) or blue light-emitting diode (BL, 470 nm) at 428 mW/cm2 in comparison with chlorhexidine (CHG) treatment. The cells were cultured anaerobically on agar plates, and the number of colony-forming units (CFU) was determined. The treated suspension was anaerobically incubated, and the cell density (OD600nm) was monitored for 24 h. Also, the viability of treated human gingival fibroblast (HGF-1) was measured using WST-8 assay. Pg morphology was observed with a scanning electron microscope. The RNA integrity number of aPDT-treated Pg was determined and gene expressions were evaluated by quantitative real-time polymerase chain reaction. RESULTS: RB + YGL (aPDT) demonstrated a significantly higher reduction of CFU, compared to RB + BL (aPDT) and CHG, furthermore the OD value rapidly decreased. Morphological changes of Pg with RB + YGL were more severe than with CHG. Although RB + YGL reduced HGF-1 viability, aPDT's impact was significantly lower than CHG's. With RB + YGL treatment, RIN values decreased; furthermore, gene expressions associated with DNA replication and cell division were remarkably decreased after 12 h. CONCLUSION: The results of this study demonstrated that a novel aPDT system using RB + YGL may have potential as a new technical modality for bacterial elimination in periodontal therapy.

The Effects of Ultraviolet Light-Emitting Diodes with Different Wavelengths on Periodontopathic Bacteria In Vitro.

Objective: The aim of this study was to examine effects of recently developed ultraviolet light-emitting diodes (UV LEDs) wavelengths on in vitro growth and gene expression of cultural periodontopathic bacteria, and on viability of experimental gingival fibroblasts. Materials and methods: Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Streptococcus oralis were irradiated by UV LEDs (265, 285, 310, 365, and 448 nm) at 600 mJ/cm2 and grown anaerobically in vitro. The colony forming units were counted after 1 week. Cell morphology was observed using a scanning electron microscope (SEM). Quantitative real-time polymerase chain reaction was performed to investigate gene expression changes by 310 nm irradiation. Viability of the irradiated human gingival fibroblasts was evaluated using WST-8 assay. Results: Both 265 and 285 nm resulted in the complete death of bacteria and fibroblasts, whereas 310 nm caused partial killing and suppression of bacterial growth and much less damage to the fibroblasts in vitro. Both 365 and 448 nm resulted in no significant change. SEM showed that P. gingivalis cells gradually degraded from day 2 or 3 and were severely destructed on day 5 for 265, 285, and 310 nm. The 310 nm irradiation transiently suppressed the transcripts of SOS response- and cell division-relative genes. Conclusions: Both 265 and 285 nm may induce powerful bactericidal effects and severe fibroblast phototoxicity, and 310 nm may induce partial killing or growth suppression of bacterial cells with much less fibroblast phototoxicity. UV lights may have potential for bacterial suppression, with situations dependent on wavelength, in periodontal and peri-implant therapy.

Immunoglobulin-like domains of the cargo proteins are essential for protein stability during secretion by the type IX secretion system.

The periodontal pathogen Porphyromonas gingivalis secretes many potent virulence factors using the type IX secretion system (T9SS). T9SS cargo proteins that have been structurally determined by X-ray crystallography are composed of a signal peptide, functional domain(s), an immunoglobulin (Ig)-like domain and a C-terminal domain. Role of the Ig-like domains of cargo proteins in the T9SS has not been elucidated. Gingipain proteases, which are cargo proteins of the T9SS, were degraded when their Ig-like domains were lacking or truncated. The degradation was dependent on the activity of a quality control factor, HtrA protease. Another T9SS cargo protein, HBP35, which has a thioredoxin domain as a functional domain, was analyzed by X-ray crystallography, revealing that HBP35 has an Ig-like domain after the thioredoxin domain and that the hydrophobic regions of the thioredoxin domain and the Ig-like domain face each other. HBP35 with substitution of hydrophobic amino acids in the Ig-like domain was degraded depending on HtrA. These results suggest that the Ig-like domain mediates stability of the cargo proteins in the T9SS.

