Effect of Gum Chewing on PFC Activity During Discomfort Sound Stimulation.

The prefrontal cortex (PFC) is sensitive to the stress exposure and involved in stress coping. And the effects of gum chewing on the stress have been studied using NIRS. However, when measuring NIRS on PFC during gum chewing, blood flows in shallow tissues (scalp, skin, muscle) might be affected. A NIRS used in the present study first, which has a short distance (1 cm) and the usual (3 cm) source-detector (S-D) regression, can allow eliminating shallow tissues effect of gum chewing. The aim of this study was to investigate the hypothesis that gum chewing activates the right prefrontal cortex (PFC) in stress coping against negative sounds (NS) from the International Affective Digitized Sounds-2 (IADS) as a mental stress task. NS showed activation in the right PFC. There was a significant difference between NS, and NS with Gum, where NS with Gum showed an increased PFC activity, increased alpha wave appearance rate, a higher value in heart rate level, and a higher VAS score indicating 'pleasant'. Gum chewing activated right PFC activity while exposed to negative sounds from IADS as a mental stress task.

Relationships Between Gum Chewing and Stroop Test: A Pilot Study.

Cognitive function tends to decrease with aging, therefore maintenance of this function in an aging society is an important issue. The role of chewing in nutrition is important. Although several studies indicate that gum chewing is thought to improve cognitive function, it remains debatable whether gum-chewing does in fact improve cognitive function. The Stroop test is a psychological tool used to measure cognition. A shorter reaction time indicates a mean higher behavioral performance and higher levels of oxy-Hb concentration. fNIRS is a powerful, non-invasive imaging technique offering many advantages, including compact size, no need for specially equipped facilities, and the potential for real-time measurement. The left dorsolateral prefrontal cortex (DLPFC) seems to be mainly involved in the Stroop task.The aim of the present study was to investigate the hypothesis that gum-chewing changes cerebral blood flow in the left DLPFC during the Stroop test, and also changes the reaction time. Fourteen healthy volunteers (mean age 26.9 years) participated in this study after providing written informed consent. A piece of tasteless gum weighing 1.0 g was used. Each session was designed in a block manner, i.e. 4 rests (30 s) and 3 blocks of task (30 s). A computerized Stroop test was used (including both congruent and incongruent Stroop tasks) which calculates a response time automatically. The Binominal test was used for comparisons (p < 0.05). The results show activation of the left DLPFC during the Stroop task and that gum chewing significantly increases responses/oxy-Hb concentration and significantly shortens the reaction time.

Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3.

STUDY QUESTION: What are the roles of the microRNA miR-210-an miRNA that is up-regulated in endometriotic cyst stromal cells (ECSCs)-in the pathogenesis of endometriosis? SUMMARY ANSWER: Up-regulated miR-210 expression in ECSCs is involved in their proliferation, resistance to apoptosis and angiogenesis through signal transducer and activator of transcription (STAT) 3. WHAT IS KNOWN ALREADY: In the pathogenesis of endometriosis, a number of roles for microRNAs (miRNAs) are becoming apparent. STUDY DESIGN, SIZE, DURATION: ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues (patients aged 24-40 years undergoing salpingo-oophorectomy or evisceration for the treatment of ovarian endometriotic cysts, n = 10) and the eutopic endometrial tissues without endometriosis (premenopausal patients aged 35-45 years undergoing hysterectomies for subserousal leiomyoma, n = 13), respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used a global gene expression microarray technique to identify downstream targets of miR-210, and we assessed the functions of miR-210 in the pathogenesis of endometriosis by using the miR-210-transfected NESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression microarray analysis revealed that one of the key target molecules of miR-210 is STAT3. In the NESCs, in comparison to the control, miR-210 transfection resulted in the induction of cell proliferation (P < 0.0005), the production of vascular endothelial cell growth factor (VEGF) (P < 0.0005) and the inhibition of apoptosis (P < 0.05) through STAT3 activation [increased levels of mRNA (P < 0.0005), and protein (P < 0.005)]. In the ECSCs, inhibitors of STAT3 inhibited the cell proliferation and VEGF production (P < 0.05), and induced the apoptosis of these cells (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The roles of aberrant miR-210 expression were investigated only in the stromal component of ectopic and eutopic endometrium. Control endometrial tissues were obtained from premenopausal patients who had subserosal leiomyoma and NESC gene expression patterns may be altered in these women. Furthermore, the effects of STAT3 inhibitors were evaluated only in ECSCs and not in NESCs. WIDER IMPLICATIONS OF THE FINDINGS: The present findings indicate that miR-210 induces NESCs to differentiate into the endometriotic phenotype and we speculate that up-regulated miR-210 expression in ECSCs is involved in the creation of the endometriosis-specific cellular dysfunctions through epigenetic mechanisms. The data indicate that STAT3 inhibitors may be promising candidates for the treatment of endometriosis. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 13237327 to K.N., no. 25861500 to Y.K. and no. 23592407 to H.N.). There are no conflicts of interest to declare.

