Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging.

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

The effect of Porphyromonas gingivalis on the gut microbiome of mice in relation to aging.

BACKGROUND AND OBJECTIVE: The translocation of oral bacteria, including Porphyromonas gingivalis, to the gut has been shown to alter gut microbiome. However, the effect of P. gingivalis on gut microbiome in relation to aging has not been demonstrated. We hypothesize that P. gingivalis has more detrimental effect on gut environment with increased age. The objective of this study is to investigate the effect of P. gingivalis on gut environment using aged mice. MATERIALS AND METHODS: C57BL/6J mice aged 4 weeks (young) or 76 weeks (old) were divided into four groups: control-young, control-old, P. gingivalis-administered young, and P. gingivalis-administered old. P. gingivalis was orally administered thrice weekly for 5 weeks. At 30 days after the last P. gingivalis administration, 16S rRNA sequencing was performed to study the gut microbiome. The mRNA and protein expression of intestinal junctional barrier molecules and the levels of the inflammatory cytokines IL-1ß and TNF-α in the serum were evaluated. RESULTS: Significant differences in the gut microbiomes between the groups, in terms of taxonomic abundance, bacterial diversity, and predicted metagenome function, were observed. A significant reduction in the alpha diversity and in the abundance of beneficial bacteria, such as Akkermansia and Clostridiaceae, in the P. gingivalis-administered old mice was observed. The mRNA and protein levels of Claudin-1 and Claudin-2 in the intestine were significantly elevated, while E-cadherin was significantly downregulated in the P. gingivalis-administered old mice, as were the serum levels of IL-1ß and TNF-α. CONCLUSION: The effect of P. gingivalis on the gut environment is more pronounced in old mice than in young mice.

The profiling and analysis of gene expression in human periodontal ligament tissue and fibroblasts.

OBJECTIVES: The periodontal ligament (PDL) is an important component of periodontium to support dental structure in the alveolar socket. Regeneration of PDL tissue is an effective treatment option for periodontal disease and the profiling of genes involved in this process will be informative. Therefore, our study aims to accurately delineate the profiling of gene expression for PDL tissue regeneration. MATERIALS AND METHODS: We isolated PDL tissues and PDL fibroblasts (PDLFs) from premolar teeth, which were extracted from healthy periodontal status patients undergoing orthodontic treatment. Messenger RNA (mRNA) expression in PDL tissue and PDLFs were analyzed using Cap analysis gene expression, which is a second-generation sequencing technique to create profiling. We also determined the protein expression using Western blot. RESULTS: Collagens (type I, III, and VI), noncollagenous proteins (periostin and osteonectin), and proteoglycans (asporin, lumican, decorin, and osteomodulin) were highly expressed in PDL tissue. Integrin, ß1 was also expressed in PDL tissue. On comparison of gene expression between PDL tissue and PDLFs, four PDL marker genes, osteopontin, asporin, periostin, and osteonectin, were decreased in PDLFs. The genes for gene regulation were also highly expressed. CONCLUSIONS: Our study demonstrated the overall profiling of mRNA expression in PDL tissue and analyzed the important genes which may be useful for providing specific information for the reconstruction of PDL. We also identified the difference in gene expression between PDL tissue and PDLFs which might provide insights towards PDL regeneration.

Inattentive Driving Detection Using Body-Worn Sensors: Feasibility Study.

This study aims to build a system for detecting a driver's internal state using body-worn sensors. Our system is intended to detect inattentive driving that occurs during long-term driving on a monotonous road, such as a high-way road. The inattentive state of a driver in this study is an absent-minded state caused by a decrease in driver vigilance levels due to fatigue or drowsiness. However, it is difficult to clearly define these inattentive states because it is difficult for the driver to recognize when they fall into an absent-minded state. To address this problem and achieve our goal, we have proposed a detection algorithm for inattentive driving that not only uses a heart rate sensor, but also uses body-worn inertial sensors, which have the potential to detect driver behavior more accurately and at a much lower cost. The proposed method combines three detection models: body movement, drowsiness, and inattention detection, based on an anomaly detection algorithm. Furthermore, we have verified the accuracy of the algorithm with the experimental data for five participants that were measured in long-term and monotonous driving scenarios by using a driving simulator. The results indicate that our approach can detect both the inattentive and drowsiness states of drivers using signals from both the heart rate sensor and accelerometers placed on wrists.

