Publications by authors named "Hiroshi Takeuchi"

84 Publications

Osteocalcin promotes proliferation, differentiation, and survival of PC12 cells.

Biochem Biophys Res Commun 2021 Jun 15;557:174-179. Epub 2021 Apr 15.

Division of Applied Pharmacology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan. Electronic address:

Involvement of the bone matrix protein osteocalcin (OC) in the development of learning and memory, and the prevention of anxiety-like behaviors in mice. However, the direct effects of OC on neurons are still unknown comparing to the mechanism how OC affects systemic energy expenditure and glucose homeostasis. In this study, we investigated the effect of OC on proliferation, differentiation, and survival of neurons using the rat pheochromocytoma cell line PC12. RT-PCR analysis for OC receptor candidates revealed that Gpr158, but not Gprc6a, mRNA was expressed in PC12 cells. The growth of PC12 cells cultured in the presence of 5-50 ng/mL of either uncarboxylated (GluOC) or carboxylated (GlaOC) OC was increased compared to cells cultured in the absence of OC. In addition, NGF-induced neurite outgrowth was enhanced by OC, and HO-induced cell death was suppressed by pretreatment with OC. All of these results were observed for both GluOC and GlaOC at comparable levels, suggesting that OC may directly affect cell proliferation, differentiation, and survival by binding to its candidate receptor, GPR158.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.146DOI Listing
June 2021

Enhanced x-ray emission coinciding with giant radio pulses from the Crab Pulsar.

Science 2021 04;372(6538):187-190

The Research Institute for Time Studies, Yamaguchi University, Yamaguchi 753-8511, Japan.

Giant radio pulses (GRPs) are sporadic bursts emitted by some pulsars that last a few microseconds and are hundreds to thousands of times brighter than regular pulses from these sources. The only GRP-associated emission outside of radio wavelengths is from the Crab Pulsar, where optical emission is enhanced by a few percentage points during GRPs. We observed the Crab Pulsar simultaneously at x-ray and radio wavelengths, finding enhancement of the x-ray emission by 3.8 ± 0.7% (a 5.4σ detection) coinciding with GRPs. This implies that the total emitted energy from GRPs is tens to hundreds of times higher than previously known. We discuss the implications for the pulsar emission mechanism and extragalactic fast radio bursts.
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http://dx.doi.org/10.1126/science.abd4659DOI Listing
April 2021

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

Eur J Pharmacol 2021 Mar 19;895:173881. Epub 2021 Jan 19.

The Center for Visiting Dental Service, Department of General Dentistry, Fukuoka Dental College, Fukuoka 8140193, Japan. Electronic address:

Objectives: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line.

Methods: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays.

Results: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane.

Conclusions: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.
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http://dx.doi.org/10.1016/j.ejphar.2021.173881DOI Listing
March 2021

Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis.

J Biol Chem 2021 Jan 8:100274. Epub 2021 Jan 8.

Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, Japan. Electronic address:

The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in wild-type mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat, high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity due to down-regulation of lipolytic enzymes such as adipose triglyceride lipase (ATGL) and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of ATGL in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.
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http://dx.doi.org/10.1016/j.jbc.2021.100274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949034PMC
January 2021

Effect of aging on bone metabolism: the involvement of complemen t C1q.

J Prosthodont Res 2020 Oct 29. Epub 2020 Oct 29.

Division of Applied Pharmacology, Kyushu Dental University, Kitakyushu.

Purpose: Impairment of normal bone remodeling affects the successful osseointegration of dental implants. Recently, it has been reported that complement C1q level increases with age and delays wound healing by modulating Wnt signaling. As Wnt signaling is known to play an essential role in bone remodeling, we hypothesized that aging-dependent increases in C1q affect bone remodeling. In this study, we examined whether C1q affects the differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts, and investigated whether C1q could modify cellular signaling, including the Wnt/β-catenin pathway in these cells.

Methods: Osteogenic differentiation of MC3T3-E1 cells was assessed using alkaline phosphatase staining. Differentiation of osteoclasts from mouse bone marrow cells was assessed using tartrate-resistant acid phosphatase staining. Activation of canonical Wnt signaling and protein phosphorylation was monitored using Western blotting.