PDK1 inhibition is a novel therapeutic target in multiple myeloma.

BACKGROUND: Cancer cells utilise the glycolytic pathway even when adequate oxygen is present, a phenomenon known as the Warburg effect. We examined whether this system is operative in multiple myeloma (MM) cells and whether glycolysis inhibition is a potential therapeutic modality. METHODS: The MM cells were purified from 59 patients using CD138-immunomagnetic beads. The expression levels of genes associated with glycolysis, c-MYC, GLUT1, LDHA, HIF1A and pyruvate dehydrogenase kinase-1 (PDK1) were determined by real-time PCR. Glucose consumption and lactate production by MM cell lines were analysed. Oxamate, an LDH inhibitor, and dichloroacetate (DCA), a PDK1 inhibitor, were employed. Inhibition of PDK1 expression was achieved using a siRNA. RESULTS: High LDHA expression was found to be an indicator of poor prognosis. It was also positively correlated with the expression of PDK1, c-MYC and GLUT1. Greater glucose consumption and lactate production in MM cells was associated with higher LDHA expression. All the glycolysis inhibitors (oxamate, DCA and PDK1 siRNA) induced apoptosis in MM cells. DCA combined with bortezomib showed additive cytotoxic effects. CONCLUSION: The present data suggest that the Warburg effect is operative in MM cells. As PDK1 is not overexpressed in normal tissues, PDK1 inhibition could serve as a novel therapeutic approach.

The role of serum adiponectin levels in women with polycystic ovarian syndrome.

PURPOSE OF INVESTIGATION: The aim of this study was to measure serum adiponectin concentrations in women with polycystic ovarian syndrome (PCOS) and to assess possible correlations between adiponectin and the hormonal or metabolic parameters of this syndrome. MATERIALS AND METHODS: Serum adiponectin levels were evaluated in 20 women with PCOS and 22 women without PCOS whose age and body mass index (BMI) matched the patients. The levels of fasting blood glucose, fasting insulin, gonadotropin, and sex steroid hormones were evaluated in both groups. The homeostasis model assessment (HOMA) score was also calculated. The serum adiponectin levels were assayed by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: Serum adiponectin levels were significantly lower in obese women than in normal-weight women, and they were also significantly lower in PCOS patients with HOMA scores greater than 1.7 compared with those with HOMA scores lower than 1.7. When the subjects were divided in two groups based on serum adiponectin levels (> 40 microg/ml, < 40 microg/ml), 65% of patients with PCOS were included in the lower adiponectin group (p < 0.05). In addition, gonadotropin levels were increased, dependent on the adiponectin levels in women with PCOS. CONCLUSION: Adiponectin is regarded as a possible link between adiposity and insulin resistance (IR). From this data, the secretions of gonadotropin are implicated in the levels of adiponectin in women with PCOS. It is suggested that adiponectin may play an important role in the pathogenesis of PCOS.

Potential role of autoantibody in severe neutropenia of a patient with Kawasaki Syndrome.