Trends and Future Prospects of the Drowsiness Detection and Estimation Technology.

Drowsiness is among the important factors that cause traffic accidents; therefore, a monitoring system is necessary to detect the state of a driver's drowsiness. Driver monitoring systems usually detect three types of information: biometric information, vehicle behavior, and driver's graphic information. This review summarizes the research and development trends of drowsiness detection systems based on various methods. Drowsiness detection methods based on the three types of information are discussed. A prospect for arousal level detection and estimation technology for autonomous driving is also presented. In the case of autonomous driving levels 4 and 5, where the driver is not the primary driving agent, the technology will not be used to detect and estimate wakefulness for accident prevention; rather, it can be used to ensure that the driver has enough sleep to arrive comfortably at the destination.

A Review of Heartbeat Detection Systems for Automotive Applications.

Many accidents are caused by sudden changes in the physical conditions of professional drivers. Therefore, it is quite important that the driver monitoring system must not restrict or interfere with the driver's action. Applications that can measure a driver's heartbeat without restricting the driver's action are currently under development. In this review, examples of heartbeat-monitoring systems are discussed. In particular, methods for measuring the heartbeat through sensing devices of a wearable-type, such as wristwatch-type, ring-type, and shirt-type devices, as well as through devices of a nonwearable type, such as steering-type, seat-type, and other types of devices, are discussed. The emergence of wearable devices such as the Apple Watch is considered a turning point in the application of driver-monitoring systems. The problems associated with current smartwatch- and smartphone-based systems are discussed, as are the barriers to their practical use in vehicles. We conclude that, for the time being, detection methods using in-vehicle devices and in-vehicle cameras are expected to remain dominant, while devices that can detect health conditions and abnormalities simply by driving as usual are expected to emerge as future applications.

Aberrant expression of DUSP4 is a specific phenomenon in betel quid-related oral cancer.

Oral cancer due to betel quid chewing habit is very common in South Asian countries. We attempted to detect the presence of a novel gene in epithelial cells stimulated with arecoline, a main component of betel quid. Human gingival epithelial progenitors were cultured and treated with a 3-day alternating regimen with/without 50 µg/ml arecoline for 1 month. DNA microarray and methylation arrays were analyzed to identify the candidate genes. Immunohistochemical staining was performed in the tissue samples. Genome-wide analyses, quantitative reverse transcription PCR and quantitative methylation-specific PCR revealed DUSP4 as the most significant and promising gene. The methylation levels of DUSP4 were significantly higher in the betel quid-related oral squamous cell carcinoma (OSCC) than those in the non-related OSCC and controls (Mann-Whitney U test, p < 0.05). The number of DUSP4 immunopositive cells in betel quid-related OSCC was significantly higher than those from the non-chewing patients and the controls (p < 0.05). Hypermethylation of DUSP4 may be considered as a specific event in betel quid-related oral cancer.

Possibility of Autonomous Estimation of Shiba Goat's Estrus and Non-Estrus Behavior by Machine Learning Methods.

Mammalian behavior is typically monitored by observation. However, direct observation requires a substantial amount of effort and time, if the number of mammals to be observed is sufficiently large or if the observation is conducted for a prolonged period. In this study, machine learning methods as hidden Markov models (HMMs), random forests, support vector machines (SVMs), and neural networks, were applied to detect and estimate whether a goat is in estrus based on the goat's behavior; thus, the adequacy of the method was verified. Goat's tracking data was obtained using a video tracking system and used to estimate whether they, which are in "estrus" or "non-estrus", were in either states: "approaching the male", or "standing near the male". Totally, the PC of random forest seems to be the highest. However, The percentage concordance (PC) value besides the goats whose data were used for training data sets is relatively low. It is suggested that random forest tend to over-fit to training data. Besides random forest, the PC of HMMs and SVMs is high. However, considering the calculation time and HMM's advantage in that it is a time series model, HMM is better method. The PC of neural network is totally low, however, if the more goat's data were acquired, neural network would be an adequate method for estimation.

Recent Research and Developing Trends of Wearable Sensors for Detecting Blood Pressure.