Results: C1q, at 5-15 µg/mL promoted osteoclast fusion, whereas it did not affect the differentiation of osteoblasts. On the other hand, a higher concentration of C1q (50 µg/mL) suppressed both bone morphogenetic protein-2-induced osteogenic differentiation and osteoclast formation. C1q did not induce an obvious activation of Wnt/ β-catenin signaling in either pre-osteoblasts or pre-osteoclasts, contrary to previous reports using other tissues. Instead, C1q upregulated the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced phosphorylation of Akt.

Conclusions: C1q could affect cellular signaling and modify the differentiation of osteoblasts and osteoclasts, depending on the concentration. Therefore, an increase in C1q with age could be one of the factors that determine the prognosis of treatment of elderly patients.
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http://dx.doi.org/10.2186/jpr.JPOR_2019_644DOI Listing
October 2020

The roles of osteocalcin in lipid metabolism in adipose tissue and liver.

Adv Biol Regul 2020 12 10;78:100752. Epub 2020 Sep 10.

Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, 814-0193, Japan. Electronic address:

Bone provides skeletal support and functions as an endocrine organ by producing osteocalcin, whose uncarboxylated form (GluOC) increases the metabolism of glucose and lipid by activating its putative G protein-coupled receptor (family C group 6 subtype A). Low doses (≤10 ng/ml) of GluOC induce the expression of adiponectin, adipose triglyceride lipase and peroxisome proliferator-activated receptor γ, and promote active phosphorylation of lipolytic enzymes such as perilipin and hormone-sensitive lipase via the cAMP-PKA-Src-Rap1-ERK-CREB signaling axis in 3T3-L1 adipocytes. Administration of high-dose (≥20 ng/ml) GluOC induces programmed necrosis (necroptosis) through a juxtacrine mechanism triggered by the binding of Fas ligand, whose expression is induced by forkhead box O1, to Fas that is expressed in adjacent adipocytes. Furthermore, expression of adiponectin and adipose triglyceride lipase in adipocytes is triggered in the same manner as following low-dose GluOC stimulation; these effects protect mice from diet-induced accumulation of triglycerides in hepatocytes and consequent liver injury through the upregulation of nuclear translocation of nuclear factor-E2-related factor-2, expression of antioxidant enzymes, and inhibition of the c-Jun N-terminal kinase pathway. Evaluation of these molecular mechanisms leads us to consider that GluOC might have potential as a treatment for lipid metabolism disorders. Indeed, there have been many reports demonstrating the negative correlation between serum osteocalcin levels and obesity or non-alcoholic fatty liver disease, a common risk factor for which is dyslipidemia in humans. The present review summarizes the effects of GluOC on lipid metabolism as well as its possible therapeutic application for metabolic diseases including obesity and dyslipidemia.
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http://dx.doi.org/10.1016/j.jbior.2020.100752DOI Listing
December 2020

Highly porous nature of a primitive asteroid revealed by thermal imaging.

Nature 2020 03 16;579(7800):518-522. Epub 2020 Mar 16.

The Graduate University for Advanced Studies, SOKENDAI, Hayama, Japan.

Carbonaceous (C-type) asteroids are relics of the early Solar System that have preserved primitive materials since their formation approximately 4.6 billion years ago. They are probably analogues of carbonaceous chondrites and are essential for understanding planetary formation processes. However, their physical properties remain poorly known because carbonaceous chondrite meteoroids tend not to survive entry to Earth's atmosphere. Here we report on global one-rotation thermographic images of the C-type asteroid 162173 Ryugu, taken by the thermal infrared imager (TIR) onboard the spacecraft Hayabusa2, indicating that the asteroid's boulders and their surroundings have similar temperatures, with a derived thermal inertia of about 300 J m s K (300 tiu). Contrary to predictions that the surface consists of regolith and dense boulders, this low thermal inertia suggests that the boulders are more porous than typical carbonaceous chondrites and that their surroundings are covered with porous fragments more than 10 centimetres in diameter. Close-up thermal images confirm the presence of such porous fragments and the flat diurnal temperature profiles suggest a strong surface roughness effect. We also observed in the close-up thermal images boulders that are colder during the day, with thermal inertia exceeding 600 tiu, corresponding to dense boulders similar to typical carbonaceous chondrites. These results constrain the formation history of Ryugu: the asteroid must be a rubble pile formed from impact fragments of a parent body with microporosity of approximately 30 to 50 per cent that experienced a low degree of consolidation. The dense boulders might have originated from the consolidated innermost region or they may have an exogenic origin. This high-porosity asteroid may link cosmic fluffy dust to dense celestial bodies.
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http://dx.doi.org/10.1038/s41586-020-2102-6DOI Listing
March 2020

Modification of TRPV4 activity by acetaminophen.