Neutropenia associated with Kawasaki Syndrome (KS) has been rarely reported, and the detailed mechanisms responsible for this state are not yet elucidated. The aim of this study was to clarify the mechanisms of neutropenia in KS. We examined antibodies to known neutrophil antigens (HNA1a, HNA1b, HNA null, HNA2, HNA3, HNA4 and non-HLA antigen 9a) in a KS patient with neutropenia. We also performed the granulocyte immunofluorescence test (GIFT) using patient or control neutrophils incubated with the patient's serum at serial time points over the patient's clinical course. No specific antibody to known neutrophil antigens was detected. Flow cytometric analysis showed that autoantibodies bound to immature CD13-positive myeloid cells, which resulted in myeloid lineage maturation arrest in the bone marrow. GIFT showed that neutrophil-specific autoantibodies were produced by the patient, and the amount of autoantibody inversely correlated with the patient's neutrophil counts. The presence of an autoantibody to a novel antigen on immature myeloid cells or neutrophils is the likely the cause of severe neutropenia in this patient with KS.

Regulation of interleukin-1alpha and tumor necrosis factor-alpha-induced interleukin-8 production by amnion-derived (WISH) cells.

PURPOSE OF INVESTIGATION: It has been reported that interleukin (IL)-8 is produced in the amnion and that its production is enhanced by the initiation of labor. The purpose of this study was to clarify the mechanism of IL-8 production by amnion-derived (WISH) cells. METHODS: Cells were cultured and treated with various concentrations of interleukin (IL)-1alpha, IL-1 receptor antagonist (ra), tumor necrosis factor (TNF)- alpha, C2-, C6-ceramide, mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor (U0126) and pyridinyl imidazole (p38 MAP kinase inhibitor, SB203580). IL-8 in the culture medium was measured by ELISA. RESULTS: The production of IL-8 was significantly increased by IL-1alpha or TNF-alpha, and the increase of IL-8 stimulated by IL-1alpha was suppressed by IL-1 ra in a dose-dependent manner. The increase in IL-8 production by IL-1alpha or TNF-alpha was further enhanced by simultaneous addition of C2-ceramide. The increase of IL-8 stimulated by IL-1alpha or TNF-alpha was also suppressed by treatment with U0126 or SB203580. The results of this study demonstrate that the production of IL-8 induced by IL-1alpha and TNF-alpha is enhanced by C2-ceramide, and suppressed by MEK inhibitor or P38 MAP kinase inhibitor. CONCLUSION: The results suggest that ceramide-mediated accumulation and MAP kinase-mediated suppression of inflammatory events in the amnion may play an important role in the maintenance of pregnancy and initiation of labor.

Melanogenic effect of dersimelagon (MT-7117), a novel oral melanocortin 1 receptor agonist.

Background: The activation of melanocortin 1 receptor (MC1R) on melanocytes stimulates the production of eumelanin. A tridecapeptide α melanocyte-stimulating hormone (αMSH) is known to induce skin pigmentation. Objectives: We characterised the properties of a novel oral MC1R agonist dersimelagon (MT-7117) with respect to its specific binding to MC1R, downstream signalling and eumelanin production in experimental models. Methods: The competitive binding and production of intracellular cyclic adenosine 3', 5'-monophosphate in cells expressing recombinant melanocortin receptors were examined. A mouse melanoma cell line B16F1 was used for the evaluation of in vitro melanin production. The in vitro activity of MT-7117 was determined with αMSH and [Nle4, D-Phe7]-αMSH (NDP-αMSH) as reference comparators. The change of coat colour and skin pigmentation were evaluated after repeat administration of MT-7117 by oral gavage to C57BL/6J-Ay/+ mice and cynomolgus monkeys, respectively. Results: MT-7117 showed the highest affinity for human MC1R compared to the other melanocortin receptors evaluated and agonistic activity for human, cynomolgus monkey and mouse MC1R, with EC50 values in the nanomolar range. In B16F1 cells, MT-7117 increased melanin production in a concentration-dependent manner. In vivo, MT-7117 (≥0.3 mg/kg/day p.o.) significantly induced coat colour darkening in mice. MT-7117 (≥1 mg/kg/day p.o.) induced significant skin pigmentation in monkeys and complete reversibility was observed after cessation of its administration. Conclusions: MT-7117 is a novel oral MC1R agonist that induces melanogenesis in vitro and in vivo, suggesting its potential application for the prevention of phototoxic reactions in patients with photodermatoses, such as erythropoietic protoporphyria and X-linked protoporphyria.