Evaluation of the child oral health promotion 'MaliMali' Programme based on schools in the Kingdom of Tonga.

INTRODUCTION: The South Pacific Medical Team (SPMT) has supported oral health care for Tongan juveniles since 1998. This voluntary activity, named the MaliMali ('smile' in Tongan) Programme, is evaluated in detail in this paper. METHODS: This evaluation was guided by the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework. The objectives were to explore: (i) whether the programme was accessible to Tongan schoolchildren (Reach); (ii) the impact of the programme on decayed, missing and filled teeth (DMFT) scores and toothbrushing habits (Effectiveness); (iii) factors that affected the adoption of the programme (Adoption); (iv) whether implementation was consistent with the programme model (Implementation); and (v) the long-term sustainability of the programme (Maintenance). RESULTS: The MaliMali Programme has grown into an international project, has spread countrywide as a uniform health promotion and is reaching children in need. Following implementation of this programme, the oral health of Tongan juveniles has improved, with a decrease in the mean DMFT index and an increase in toothbrushing. To provide training that will allow Tongans to assume responsibility for the MaliMali Programme in the future, dental health education literature was prepared and workshops on oral hygiene and the MaliMali Programme were held frequently. At present, the programme is predominantly managed by Tongan staff, rather than by Japanese staff. CONCLUSIONS: This evaluation found the MaliMali Programme to be feasible and acceptable to children and schools in the Kingdom of Tonga. The programme promotes oral health and provides accessible and improved oral health care in the school setting, consistent with the oral health-promoting school framework.

Effect of the surface morphology of silk fibroin scaffolds for bone regeneration.

Existing scaffolds cannot adequately satisfy the simultaneous requirements for the regeneration of bone. The challenge remains of how to improve the integration of newly formed bone with the surrounding tissues. The purpose of this study was to investigate the effects of two silk fibroin scaffolds, a hexafluoro isopropanol-based silk fibroin (HFIP-F) and an aqueous-based silk fibroin (A-F), for their osteoinductive potentials in large critical size bone defects in vivo. ß-tricalcium phosphate (ß-TCP) was used as a positive control. After implantation into defects created in the knee joints of rabbits for 1 and 2 weeks, hematoxylin and eosin (H-E) and Azan staining revealed that the A-F scaffold as well as ß-TCP had stronger osteoinductive ability than the HFIP-F scaffold. The A-F scaffold exhibited prominent areas of neo-tissue containing bone-like nodules. Furthermore, induced osteointegration was observed between native and neo-tissue within the osteo defects in the knee joints of rabbits. Immunohistochemical staining showed the highest expression of Runx2, BMP-2, BMP-7, Smad1/5/9 and Phospho-Smad in the A-F scaffold implants. Osteoinduction of the porous A-F scaffold might be influenced by the amount of BMP signaling present in the local microenvironment in the implants. This study opens a new avenue to use A-F silk fibroin scaffolds for the regeneration of bone defects.

Effect of antimicrobial photodynamic therapy using rose bengal and blue light-emitting diode on Porphyromonas gingivalis in vitro: Influence of oxygen during treatment.