Heliyon 2020 Jan 31;6(1):e03301. Epub 2020 Jan 31.

Division of Applied Pharmacology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, 803-8580, Japan.

-Acetyl-p-aminophenol (APAP/acetaminophen) is a widely used analgesic/antipyretic with weaker inhibitory effects on cyclooxygenase compared to those of non-steroidal anti-inflammatory drugs. The effect of APAP is mediated by its metabolites, -arachidonoyl-phenolamine and -acetyl--benzoquinone imine, which activate transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or cannabinoid receptor type 1. However, the exact molecular mechanism underlying the cellular actions of APAP remains unclear. Recently, we observed that APAP promotes cell migration through TRPV4; in this study, we examined the effect of APAP on Ca-channel activity of TRPV4. In the rat cell line PC12 expressing TRPV4, GSK1016790A (GSK), a TRPV4 agonist, stimulated an increase in [Ca]; these effects were abrogated by HC-067047 treatment. This GSK-induced Ca entry through TRPV4 was inhibited by APAP in a dose-dependent manner, whereas APAP alone did not affect [Ca]. The specificity of the effect of APAP on TRPV4 was further confirmed using HeLa cells, which lack endogenous TRPV4 but stably express exogenous TRPV4 (HeLa-mTRPV4). GSK-induced [Ca] elevation was only observed in HeLa-mTRPV4 cells compared to that in the control HeLa cells, indicating the specific action of GSK on TRPV4. APAP dose-dependently suppressed this GSK-induced Ca entry in HeLa-mTRPV4. However, it is unlikely that the metabolites of APAP were involved in these effects as the reaction in this study was rapid. The results suggest that APAP suppresses the newly identified target TRPV4 without being metabolized and exerts antipyretic/analgesic and/or other effects on TRPV4-related phenomena in the body. The effect of APAP on TRPV4 was opposite to that on TRPV1 or TRPA1, as the latter is activated by APAP.
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http://dx.doi.org/10.1016/j.heliyon.2020.e03301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002858PMC
January 2020

GLP-1 signaling is required for improvement of glucose tolerance by osteocalcin.

J Endocrinol 2020 02;244(2):285-296

Oral Medicine Research Center, School of Dental Medicine, Fukuoka Dental College, Fukuoka, Japan.

Osteocalcin is a bone-derived hormone that in its uncarboxylated form (GluOC) plays an important role in glucose and energy metabolism by stimulating insulin secretion and pancreatic β-cell proliferation through its putative receptor GPRC6A. We previously showed that the effect of GluOC on insulin secretion is mediated predominantly by glucagon-like peptide-1 (GLP-1) released from intestinal endocrine cells in response to GluOC stimulation. Moreover, oral administration of GluOC was found to reduce the fasting blood glucose level, to improve glucose tolerance, and to increase the fasting serum insulin concentration and β-cell area in the pancreas in wild-type mice. We have now examined the effects of oral GluOC administration for at least 4 weeks in GLP-1 receptor-knockout mice. Such administration of GluOC in the mutant mice triggered glucose intolerance, enhanced gluconeogenesis and promoted both lipid accumulation in the liver as well as adipocyte hypertrophy and inflammation in adipose tissue. Furthermore, inactivation of GLP-1 receptor signaling in association with GluOC administration induced activation of the transcription factor FoxO1 and expression of its transcriptional coactivator PGC1α in the liver, likely accounting for the observed upregulation of gluconeogenic gene expression. Our results thus indicate that the beneficial metabolic effects of GluOC are dependent on GLP-1 receptor signaling.
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http://dx.doi.org/10.1530/JOE-19-0288DOI Listing
February 2020

Determination of sterigmatocystin in foods in Japan: method validation and occurrence data.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2019 Sep 26;36(9):1404-1410. Epub 2019 Jun 26.