Assessment of the albumin-bilirubin grade as a prognostic factor in patients with non-small-cell lung cancer receiving anti-PD-1-based therapy.

INTRODUCTION: The albumin-bilirubin (ALBI) grade is a novel indicator of the liver function. Some studies showed that the ALBI grade was a prognostic and predictive biomarker for the efficacy of chemotherapy in cancer patients. The association between the ALBI grade and outcomes in patients with non-small-cell lung cancer (NSCLC) treated with cancer immunotherapy, however, is poorly understood. METHODS: We retrospectively enrolled 452 patients with advanced or recurrent NSCLC who received anti-programmed cell death protein 1 (PD-1)-based therapy between 2016 and 2019 at three medical centers in Japan. The ALBI score was calculated from albumin and bilirubin measured at the time of treatment initiation and was stratified into three categories, ALBI grade 1-3, with reference to previous reports. We examined the clinical impact of the ALBI grade on the outcomes of NSCLC patients receiving anti-PD-1-based therapy using Kaplan-Meier survival curve analysis with log-rank test and Cox proportional hazards regression analysis. RESULTS: The classifications of the 452 patients were as follows: grade 1, n = 158 (35.0%); grade 2, n = 271 (60.0%); and grade 3, n = 23 (5.0%). Kaplan-Meier survival curve analysis showed that the ALBI grade was significantly associated with progression-free survival and overall survival. Moreover, Cox regression analysis revealed that the ALBI grade was an independent prognostic factor for progression-free survival and overall survival. CONCLUSION: The ALBI grade was an independent prognostic factor for survival in patients with advanced or recurrent NSCLC who receive anti-PD-1-based therapy. These findings should be validated in a prospective study with a larger sample size.

A pathogenic autoimmune process targeted at a surrogate epitope.

Immunization with the retinal interphotoreceptor retinoid-binding protein (IRBP) induces in a variety of animals an inflammatory eye disease, experimental autoimmune uveoretinitis (EAU). We have previously shown that sequence 1181-1191 of bovine IRBP (BOV 1181-1191) is immunodominant and highly uveitogenic and immunogenic in Lewis rats. Sequence 1181-1191 of the rat IRBP (RAT 1181-1191) differs from BOV 1181-1191 by two residues, at positions 1188 and 1190, that are pivotal for the immunological activity of the bovine epitope. Here we show that, unlike its bovine homologue, RAT 1181-1191 did not induce EAU or an immune response in Lewis rats. The immunological inactivity of RAT 1181-1191 in Lewis rats is due at least in part to its poor affinity toward the antigen-presenting cells of these rats, as shown by its failure to compete with binding of BOV 1181-1191 to Lewis adherent spleen cells. Moreover, unlike all other known autologous homologues of immunopathogenic epitopes, RAT 1181-1191 was not recognized by lymphocytes sensitized against BOV 1181-1191 and failed to stimulate proliferation, uveitogenic capacity or signal transduction in these cells. These findings thus suggest that RAT 1181-1191 is not a likely target for lymphocytes sensitized against BOV 1181-1191 in the process in which these cells recognize IRBP in the rat eye and trigger the inflammatory reaction of EAU. Our data further suggest that the target for the disease-inducing lymphocytes is sequence 273-283 of the rat IRBP: (a) sequence 273-283 is highly conserved and is identical in the bovine and rat proteins; (b) determinant 273-283 is a "repeat" of 1181-1191 in the fourfold structure of IRBP and shares seven residues with BOV 1181-1191; (c) rat peptide 273-283 is recognized by lymphocytes sensitized against BOV 1181-1191 and stimulates them for proliferation and for acquisition of uveitogenicity; and (d) moreover, sequence 273-283 is superior to BOV 1181-1191 in its capacity to generate uveitogenicity in lymphocytes sensitized against this bovine peptide. The present study thus describes for the first time a system in which an autologous homologue of an immunopathogenic epitope is inactive and a "surrogate" determinant apparently serves as the target for lymphocytes sensitized against the immunopathogenic peptide.

