Junji Ohnishi

NARF, an nemo-like kinase (NLK)-associated ring finger protein regulates the ubiquitylation and degradation of T cell factor/lymphoid enhancer factor (TCF/LEF).

β-Catenin is a key player in the Wnt signaling pathway, and interacts with cofactor T cell factor/lymphoid enhancer factor (TCF/LEF) to generate a transcription activator complex that activates Wnt-induced genes. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via phosphorylation of TCF/LEF. To further evaluate the physiological roles of NLK, we performed yeast two-hybrid screening to identify NLK-interacting proteins. From this screen, we isolated a novel RING finger protein that we term NARF (NLK associated RING finger protein). Here, we show that NARF induces the ubiquitylation of TCF/LEF in vitro and in vivo, and functions as an E3 ubiquitin-ligase that specifically cooperates with the E2 conjugating enzyme E2-25K. We found that NLK augmented NARF binding and ubiquitylation of TCF/LEF, and this required NLK kinase activity. The ubiquitylated TCF/LEF was subsequently degraded by the proteasome. Furthermore, NARF inhibited formation of the secondary axis induced by the ectopic expression of β-catenin in Xenopus embryos. Collectively, our findings raise the possibility that NARF functions as a novel ubiquitin-ligase to suppress the Wnt-β-catenin signaling.

Nemo-Like Kinase-Myocyte Enhancer Factor 2A Signaling Regulates Anterior Formation in Xenopus Development

ABSTRACT The development of anterior neural structure in Xenopus laevis requires the inhibition of bone morphogenic protein 4 and Wnt signaling. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via the phosphorylation of T-cell factor/lymphoid enhancer factor. However, the molecular events occurring downstream of NLK pathways in early neural development remain unclear. In the present study, we identified the transcription factor myocyte enhancer factor 2A (MEF2A) as a novel substrate for NLK. NLK regulates the function of Xenopus MEF2A (xMEF2A) via phosphorylation, and this modification can be inhibited by the depletion of endogenous NLK. In Xenopus embryos, the depletion of either NLK or MEF2A results in a severe defect in anterior development. The endogenous expression of anterior markers was blocked by the depletion of endogenous Xenopus NLK (xNLK) or xMEF2A but, notably, not by the depletion of other xMEF2 family proteins, xMEF2C and xMEF2D. Defects in head formation or the expression of the anterior marker genes caused by the depletion of endogenous xMEF2A could be eliminated by the expression of wild-type xMEF2A, but not xMEF2A containing mutated xNLK phosphorylation sites. Furthermore, the expression of xNLK-induced anterior markers was efficiently blocked by the depletion of endogenous xMEF2A in animal pole explants. These results show that NLK specifically regulates the MEF2A activity required for anterior formation in Xenopus development.

Evaluation of mental stress by physiological indices derived from finger plethysmography.

BackgroundQuantitative evaluation of mental stress is important to prevent stress-related disorders. Finger plethysmography (FPG) is a simple noninvasive method to monitor peripheral circulation, and provides many physiological indices. Our purpose is to investigate how FPG-derived indices reflect on mental stress, and to clarify any association between these physiological indices and subjective indices of mental stress.MethodsThirty-one healthy women (mean age, 22 years ± 2) participated. The participants rested by sitting on a chair for 10 min. They then performed a computerized version of the Stroop color-word conflict test (CWT) for 10 min. Finally, they rested for 10 min. FPG was recorded throughout the experiment. The participants completed a brief form of the Profile of Mood States (POMS) questionnaire before and after the test. Using the FPG data, we conducted chaos analysis and fast Fourier transform analysis, and calculated chaotic attractors, the largest Lyapunov exponent, a high-frequency (HF) component, a low-to-high-frequency (LF/HF) ratio, finger pulse rate and finger pulse wave amplitude.ResultsThe HF component decreased and the LF/HF ratio increased significantly during the test (P < 0.01), while the confusion subscale of POMS increased after the test (P < 0.05). During testing, finger pulse rate significantly increased (P < 0.001), and the finger pulse wave amplitude decreased (P < 0.001). The attractor size reduced during testing and returned to a baseline level afterwards. Although the largest Lyapunov exponent showed no significant change during testing, significant negative correlation with the tension-anxiety subscale of POMS was observed at the beginning (P < 0.01). A significant negative correlation between the LF/HF ratio and two subscales was also observed in the beginning and middle of the test (P < 0.05). There were no correlations during the rest periods.ConclusionsThe physiological indices derived from FPG were changed by mental stress. Our findings indicate that FPG is one of the easiest methods to evaluate mental stress quantitatively. In particular, the largest Lyapunov exponent and the LF/HF ratio might be associated with acute mental stress. Farther examination is needed to find any association between the physiological indices and various types of mental stress.