Department of Food and Life Sciences, Azabu University , Kanagawa , Japan.

A survey of the contamination of foods by sterigmatocystin (STC) was performed by an analytical method based on LC-MS/MS. STC was extracted from samples with acetonitrile/water (85/15, v/v) and then purified with immunoaffinity columns. The method was validated by a small-scale inter-laboratory study using spiked wheat samples. Mean recoveries of STC were 100.3% and 92.5% from two samples spiked at 0.5 and 5.0 µg/kg, respectively. A total of 583 samples were analysed between 2016 and 2018, and STC was detected in 19.9% of all samples at >0.05 μg/kg (limit of quantification). The foods that were contaminated by STC were wheat flour, Job's tears products, rye flour, rice, buckwheat flour, white sorghum, barley products, azuki bean and corn flour. STC was not found in beer or wine. The occurrence of STC in domestic wheat flour (44.4%), Job's tears products (41.7%) and rye flour (29.9%) accounted for the three highest values. The highest mean concentrations were obtained for Job's tears products (0.3 μg/kg) and rye flour (0.3 μg/kg). The maximum contamination level was present in a sample of rye flour (7.1 μg/kg). Although the contamination levels were low, these results indicate that STC frequently contaminates Japanese retail foods. A continuous survey is required to assess exposure to STC in Japan.
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http://dx.doi.org/10.1080/19440049.2019.1628359DOI Listing
September 2019

Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300.

Cell Death Dis 2018 12 13;9(12):1194. Epub 2018 Dec 13.

School of Dental Medicine, Fukuoka Dental College, Fukuoka, 814-0193, Japan.

The uncarboxylated form of osteocalcin (GluOC) regulates glucose and lipid metabolism in mice. We previously showed that low-dose (≤10 ng/ml) GluOC induces the expression of adiponectin and peroxisome proliferator-activated receptor γ (PPARγ) via a cAMP-PKA-ERK-CREB signaling pathway in 3T3-L1 adipocytes. We also noticed that high-dose (≥20 ng/ml) GluOC inhibits the expression of adiponectin and PPARγ in these cells. We have here explored the mechanism underlying these effects of high-dose GluOC. High-dose GluOC triggered morphological changes in 3T3-L1 adipocytes suggestive of the induction of cell death. It activated the putative GluOC receptor GPRC6A and thereby induced the production of cAMP and activation of protein kinase A (PKA), similar to signaling by low-dose GluOC with the exception that the catalytic subunit of PKA also entered the nucleus. Cytosolic PKA induced phosphorylation of cAMP response element-binding protein (CREB) at serine-133 via extracellular signal-regulated kinase (ERK). Nuclear PKA appeared to mediate the inhibitory phosphorylation of salt-inducible kinase 2 (SIK2) at serine-358 and thereby to alleviate the inhibitory phosphorylation of the CREB co-activator p300 at serine-89. The activation of CREB and p300 resulted in increased expression of the transcription factor FoxO1 and consequent upregulation of Fas ligand (FasL) at the plasma membrane. The interaction of FasL with Fas on neighboring adipocytes triggered the phosphorylation at threonine-357/serine-358 and homotrimerization of mixed-lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, as well as Ca influx via transient receptor potential melastatin 7 (TRPM7), the generation of reactive oxygen species and lipid peroxides, and dephosphorylation of dynamin-related protein 1 (DRP1) at serine-637, resulting in mitochondrial fragmentation. Together, our results indicate that high-dose GluOC triggers necroptosis through upregulation of FasL at the plasma membrane in a manner dependent of activation of CREB-p300, followed by the activation of Fas signaling in neighboring adipocytes.
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http://dx.doi.org/10.1038/s41419-018-1257-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294257PMC
December 2018

Brief multifamily Psychoeducation for family members of patients with chronic major depression: a randomized controlled trial.

BMC Psychiatry 2018 06 22;18(1):207. Epub 2018 Jun 22.

Department of Health Promotion and Human Behavior, Kyoto University Graduate School of Medicine/School of Public Health, Yoshida Konoe-cho, Sakyo-ku, Kyoto, Japan.