Influence of HLA-DRB1 alleles on the susceptibility and resistance to multiple sclerosis in Japanese patients with respect to anti-aquaporin 4 antibody status.

BACKGROUND: Epistatic interactions between human leukocyte antigen (HLA)-DRB1 alleles alter multiple sclerosis (MS) risk in Caucasians. Such interactions have never been studied in Asian MS patients. OBJECTIVE: To investigate the influence of HLA-DRB1 alleles, including epistatic interactions at this locus, in Japanese MS patients with and without the anti-aquaporin 4 (AQP4) antibody. METHODS: The HLA-DRB1 locus was genotyped in 108 MS patients and 127 healthy controls. MS patients were further classified into two groups according to anti-AQP4 antibody status (27 positive and 81 negative). RESULTS: HLA-DRB1*09 (adjusted odds ratio (OR) = 0.243, 95% confidence interval (CI) 0.099-0.533) and HLA-DRB1*01 (adjusted OR = 0.327, 95% CI 0.103-0.873) decreased the incidence of anti-AQP4 antibody-negative MS. By contrast, HLA-DRB1*12 increased the risk of anti-AQP4 antibody-positive MS (adjusted OR = 3.691, 95% CI 1.233-10.565). Individuals with HLA-DRB1*09/15 decreased the risk of anti-AQP4 antibody-negative MS (adjusted OR = 0.164, 95% CI 0.026-0.593), while those with HLA-DRB1*12/15 increased the risk of anti-AQP4 antibody-positive MS (adjusted OR = 10.870, 95% CI 2.004-81.752). CONCLUSIONS: The ability of HLA-DRB1*09 to reduce the risk of anti-AQP4 antibody-negative MS may arise from an interaction with HLA-DRB1*15. By contrast, HLA-DRB1*12 increases susceptibility to anti-AQP4 antibody-positive MS, possibly via an interaction with HLA-DRB1*15.

Comparison of oestrous intensity between natural oestrus and oestrus induced with ovsynch based treatments in Japanese black cows.

The aim of this study was to examine the possible differences of oestrous intensity between natural oestrus and induced oestrus using the walking activity measuring device. Walking activity was used as an evaluation index of oestrous intensity. A total of 27 Japanese Black cows, more than 40 days after calving and clinically normal, were randomly assigned to three groups. Walking activity was recorded using a commercially available computerized pedometer system. The treatment groups consisted of an Ovsynch (n = 8) and a controlled internal drugs releasing device (CIDR) + Ovsynch (n = 9) group. The control group (n = 10) received no treatment. Walking activity was examined in all groups. Timed artificial insemination (timed AI) was performed at 16 hours after the onset of oestrus in the control group and at 24 h after second administration of GnRH in the treatment groups. Duration of oestrus had a tendency to be shorter in both the Ovsynch and the CIDR + Ovsynch groups when compared with the control group. The time required from the onset of oestrus to the time showing the highest number of steps of walking (the time to peak) showed a tendency to be shorter in CIDR + Ovsynch group. The number of steps of walking at peak and overall walking activities were significantly lower in both treatment groups than in the control group. Both activity and super-activity periods of time in the treatment groups were shorter than the control group. No difference was observed in the conception rate between the control (50.0%; 10/20), Ovsynch (50.0%; 4/8) and CIDR + Ovsynch groups (66.7%; 6/9). This study demonstrates that the oestrous intensity of cows in oestrus was different between natural oestrus and induced oestrus and also between the methods of the synchronization, but no difference was observed in the conception rate among the three groups.

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer.

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/beta-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised beta-catenin or beta-catenin shRNA, suggesting it does not involve Wnt/beta-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca(2+) and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of beta-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer.

Association of the HLA-DPB1*0501 allele with anti-aquaporin-4 antibody positivity in Japanese patients with idiopathic central nervous system demyelinating disorders.