Nemo-Like Kinase, an Essential Effector of Anterior Formation, Functions Downstream of p38 Mitogen-Activated Protein Kinase

ABSTRACT Nemo-like kinase (NLK) is known to function as a mitogen-activated protein kinase (MAPK)-like kinase. However, the upstream molecules and molecular mechanisms that regulate NLK activity remain unclear. In the present study, we identified p38 MAPK as an upstream kinase and activator of NLK. p38 regulates the function of NLK via phosphorylation, and this modification can be abrogated by depletion of endogenous p38. In Xenopus laevis embryos, depletion of either p38β or NLK by antisense morpholino oligonucleotides results in a severe defect in anterior development and impaired expression of endogenous anterior markers. It is notable that morphants of Xenopus p38α, another isoform of the p38 MAPK family, exhibited no obvious defects in anterior development. Defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous p38β expression could be rescued by expression of wild-type NLK but not by expression of mutant NLK lacking the p38β phosphorylation site. In contrast, defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous NLK expression could not be rescued by expression of p38. These results provide the first evidence that p38 specifically regulates NLK function, which is required for anterior formation in Xenopus development.

Tickling increases dopamine release in the nucleus accumbens and 50 kHz ultrasonic vocalizations in adolescent rats.

Adolescent rats emit 50 kHz ultrasonic vocalizations, a marker of positive emotion, during rough-and-tumble play or on tickling stimulation. The emission of 50 kHz ultrasonic vocalizations in response to tickling is suggested to be mediated by dopamine release in the nucleus accumbens; however, there is no direct evidence supporting this hypothesis. The present study aimed to elucidate whether play behavior (tickling) in adolescent rats can trigger dopamine release in the nucleus accumbens with hedonic 50 kHz ultrasonic vocalizations. The effect of tickling stimulation was compared with light-touch stimulation, as a discernible stimulus. We examined 35–40-day-old rats, which corresponds to the period of midadolescence. Tickling stimulation for 5 min significantly increased dopamine release in the nucleus accumbens (118±7% of the prestimulus control value). Conversely, light-touch stimulation for 5 min did not significantly change dopamine release. In addition, 50 kHz ultrasonic vocalizations were emitted during tickling stimulation but not during light-touch stimulation. Further, tickling-induced 50 kHz ultrasonic vocalizations were significantly blocked by the direct application of SCH23390 (D1 receptor antagonist) and raclopride (D2/D3 receptor antagonist) into the nucleus accumbens. Our study demonstrates that tickling stimulation in adolescent rats increases dopamine release in the nucleus accumbens, leading to the generation of 50 kHz ultrasonic vocalizations.

Tickling during adolescence alters fear-related and cognitive behaviors in rats after prolonged isolation

Social interactions during adolescence are important especially for neuronal development and behavior. We recently showed that positive emotions induced by repeated tickling could modulate fear-related behaviors and sympatho-adrenal stress responses. In the present study, we examined whether tickling during early to late adolescence stage could reverse stress vulnerability induced by socially isolated rearing. Ninety-five male Fischer rats were reared under different conditions from postnatal day (PND) 21 to 53: group-housed (three rats/cage), isolated-nontickled (one rat/cage) and isolated-tickled (received tickling stimulation for 5min a day). Auditory fear conditioning was then performed on the rats at PND 54. Isolated-tickled rats exhibited significantly lower freezing compared with group-housed rats in the first retention test performed 48h after conditioning and compared with isolated-nontickled rats in the second retention test performed 96h after conditioning. Moreover, group-housed and isolated-tickled rats tended to show a significant decrease in freezing responses in the second retention test; however, isolated-nontickled rats did not. In the Morris water maze task that was trained in adulthood (PND 88), but not in adolescence (PND 56), isolated-nontickled rats showed slower decrease of escape latency compared to group-housed rats; however, tickling treatment significantly improved this deficit. These results suggest that tickling stimulation can alleviate the detrimental effects of isolated rearing during adolescence on fear responses and spatial learning.

Effects of repeated tickling on conditioned fear and hormonal responses in socially isolated rats.