Aims: A combination of rose bengal (RB) and blue LED (BL) has emerged as a new technical modality for antimicrobial photodynamic therapy (a-PDT). The purpose of this study was to clarify the influence of oxygen on the antimicrobial effect of RB + BL treatment on Porphyromonas gingivalis in vitro.Materials and Methods:P. gingivalis cells were treated with RB, BL (450-470 nm; 1 W/cm2, 5 s), or RB + BL under anaerobic/aerobic conditions. Cells were incubated anaerobically, and the cell density (OD600 nm) was measured after 6-48 h. Additionally, cells were cultured anaerobically on blood agar plates for 9 days, and the resulting colonies were observed. Bacterial growth within 1 h of aerobic RB + BL treatment was examined, and RNA degradation due to anaerobic/aerobic RB + BL treatment was measured after 3 h of culture. Results: Under anaerobic conditions, RB + BL significantly suppressed bacterial growth after 18 h; however, the growth after 48 h and the number of colonies after 9 days were similar to those of the untreated control. RNA degradation in the anaerobic-treatment group was not significantly different from that in the control. Under aerobic conditions, RB + BL immediately affected bacterial growth and completely inhibited growth for up to 48 h. Few colonies were detected even after 9 days of culture, and RNA was completely degraded. Conclusions: Unlike the bacteriostatic effect of anaerobic treatment, aerobic RB + BL treatment may have a bactericidal action via a-PDT effect, resulting in the destruction of RNA and bacterial cells within a short period.

Blue LED inhibits the growth of Porphyromonas gingivalis by suppressing the expression of genes associated with DNA replication and cell division.

BACKGROUND AND OBJECTIVES: Blue light has been employed or investigated in both the medical and dental fields. Many studies have so far been reported a bactericidal effect of blue light emitting diodes (LED). However, it is still unclear whether exposure to blue LED kills or inhibits the growth of bacteria. We therefore investigated the effect of blue LED irradiation on the growth of Porphyromonas gingivalis compared with the effects of red LED. MATERIALS AND METHODS: P. gingivalis cell suspensions were irradiated with blue or red LED (135 J/cm2) anaerobically, incubated for various lengths of time, and then the total RNAs were isolated. The RNA degradation and gene expression levels of stress-related proteins in blue or red LED-irradiated samples were examined using the RNA integrity number (RIN) and RT-PCR, respectively. Quantitative RT-PCR was done to investigate the gene expression profiles associated with chromosome replication and cell division. RESULTS: Exposure to blue LED delayed the growth of P. gingivalis, while red LED did not. The RIN value indicated no RNA degradation in either the blue or red LED-irradiated samples. In addition, the gene expression levels of stress-related molecules remained either constant or increased 15 minutes after the blue LED irradiation compared to that before irradiation, thus suggesting that blue LED may not kill P. gingivalis cells. However, the blue LED irradiation did lead to a remarkably decreased expression of genes associated with chromosomal DNA replication and cell division after 5 minutes; exposure to the red LED did not. CONCLUSION: The inhibition of the growth of P. gingivalis by blue LED may therefore be induced not by a bactericidal effect, but instead due to a bacteriostatic effect mediated by the suppression of the genes associated with chromosomal DNA replication and cell division at the transcriptional level.

Expression of Ten-m/Odz3 in the fibrous layer of mandibular condylar cartilage during postnatal growth in mice.

It has been speculated that the mandibular condyle develops via the differentiation of the fibroblast-like cells covering the condyle into chondrocytes; however, the developmental mechanisms behind this process have not been revealed. We used laser-capture microdissection and cDNA microarray analysis to elucidate the genes that are highly expressed in these fibroblast-like cells. Among these genes, the transcription of Ten-m/Odz3 was significantly increased in the fibroblast-like cells compared with other cartilage tissues. For the first time, we describe the temporal and spatial expression of Ten-m/Odz3 mRNA in relation to the expression of type I, II, and X collagen mRNA, as determined by in-situ hybridization in mouse mandibular condylar cartilage and mouse femoral cartilage during the early stages of development. Ten-m/Odz3 was expressed in the fibrous layer and the proliferating and mature chondrocyte layers, which expressed type I and II collagen, respectively, but was not detected in the hypertrophic chondrocyte layer. Furthermore, we evaluated the in-vitro expression of Ten-m/Odz3 using ATDC5 cells, a mouse chondrogenic cell line. Ten-m/Odz3 was expressed during the early stage of the differentiation of mesenchymal cells into chondrocytes. These findings suggest that Ten-m/Odz3 is involved in the differentiation of chondrocytes and that it acts as a regulatory factor in the early stages of the development of mandibular condylar cartilage.