Background: Major depressive disorder (MDD) is a common and often chronic problem. Patients with chronic MDD often have negative impacts on the health of their families. Family psychoeducation is recognized as part of the optimal treatment for patients with psychotic disorder, and has been shown to reduce the rate of relapse in individuals with schizophrenia and to reduce the burden on their caregivers. Thus, we predict that family psychoeducation has the potential to reduce the burden on the caregivers of patients with chronic MDD. In the present study, we aimed to investigate the effects of brief multifamily psychoeducation (BMP) on the mental health status of family members of patients with chronic MDD.

Methods: We conducted a clinical trial consisting of 49 chronic MDD patients and their families. Each family was randomly assigned to either the BMP intervention group or the control group. The intervention group received four BMP sessions, once every two weeks for eight weeks. The control group received one counseling session administered by a nurse. All patients received standard treatment administered by physicians. The primary outcome measurement was the Kessler Screening Scale for Psychological Distress (K6) score of family members at 16- weeks after the first BMP session. Secondary outcomes were depressive symptoms of both family members and patients at multiple time points, as well as family functioning as evaluated by the patients. Intention-to-treat analyses were conducted.

Results: There was no statistically significant effect of BMP on K6 scores at 16- weeks (mean difference 1.17, 95% confidence interval: - 0.63 to 2.98, P = 0.19). Exploratory analyses revealed that BMP reduced depressive symptoms in family members at 8- weeks (difference = - 3.37, 95%CI -6.32 to - 0.43, P = 0.02) and improved family functioning at multiple time points (Role; 8 W, difference = - 0.13, 95%CI -0.26 to - 0.00, P = 0.04, Affective Responsiveness; 8 W, difference = - 0.24, 95%CI -0.43 to - 0.05, P = 0.01, 32 W, difference = - 0.22, 95%CI -0.41 to - 0.03, P = 0.02, Behavior Control; 16 W, difference = - 0.17, 95%CI -0.34 to - 0.00, P = 0.04).

Conclusions: Four BMP sessions did not significantly reduce the psychological distress of family members of patients with chronic MDD.

Trial Registration: Clinical Trials. gov NCT01734291 , retrospectively registered (Registration date: November 21, 2012).
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http://dx.doi.org/10.1186/s12888-018-1788-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013880PMC
June 2018

Size-guided multi-seed heuristic method for geometry optimization of clusters: Application to benzene clusters.

Authors:
Hiroshi Takeuchi

J Comput Chem 2018 Aug 8;39(22):1738-1746. Epub 2018 May 8.

Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.

Since searching for the global minimum on the potential energy surface of a cluster is very difficult, many geometry optimization methods have been proposed, in which initial geometries are randomly generated and subsequently improved with different algorithms. In this study, a size-guided multi-seed heuristic method is developed and applied to benzene clusters. It produces initial configurations of the cluster with n molecules from the lowest-energy configurations of the cluster with n - 1 molecules (seeds). The initial geometries are further optimized with the geometrical perturbations previously used for molecular clusters. These steps are repeated until the size n satisfies a predefined one. The method locates putative global minima of benzene clusters with up to 65 molecules. The performance of the method is discussed using the computational cost, rates to locate the global minima, and energies of initial geometries. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcc.25349DOI Listing
August 2018

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

J Cell Physiol 2018 09 16;233(9):7356-7366. Epub 2018 Apr 16.

Department of Health Promotion, Kyushu Dental University, Fukuoka, Japan.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.
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http://dx.doi.org/10.1002/jcp.26571DOI Listing
September 2018

Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

Pharmacol Rep 2018 Feb 15;70(1):29-36. Epub 2017 Jul 15.

Division of Applied Pharmacology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Japan. Electronic address:

Background: N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1.

Methods: Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively.

Results: APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression.

Conclusion: APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions.
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http://dx.doi.org/10.1016/j.pharep.2017.07.006DOI Listing
February 2018

Uncarboxylated Osteocalcin Induces Antitumor Immunity against Mouse Melanoma Cell Growth.

J Cancer 2017 2;8(13):2478-2486. Epub 2017 Aug 2.

Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.