Positive emotional states have been reported to modify human resilience to fear and anxiety, but few animal models are available to elucidate underlying mechanisms. In the current study, we examined whether 2 weeks of tickling, which is considered to evoke positive emotions, alters conditioned fear and hormonal reactions in Fischer rats. We conditioned rats to fear an auditory tone which was initially paired with a mild foot-shock (0.2mA), and retention test was conducted 48h and 96h after conditioning. During these tests, we found that prior tickling treatment significantly diminished fear-induced freezing. To examine the effects of tickling on sympatho-adrenal and hypothalamic-pituitary-adrenal responses associated with conditioned fear, we measured plasma catecholamine and corticosterone levels in the retention test 96h after conditioning. The plasma catecholamine concentration of non-tickled rats was higher than basal levels, whereas tickled rats showed significantly reduced concentrations of both plasma adrenaline and noradrenaline. No significant differences in plasma corticosterone levels were observed between tickled and non-tickled rats. These results suggest that repeated exposure to tickling can modulate fear-related behavior and sympatho-adrenal stress responses.

Tickling stimulation causes the up-regulation of the kallikrein family in the submandibular gland of the rat

We recently showed that tactile stimulation (tickling) accompanied by positive emotion altered the expression of many genes in the rat hypothalamus (Hori et al., 2009 [15]). In this study, the effect of repeated tickling on gene expressions of the rat salivary gland was examined. After 4-week stimulation, several genes of the kallikrein (Klk) family were remarkably up-regulated and the alpha-amylase (amylase) gene was down-regulated in DNA microarray analysis. In quantitative analysis using real-time PCR of the submandibular gland of the rats tickled for 2 weeks, mRNAs of Klk1, Klk2 (Klk1c2, Tonin), Klk7 (Klk1l), Klk1b3 (Nerve growth factor, gamma), Klk1c10, Klks3 (Klk1c9) and GK11 were significantly 2-5-fold increased among 18 members of the Klk gene family examined and the submandibular amylase was decreased compared with the lightly touched and untouched control rats. In immunoblot analysis the increase in Klk7 protein was observed in the whole cell lysate fraction of the submandibular gland. In immunohistochemical analysis with anti-Klk7 polyclonal antibody, the immunostain was increased in duct cells of the submandibular gland of the tickled rat when compared with the lightly touched and untouched control rats. These results suggest that tactile sensory processing in the central nervous system affects the gene expression in the peripheral tissue probably via hormonal and/or autonomic neural activities. Submandibular Klks may be biochemical markers indicating positive emotional states.

Mucin 3 is involved in intestinal epithelial cell apoptosis via N-(3-oxododecanoyl)-l-homoserine lactone-induced suppression of Akt phosphorylation

N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.

Distinct transcriptional and metabolic profiles associated with empathy in Buddhist priests: a pilot study

BackgroundGrowing evidence suggests that spiritual/religious involvement may have beneficial effects on both psychological and physical functions. However, the biological basis for this relationship remains unclear. This study explored the role of spiritual/religious involvement across a wide range of biological markers, including transcripts and metabolites, associated with the psychological aspects of empathy in Buddhist priests.MethodsTen professional Buddhist priests and 10 age-matched non-priest controls were recruited. The participants provided peripheral blood samples for the analysis of gene expression and metabolic profiles. The participants also completed validated questionnaires measuring empathy, the Health-Promoting Lifestyle Profile-II (HPLP-II), and a brief-type self-administered diet history questionnaire (BDHQ).ResultsThe microarray analyses revealed that the distinct transcripts in the Buddhist priests included up-regulated genes related to type I interferon (IFN) innate anti-viral responses (i.e., MX1, RSAD2, IFIT1, IFIT3, IFI27, IFI44L, and HERC5), and the genes C17orf97 (ligand of arginyltranseferase 1; ATE1), hemoglobin γA (HBG1), keratin-associated protein (KRTAP10-12), and sialic acid Ig-like lectin 14 (SIGLEC14) were down-regulated at baseline. The metabolomics analysis revealed that the metabolites, including 3-aminoisobutylic acid (BAIBA), choline, several essential amino acids (e.g., methionine, phenylalanine), and amino acid derivatives (e.g., 2-aminoadipic acid, asymmetric dimethyl-arginine (ADMA), symmetric dimethyl-arginine (SMDA)), were elevated in the Buddhist priests. By contrast, there was no significant difference of healthy lifestyle behaviors and daily nutrient intakes between the priests and the controls in this study. With regard to the psychological aspects, the Buddhist priests showed significantly higher empathy compared with the control. Spearman’s rank correlation analysis showed that empathy aspects in the priests were significantly correlated with the certain transcripts and metabolites.ConclusionsWe performed in vivo phenotyping using transcriptomics, metabolomics, and psychological analyses and found an association between empathy and the phenotype of Buddhist priests in this pilot study. The up-regulation of the anti-viral type I IFN responsive genes and distinct metabolites in the plasma may represent systemic biological adaptations with a unique signature underlying spiritual/religious practices for Buddhists.