Hemin-binding protein 35 (HBP35) plays an important role in bacteria-mammalian cells interactions in Porphyromonas gingivalis.

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.

Role of the hemin-binding protein 35 (HBP35) of Porphyromonas gingivalis in coaggregation.

Hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis is one of the outer membrane proteins and has been reported to be a non-fimbrial coaggregation factor. In this study, a P. gingivalis HBP35-deficient mutant (MD774) was constructed from wild-type strain FDC381 by insertion mutagenesis in order to provide a better understanding of this protein's role in coaggregation. The intact cells and vesicles in FDC381 were found to have strong aggregation activities with Gram-positive bacteria. But neither the vesicles nor the intact cells showed aggregation activity in MD774. In addition, MD774 reduced autoaggregation activity. Immunoblot analysis of MD774 showed the presence of a non-maturated 45-kDa fimbrillin protein. Electron microscopy showed that the MD774 had no long fimbriae on the cell surface. Arg- and Lys-gingipain activity in MD774 was significantly decreased, compared with FDC381. Real-time RT-PCR demonstrated a significant reduction in the expression of gingipain-associated genes rgpA, rgpB, and kgp. In conclusion, we suggest that the reduction in coaggregation was caused by the combined reduction of a variety of molecules, including HBP35, gingipains, and fimbriae. Our results suggest that the HBP35 protein directly influences not only coaggregation as an adhesion molecule but also indirectly influences the expression of other coaggregation factors.

Oligonucleotide array analysis of cyclic tension-responsive genes in human periodontal ligament fibroblasts.

Mechanical stress results in differential gene expression that is critical to convert the stimulus into biochemical signals. Under physiological stress such as occlusal force, human periodontal ligament fibroblasts (HPLF) are associated with homeostasis of periodontal tissues however the changes in response to mechanotransduction remain uncharacterized. We hypothesized that cyclic tension-responsive (CT) genes may be used to identify a set of fundamental pathways of mechanotransduction. Our goal was to catalogue CT genes in cultured HPLF. HPLF were subjected to cyclic tension up to 16h, and total RNA was isolated from both tension-loaded and static HPLF. The oligonucleotide arrays analysis revealed significant changes of mRNA accumulation for 122 CT genes, and their kinetics were assigned by the K-means clustering methods. Ingenuity Pathway Analysis was completed for HPLF mechanotransduction using 50 CT genes. This analysis revealed that cyclic tension immediately down-regulated all nuclear transcription factors except v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) reacting as an early responsive gene. In turn, transcription factors such as tumor protein p53 binding protein 2 (TP53BP2), and extra-nuclear molecules such as adrenergic receptor beta2 (ADRB2) were up-regulated after 1-2h, which may result in fundamental HPLF functions to adapt to cyclic tension. Subsequent inhibition assays using Y27632, a pharmacologic inhibitor of Rho-associated kinase (ROCK), suggested that HPLF has both ROCK-dependent and ROCK-independent CT genes. Mechanical stress was found to effect the expression of numerous genes, in particular, expression of an early responsive gene; FOS initiates alteration of HPLF behaviors to control homeostasis of the periodontal ligament.

Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus.

BACKGROUND: To understand the immunopathological features of oral lichen planus (OLP), we analyzed the expression of chemokines in the epithelial cell layers. METHODS: Epithelia from OLP or healthy gingiva were collected by laser microdissection. The chemokine and chemokine receptor expressions in the epithelia were analyzed by DNA microarray. RESULTS: High levels of MIP-3alpha/LARC/CCL20 and its receptor CCR6 were expressed in the lesional epithelia. Furthermore, DC-CK1/CCL18, ELC/CCL19, SDF-1/CXCL12 and CXCR4 expressions were also increased. Immunohistologial analysis showed that high numbers of Langerhans cells (LCs) were present in the epithelia of OLP. Lesional epithelia also expressed high levels of the ligands specific for CXCR3 (e.g. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) and CCR5 (e.g. RANTES/CCL5). CONCLUSIONS: Infiltration of LCs is orchestrated by CCR6. Further, LCs residing in the lesional epithelia may be a mature phenotype. Moreover, infiltration of T cells in OLP could be mediated by signaling pathways through CXCR3 and CCR5.