Because of the poor response to chemotherapy and radiation therapy, new treatment approaches by immune-based therapy involving activated T cells are required for melanoma. We previously reported that the uncarboxylated form of osteocalcin (GluOC), derived from osteoblasts, potentially suppresses human prostate cancer cell proliferation by direct suppression of cell growth. However, the mechanisms have not been elucidated. In this study, we found that GluOC suppressed tumor growth of B16 mouse melanoma transplants in C57Bl/6N wild-type mice. Our data demonstrated that GluOC suppressed cell growth by downregulating phosphorylation levels of receptor tyrosine kinases and inducing apoptosis . Additionally, stimulation of primary mouse splenocytes with concanavalin A, a polyclonal T-cell mitogen, in the presence of GluOC increased T cell proliferation and their interferon-γ production. Taken together, we demonstrate that GluOC exerts multiple antitumor effects not only , but also through cellular immunostimulatory effects against B16 mouse melanoma cells.
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http://dx.doi.org/10.7150/jca.18648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595077PMC
August 2017

Celecoxib inhibits osteoblast differentiation independent of cyclooxygenase activity.

Clin Exp Pharmacol Physiol 2018 Jan 20;45(1):75-83. Epub 2017 Sep 20.

Division of Applied Pharmacology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Japan.

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects primarily by inhibiting the activity of cyclooxygenase (COX), thus suppressing prostaglandin synthesis. Some NSAIDs are known to perform functions other than pain control, such as suppressing tumour cell growth, independent of their COX-inhibiting activity. To identify NSAIDs with COX-independent activity, we examined various NSAIDs for their ability to inhibit osteoblastic differentiation using the mouse pre-osteoblast cell line MC3T3-E1. Only celecoxib and valdecoxib strongly inhibited osteoblastic differentiation, and this effect was not correlated with COX-inhibiting activity. Moreover, 2,5-dimethyl (DM)-celecoxib, a celecoxib analogue that does not inhibit COX activity, also inhibited osteoblastic differentiation. Celecoxib and DM-celecoxib inhibited osteoblastic differentiation induced by bone morphogenetic protein (BMP)-2 in C2C12 mouse myoblast cell line. Although celecoxib suppresses the growth of some tumour cells, the viability and proliferation of MC3T3-E1 cells were not affected by celecoxib or DM-celecoxib. Instead, celecoxib and DM-celecoxib suppressed BMP-2-induced phosphorylation of Smad1/5, a major downstream target of BMP receptor. Although it is well known that COX plays important roles in osteoblastic differentiation, these results suggest that some NSAIDs, such as celecoxib, have targets other than COX and regulate phospho-dependent intracellular signalling, thereby modifying bone remodelling.
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http://dx.doi.org/10.1111/1440-1681.12846DOI Listing
January 2018

Suppression of cell migration by phospholipase C-related catalytically inactive protein-dependent modulation of PI3K signalling.

Sci Rep 2017 07 14;7(1):5408. Epub 2017 Jul 14.

Department of Cellular and Molecular Pharmacology, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.

The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] into PI(3,4,5)P and the subsequent PI(3,4,5)P signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo. Overexpression of the PRIP pleckstrin homology domain, a PI(4,5)P binding motif, in MCF-7 cells caused significant suppression of cell migration. Consistent with these results, in comparison with wild-type cells, Prip-deficient mouse embryonic fibroblasts exhibited increased cell migration, and this was significantly attenuated upon transfection with a siRNA targeting p110α, a catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI(3,4,5)P production was decreased in Prip-overexpressing MCF-7 and BT-549 cells. PI3K binding to PI(4,5)P was significantly inhibited by recombinant PRIP in vitro, and thus the activity of PI3K was downregulated. Collectively, PRIP regulates the production of PI(3,4,5)P from PI(4,5)P by PI3K, and the suppressor activity of PRIP in PI(4,5)P metabolism regulates the tumour migration, suggesting PRIP as a promising target for protection against metastatic progression.
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http://dx.doi.org/10.1038/s41598-017-05908-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511194PMC
July 2017

Two Perturbations for Geometry Optimization of Off-lattice Bead Protein Models.

Authors:
Hiroshi Takeuchi

Mol Inform 2017 08 27;36(8). Epub 2017 Apr 27.

Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, 060-0810, Japan.