Blood pressure is considered an index to measure a person's health or state. The IEEE published a standard for wearable cuffless blood pressure measuring devices, which was certified as IEEE1708 on 26 August 2014, and, according to this standard, the development of wearable devices based on blood pressure is expected in the future. Considering this, blood pressure should be detectable all the time and everywhere, and this can help improve health consciousness. In this review, we introduce the recent development of wearable blood pressure measuring devices and research trends, and present the future prospects for blood pressure measuring devices.

Immunohistochemical evaluation of Klotho and DNA methyltransferase 3a in oral squamous cell carcinomas.

Carcinoma follows a course of multiple changes that are affected by several important factors, with epigenetic silencing of the promoter gene being one of them. A series of studies have suggested that epigenetic changes in the anti-aging gene Klotho may be one of the emerging areas of concern in the study of carcinogenesis. We hypothesized that epigenetic silencing of Klotho due to hypermethylation of DNMT3a may be one of the causes of carcinoma in the oral and maxillofacial region. In this study, we analyzed the immunohistochemical expressions of Klotho and DNMT3a in tissues obtained from oral dysplasia and oral squamous cell carcinoma. Our results showed increased immune expression of DNMT3a, and decreased expression of Klotho in cells of the cancer tissues when compared with those in the dysplasia and healthy control samples. Chi-square tests complemented by adjusted residual analysis revealed significantly higher number of Klotho-positive and DNMT3a-negative cases in healthy controls, Klotho-negative and DNMT3a-negative cases in ODL, and Klotho-negative and DNMT3a-positive cases in OSCC when compared with the other types among the three groups (X 2 = 46.66, p < 0.001). Thus, downregulation of Klotho may be associated with the overexpression of DNMT3a in cancer tissues.

Automated Estimation of Mouse Social Behaviors Based on a Hidden Markov Model.

Recent innovations in sensing and Information and Communication Technology (ICT) have enabled researchers in animal behavior to collect an enormous amount of data. Consequently, the development of an automated system to substitute for some of the observations and analyses that are performed currently by expert researchers is becoming a crucial issue so that the vast amount of accumulated data can be processed efficiently. For this purpose, we introduce a process for the automated classification of the social interactive status of two mice in a square field on the basis of a Hidden Markov model (HMM). We developed two models: one for the classification of two states, namely, indifference and interaction, and the other for three states, namely, indifference, sniffing, and following. The HMM was trained with data from 50 pairs of mice as provided by expert human observers. We measured the performance of the HMM by determining its rate of concordance with human observation. We found that sniffing behavior was segmented well by the HMM; however, following behavior was not segmented well by the HMM in terms of percentage concordance. We also developed software called DuoMouse, an automated system for the classification of social interactive behavior of mice, that was based on the HMM. Finally, we compared two implementations of the HMM that were based on a histogram and a Gaussian mixture model.

Cementocyte cell death occurs in rat cellular cementum during orthodontic tooth movement.

OBJECTIVE: To clarify the mechanism of root resorption during orthodontic treatment, we examined cementocyte cell death and root resorption in the cellular cementum on the pressure side during experimental tooth movement. MATERIALS AND METHODS: Using 8-week-old male Wistar rats, the right first molar was pushed mesiobuccally with a force of 40 g by a Ni-Ti alloy wire while the contralateral first molar was used as a control. Localization and number of cleaved caspase-3-positive and single-stranded DNA (ssDNA) - positive cells were evaluated using dual-label immunohistochemistry with anticleaved caspase-3 and anti-ssDNA antibodies. In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the cellular cementum were evaluated using TRAP histochemical staining. RESULTS: Caspase-3- and ssDNA-positive cells appeared at 12 hours, but were restricted to the compressed periodontal ligament (PDL) and not the cellular cementum. Cleaved caspase-3-positive cementocytes were observed in the cellular cementum adjacent to the compressed PDL on day 1. From days 2 to 4, the number of caspase-3- and ssDNA-positive cementocytes increased. TRAP-positive cells appeared on the cellular cementum at the periphery of the hyalinized tissue on day 7, and resorption progressed into the broad surface of the cementum by day 14. CONCLUSION: Cementocytes adjacent to the hyalinized tissue underwent apoptotic cell death during orthodontic tooth movement, which might have been associated with subsequent root resorption.