There are two subtypes of multiple sclerosis (MS) in Asians: the opticospinal (OSMS) form that shows a selective involvement of the optic nerve and the spinal cord and the conventional (CMS) form that has disseminated lesions in the central nervous system including the cerebrum, cerebellum and brainstem. Both show distinct human leukocyte antigen (HLA) class II associations. OSMS has similar features to the relapsing form of neuromyelitis optica (NMO) in Western populations. Recently, it was shown that antibodies to aquaporin-4 (AQP4) are specifically detected in NMO patients and in some Japanese patients with OSMS or recurrent optic neuritis or myelitis. To clarify the immunogenetic background of anti-AQP4 antibody production, we studied HLA-DRB1 and -DPB1 gene polymorphisms in anti-AQP4 antibody-positive and -negative patients with idiopathic demyelinating diseases, such as MS, recurrent optic neuritis and recurrent myelitis. The phenotypic frequency of the HLA-DPB1*0501 allele was significantly increased in anti-AQP4 antibody-positive patients (89.5%, odds ratio = 4.8; 95% confidence interval = 1.6-14.3, n = 38, P(corr) = 0.032) compared with controls (64.0%, n = 125) but not in either anti-AQP4 antibody-negative OSMS (75.0%, n = 32) or CMS (69.2%, n = 52) patients. There was no significant correlation between any HLA-DRB1 allele and the existence of anti-AQP4 antibody. These findings suggest that the emergence of anti-AQP4 antibody is reinforced by the presence of the HLA-DPB1*0501 allele in Japanese.

Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility.

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. Here we identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized alpha-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.

Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.

Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway.

CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate-responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

Heterozygous deletion of ITPR1, but not SUMF1, in spinocerebellar ataxia type 16.

We have previously mapped autosomal dominant spinocerebellar ataxia (SCA) 16 to 3p26, overlapping with the locus of SCA15. Recently, partial deletions of ITPR1 and the neighbouring SUMF1 in the SCA15 and two additional families were reported. In the present study we determined the copy number of these genes by real time quantitative polymerase chain reaction (PCR) and found a heterozygous deletion of exons 1-48 of ITPR1, but not SUMF1 in SCA16. Breakpoint analysis revealed that the size of the deletion is 313,318 bp and the telomeric breakpoint is located in the middle of their intergenic region. Our data provide evidence that haploinsufficiency of ITPR1 alone causes SCA16 and SCA15.

Influence of photoactivation protocol and light guide distance on conversion and microleakage of composite restorations.

PURPOSE: To evaluate the effect of light guide distance and the different photoactivation methods on the degree of conversion (DC) and microleakage of a composite. METHODS AND MATERIALS: Three photoactivation protocols (600 mW/cm2 x 40 seconds; 400 mW/cm2 x 60 seconds or 200 mW/cm2 x 20 seconds, followed by 500 mW/cm2 x 40 seconds) and three distances from the light source (0, 3 or 7 mm) were tested. Cylindrical specimens (5 mm diameter; 2 mm tall; n=3) were prepared for the DC test (FT-Raman). Class V cavities were made in 90 bovine incisors to conduct the microleakage test. The specimens were conditioned for 15 seconds with phosphoric acid (37%), followed by application of the adhesive system Prime & Bond NT (Dentsply/Caulk). The preparations were restored in bulk. The specimens were stored for 24 hours in distilled water (37 degrees C) before being submitted to the silver-nitrate microleakage protocol. The restorations were sectioned and analyzed under 25x magnification. RESULTS: Statistical analyses (two-way ANOVAs and Tukey test, alpha=0.05) found significance only for the factor distance (p=0.015) at the top of the composite for the DC test. Conversion was statistically lower for the 7 mm groups compared to the 0 and 3 mm groups, which were equivalent to each other. At the bottom of the specimens, none of the factors or interactions was significant (p<0.05). The Kruskal-Wallis test showed that, in general, the soft-start method led to lower microleakage scores when compared to the continuous modes, mainly when associated with a distancing of 7 mm (p<0.01). With the exception of specimens irradiated with 400 mW/cm2 that did not demonstrate variations on scores for the distances tested, higher microleakage was observed for shorter distances from the light source. CONCLUSIONS: Soft-start methods may reduce microleakage when the light guide distancing provides a low level of irradiance, which also causes a discrete reduction in the DC.