Referring to the optimization algorithm previously developed for atomic clusters, the present author develops an efficient method for geometry optimization of a coarse-grained protein model expressed with two kinds of beads (hydrophilic and hydrophobic ones). In the method, two types of geometrical perturbations, center-directed bead move and one bead rotation, are used to explore new configurations and local optimizations are performed after the perturbations. The center-directed bead move is used for hydrophobic beads and the one bead rotation is performed for both hydrophobic and hydrophilic beads. The optimization method was applied to protein models consisting of 13, 20, 21, and 34 beads. The present method produced the global minima of the 13-, 21-, and 34-bead models reported in the literature and updated the lowest energies of the protein models with 20 beads. These results indicate that the present method is efficient for searching for optimal structures of proteins.
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http://dx.doi.org/10.1002/minf.201600096DOI Listing
August 2017

Genome-Wide Association Study Identifies ZNF354C Variants Associated with Depression from Interferon-Based Therapy for Chronic Hepatitis C.

PLoS One 2016 10;11(10):e0164418. Epub 2016 Oct 10.

Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

The therapeutic use of interferon (IFN) is known to cause depression that frequently interrupts treatment. To identify genetic variants associated with IFN-induced depression, we conducted a genome-wide association study (GWAS) of 224 Japanese chronic hepatitis C patients receiving IFN-based therapy in a multicenter prospective study and stratified them into two groups according to the Beck Depression Inventory, Second Edition (BDI-II) score. In the GWAS stage, we selected 42 candidate single nucleotide polymorphisms (SNPs) to perform replication analysis in an independent set of 160 subjects. The SNP rs1863918 in strong linkage disequilibrium with SNPs located around the Zinc finger 354C (ZNF354C) gene on chromosome 5 showed a significant association when the results of GWAS and replication were combined (odds ratio = 2.55, P = 7.89×10-8 in the allele frequency model), suggesting that the rs1863918 T allele was associated with IFN-induced depression. Furthermore, logistic regression analysis showed that rs1863918 T allele, a history of depression, and younger age were independent predictive factors for IFN-induced depression. Interestingly, western blotting and immunofluorescence showed that ZNF354C was highly expressed in the hippocampus in mice, a region implicated in the pathology of psychiatric symptoms. In conclusion, we identified rs1863918 as significantly associated with IFN-induced depression, and revealed that the candidate gene ZNF354C is highly expressed in the hippocampus of mice. Our data might be useful for elucidating the pathogenic mechanisms of depression induced by drugs including IFN.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164418PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056723PMC
April 2017

Differential Roles of Carboxylated and Uncarboxylated Osteocalcin in Prostate Cancer Growth.

J Cancer 2016 18;7(12):1605-1609. Epub 2016 Jul 18.

1. Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.

Serum levels of osteocalcin (OC), a bone matrix non-collagenous protein secreted by osteoblasts, are correlated with pathological bone remodeling such as the bone metastasis of cancer, as well as physiological bone turnover. The pathological roles in prostate cancer growth of the two existing types of serum OC, γ-carboxylated (GlaOC) and lower- (or un-) carboxylated (GluOC), have not yet been discriminatively examined. In the present study, we demonstrate that normal prostate epithelial cell growth was promoted by both types of OC, while growth of cancer cells in the prostate was accelerated by GlaOC but suppressed by GluOC. We suggest that OC regulates prostate cancer growth depending on the γ-carboxylation, in part by triggering reduced phosphorylation of receptor tyrosine kinases.
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http://dx.doi.org/10.7150/jca.15523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039381PMC
July 2016

An extract from pork bones containing osteocalcin improves glucose metabolism in mice by oral administration.

Biosci Biotechnol Biochem 2016 Nov 27;80(11):2176-2183. Epub 2016 Jul 27.

a Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science , Kyushu University , Fukuoka , Japan.

Osteocalcin (OC) is a bone-derived hormone that regulates energy metabolism. OC exists in two forms, carboxylated (GlaOC) and uncaboxylated (GluOC), but only the latter appears to have an endocrine function. In this study, we prepared an extract containing both Gla- and GluOC from boiled pork bone using 0.2 M carbonate buffer at pH 9.5, and tested whether the extract had beneficial effects on improving metabolic parameters in obese mice. The extract equivalent of 1.2 μg of GluOC/mouse was orally administrated to C57BL/6 female mice fed a high-fat, high-sucrose diet. Daily oral administration of the extract for four weeks decreased blood glucose levels and promoted glucose tolerance as well as insulin sensitivity. Our study shows for the first time that boiled pork bones are a source material for osteocalcin in the large-scale production of supplements designed to improve glucose metabolism.
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http://dx.doi.org/10.1080/09168451.2016.1214530DOI Listing
November 2016

Maternal oral administration of osteocalcin protects offspring from metabolic impairment in adulthood.