Video tracking analysis of behavioral patterns during estrus in goats.

Here, we report a new method for measuring behavioral patterns during estrus in goats based on video tracking analysis. Data were collected from cycling goats, which were in estrus (n = 8) or not in estrus (n = 8). An observation pen (2.5 m × 2.5 m) was set up in the corner of the female paddock with one side adjacent to a male paddock. The positions and movements of goats were tracked every 0.5 sec for 10 min by using a video tracking software, and the trajectory data were used for the analysis. There were no significant differences in the durations of standing and walking or the total length of movement. However, the number of approaches to a male and the duration of staying near the male were higher in goats in estrus than in goats not in estrus. The proposed evaluation method may be suitable for detailed monitoring of behavioral changes during estrus in goats.

Dioscorea japonica extract down-regulates prostaglandin E2 synthetic pathway and induces apoptosis in lung cancer cells.

Prostaglandin E2 plays a role in an array of pathophysiological responses, including inflammation, carcinogenesis and so on. Prostaglandin E2 is synthesized from arachidonic acid by the enzymes cyclooxygenase and prostaglandin E synthase. In some pathological conditions, the isozymes cyclooxygenase-2 and microsomal prostaglandin E synthase-1 are transiently induced, leading to prostaglandin E2 overproduction. The present study showed that Dioscorea japonica extract suppresses mRNA expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in human non-small-cell lung carcinoma A549 cells in a dose-dependent manner. The suppressive effects of Dioscorea japonica extract on the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 were confirmed by Western blotting, cyclooxygenase activity and prostaglandin E2 production. Dioscorea japonica extract induced the translocation of nuclear factor-κB from the nucleus to the cytosol and inhibited the activity of the cyclooxygenase-2 promoter. Furthermore Dioscorea japonica extract suppressed the expression of the anti-apoptotic factor B-cell chronic lymphocytic leukemia/lymphoma 2 and enhanced apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive intensity in A549 cells. These results suggest that Dioscorea japonica extract suppresses the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, with the regulation of the transcriptional activity of cyclooxygenase-2, and induces apoptosis in cancer cells. Thus, Dioscorea japonica may contribute to the prevention of prostaglandin E2-mediated pathophysiological responses such as carcinogenesis and inflammation.

HBD-2 is downregulated in oral carcinoma cells by DNA hypermethylation, and increased expression of hBD-2 by DNA demethylation and gene transfection inhibits cell proliferation and invasion.

Human ß-defensin-2 (hBD-2) is a type of epithelial antimicrobial peptide. The expression level of hBD-2 mRNA is lower in oral carcinoma cells (OCCs) than in healthy oral epithelium. Yet, it is still unknown how hBD-2 expression is downregulated in OCCs. The present study investigated DNA hypermethylation of hBD-2 in OCCs and the effect of the demethylation and increased expression of hBD-2 on cell proliferation and invasion. Six different types of oral carcinoma cell lines (OSC-19, BSC-OF, SAS, HSC-2, HSC-4 and HSY) and normal oral keratinocytes (NOKs) were used. The expression levels of hBD-2 in all OCCs were significantly lower than that in the NOKs. Treatment with DNA methyltransferase inhibitor, 5-aza-dC, at the concentration of 50 µM significantly induced upregulation of expression of hBD-2 in the OCCs. Using methylation-specific PCR, DNA hypermethylation was observed in all OCCs. These results suggest that DNA hypermethylation is, at least in part, involved in the decreased expression of hBD-2 in OCCs. We examined the effect of 5-aza-dC on the cell proliferation and invasive ability of OCCs. The cell invasion assays showed that the number of OCCs treated with 5-aza-dC on the filters was significantly lower than that of the controls. We examined whether increased expression of hBD-2 generated by gene transfection inhibited the proliferation and invasion of SAS cells. The number of SAS cells exhibiting increased expression of hBD-2 on the filters in the invasion assay were significantly lower on day 7 when compared with the control. hBD-2 may function as a tumor suppressor. Increased expression of hBD-2 induced by demethylation or increased expression generated by gene transfection may be useful therapeutic methods for oral carcinoma.

A male-specific QTL for social interaction behavior in mice mapped with automated pattern detection by a hidden Markov model incorporated into newly developed freeware.