Gene expression profiling and characterization under hemin limitation in Porphyromonas gingivalis.

Hemin is an important nutrient for Porphyromonas gingivalis growth and pathogenicity. We examined the gene expression profile of P. gingivalis, including genes involved in its pathogenicity, at various growth stages under hemin-standard and limited conditions by using a custom-made microarray. The transcription of many genes decreased after late-log and mid-log phases under hemin-standard and limited conditions, respectively. We focused on two groups of genes while comparing gene expression profiles under hemin-standard and limited conditions by gene tree analysis. Genes belonging to group A maintained high transcriptional levels, whereas genes in group B were expressed at low levels under standard hemin conditions. However, group B genes increased remarkably under hemin-limited conditions. Groups A and B contained genes involved in regulatory functions and protein fate, respectively. Genes related to energy metabolism, transport, and protein binding were present in both groups. Our results suggest that P. gingivalis experienced severe stress under hemin-limited conditions, and growth phase-dependent changes in transcription levels were observed for many genes. Moreover, increased expression of genes involved in energy metabolism suggests that hemin is related not only to pathogenicity, but also energy metabolism.

Monitoring of dnaK gene expression in Porphyromonas gingivalis by oxygen stress using DNA microarray.

Porphyromonas gingivalis, a Gram-negative anaerobe associated with adult periodontitis, expresses numerous potential virulence factors. dnaK, a member of the heat shock protein family, functions as a molecular chaperone and plays a role in microbial pathogenicity. However, little is known regarding its gene expression caused by oxygen stress in P. gingivalis. In the present study, a custom-made DNA microarray was designed and used to monitor dnaK gene expression in P. gingivalis caused by oxygen stress. The results demonstrated that dnaK mRNA was up-regulated in a short time, and the DNA microarray results were confirmed by real-time polymerase chain reaction analysis. These findings suggest that oxygen stress stimulates gene expression of dnaK and may have a relationship to the aerotolerance activity of this organism as well as its expression of pathogenesis.

Age-related changes in IGF-1 expression in submandibular glands of senescence-accelerated mice.

Saliva is known to play important roles in such functions as swallowing, mastication, speech, and taste. Furthermore, salivary glands synthesize and secrete a number of growth factors involved in cell/tissue homeostasis. It has been demonstrated that IGF-1, which is structurally analogous to insulin, has been shown to be expressed in mouse submandibular glands, and that IGF-1 stimulates DNA synthesis, amino acid uptake, protein synthesis, and glucose transport in various cells. Diminished function of the salivary glands is thought to lead to increased dental caries and periodontal diseases, which are commonly associated with aging. However, very little is known regarding the effects of age on IGF-1 expression in submandibular glands. The senescence-accelerated mouse (SAM), an experimental murine model of accelerated aging, has been extensively used to examine the mechanisms responsible for aging. In the present study, IGF-1 production and mRNA levels in the submandibular glands of SAM-P1 mice were examined. IGF-1 levels were determined by radioimmunoassay and IGF-1 mRNA levels by semi-quantitative RT-PCR. We found that IGF-1 protein levels in homogenates and IGF-1 mRNA levels decreased with age in SAMP1 mice. These findings suggest that IGF-1 synthesis in submandibular glands decreases with aging, and this may result in lower levels of cellular proliferation, regeneration and wound healing in aged oral tissues.

Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.