BACKGROUND: Owing to their complex nature, social interaction tests normally require the observation of video data by a human researcher, and thus are difficult to use in large-scale studies. We previously established a statistical method, a hidden Markov model (HMM), which enables the differentiation of two social states ("interaction" and "indifference"), and three social states ("sniffing", "following", and "indifference"), automatically in silico. NEW METHOD: Here, we developed freeware called DuoMouse for the rapid evaluation of social interaction behavior. This software incorporates five steps: (1) settings, (2) video recording, (3) tracking from the video data, (4) HMM analysis, and (5) visualization of the results. RESULTS: Using DuoMouse, we mapped a genetic locus related to social interaction. We previously reported that a consomic strain, B6-Chr6C(MSM), with its chromosome 6 substituted for one from MSM/Ms, showed more social interaction than C57BL/6 (B6). We made four subconsomic strains, C3, C5, C6, and C7, each of which has a shorter segment of chromosome 6 derived from B6-Chr6C, and conducted social interaction tests on these strains. DuoMouse indicated that C6, but not C3, C5, and C7, showed higher interaction, sniffing, and following than B6, specifically in males. COMPARISON WITH EXISTING METHOD: The data obtained by human observation showed high concordance to those from DuoMouse. The results indicated that the MSM-derived chromosomal region present in C6-but not in C3, C5, and C7-associated with increased social behavior. CONCLUSIONS: This method to analyze social interaction will aid primary screening for difference in social behavior in mice.

Tracing the fates of site-specifically introduced DNA adducts in the human genome.

We developed a system for tracing DNA adducts in targeted mutagenesis (TATAM) and investigated the prevalence and types of consequent mutations. Targeted mutagenesis methods site-specifically replace endogenous DNA bases with bases carrying synthetic adducts using targeting vectors. The TATAM system was enabled by introduction of site-specific DNA double strand breaks (DSB), which strongly enhanced targeting efficiency through homologous recombination (HR), and a new polymerase chain reaction-based technique, which gives high yields of the target vectors carrying DNA adducts. Human lymphoblastoid TSCER122 cells are compound heterozygous for the thymidine kinase gene (TK-/-), and have a homing endonuclease I-SceI site in intron 4 of the TK gene. The TATAM system enabled targeting of the TK- allele with the I-SceI site using a synthetic TK+ allele containing an 8-oxo-7,8-dihydroguanine (8-oxoG) adduct, a typical product of oxidative DNA damage. The targeted clones (TK+/-) were then isolated by drug selection. Site-specific HR for DSB induced by I-SceI improved targeted integration of the synthetic allele by five orders of magnitude (from 10(-7) to 10(-2)). Subsequent analyses of approximately 800 target clones revealed that 8-oxoG was restored to G in 86% clones, probably reflecting base excision repair or translesion synthesis without mutation. Lesions of the remaining clones (14%) were associated with mutations. The mutation spectrum corresponded closely with that of oxidative DNA damage inducers reported, in which G:C to T:A transversions (5.9%) were predominant. Over-expression of MutY homologs in cells, which prevents G:C to T:A transversions by removing 8-oxoG:A mispairing, significantly decreased the frequency of mutations to 2.6%, indicating that the 8-oxoG adducts introduced by the TATAM system are processed in the same manner as those generated by oxidative DNA damage.

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

AIM: The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. METHODS: Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. RESULTS: Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. CONCLUSION: Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement.

VAMP4 is required to maintain the ribbon structure of the Golgi apparatus.

The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.

Isoproterenol stimulates transient SNAP23-VAMP2 interaction in rat parotid glands.

The exocytosis of salivary proteins is mainly regulated by cAMP, although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which mediate cAMP-dependent exocytic membrane fusion, have remained unidentified. Here we examined the effect of isoproterenol (ISO) and cytochalasin D (CyD) on the level of SNARE complexes in rat parotid glands. When SNARE complexes were immunoprecipitated by anti-SNAP23, the coprecipitation of VAMP2 was significantly increased in response to ISO and/or CyD, although the coprecipitation of VAMP8 or syntaxin 4 was scarcely augmented. These results suggest that the SNAP23-VAMP2 interaction plays a key role in cAMP-mediated exocytosis from parotid glands.