Obesity (Silver Spring) 2016 Apr 4;24(4):895-907. Epub 2016 Mar 4.

Laboratory of Molecular and Cellular Biochemistry, Kyushu University, Japan.

Objective: Maternal diet during pregnancy has been found to influence the health of offspring. However, strategies for modulation of maternal energy metabolism without an adverse effect on the fetus have remained limited. It was recently shown that oral administration of uncarboxylated osteocalcin (GluOC) improves metabolic status in adult female mice. Whether maternal GluOC administration during gestation might improve the metabolic status of offspring was investigated.

Methods: Female C57BL/6 mice were fed a normal diet (ND) or high-fat, high-sucrose diet (HFS) and were given saline or GluOC by oral administration during pregnancy. The resulting offspring were in turn assigned to ND- or HFS-fed groups immediately after weaning, and their body weight, glucose metabolism, serum lipid parameters, and level of adipose tissue inflammation were subsequently assessed.

Results: Maternal HFS feeding during gestation had adverse effects on glucose and lipid parameters, body weight, and adipose tissue inflammation in female offspring fed the same diet, and these effects were attenuated by maternal oral GluOC administration.

Conclusions: Maternal oral administration of GluOC protects HFS-fed female offspring from metabolic disorders induced by maternal obesity.
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http://dx.doi.org/10.1002/oby.21447DOI Listing
April 2016

Long-term oral administration of osteocalcin induces insulin resistance in male mice fed a high-fat, high-sucrose diet.

Am J Physiol Endocrinol Metab 2016 04 16;310(8):E662-E675. Epub 2016 Feb 16.

Laboratory of Molecular and Cellular Biochemistry,

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion, pancreatic β-cell proliferation, and adiponectin expression in adipocytes. Previously, we showed that long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level, improved glucose tolerance, and increased the fasting serum insulin concentration as well as pancreatic β-cell area in female mice fed a normal or high-fat, high-sucrose diet. We have now performed similar experiments with male mice and found that such GluOC administration induced glucose intolerance, insulin resistance, and adipocyte hypertrophy in those fed a high-fat, high-sucrose diet. In addition, GluOC increased the circulating concentration of testosterone and reduced that of adiponectin in such mice. These phenotypes were not observed in male mice fed a high-fat, high-sucrose diet after orchidectomy, but they were apparent in orchidectomized male mice or intact female mice that were fed such a diet and subjected to continuous testosterone supplementation. Our results thus reveal a sex difference in the effects of GluOC on glucose homeostasis. Given that oral administration of GluOC has been considered a potentially safe and convenient option for the treatment or prevention of metabolic disorders, this sex difference will need to be taken into account in further investigations.
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http://dx.doi.org/10.1152/ajpendo.00334.2015DOI Listing
April 2016

Differential role of SNAP-25 phosphorylation by protein kinases A and C in the regulation of SNARE complex formation and exocytosis in PC12 cells.

Cell Signal 2016 May 23;28(5):425-437. Epub 2015 Dec 23.

Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address:

The final step of regulated exocytosis, membrane fusion, is mediated by formation of the SNARE complex by syntaxin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and VAMP (vesicle-associated membrane protein). Phosphorylation of SNARE and accessory proteins contributes to regulation of exocytosis. We previously identified residues of SNAP-25 phosphorylated by protein kinase A (PKA) and PKC. However, the physiological role of SNAP-25 phosphorylation in exocytosis, in particular with regard to SNARE complex formation, has remained elusive. SNARE complex formation by purified recombinant SNAP-25, syntaxin-1, and VAMP-2 in vitro was inhibited or promoted as a result of the phosphorylation at Thr(138) by PKA or at Ser(187) by PKC, respectively. SNARE complex formation in intact PC12 cells was similarly inhibited by forskolin (activator of PKA) and promoted by phorbol 12-myristate 13-acetate (PMA, activator of PKC). Noradrenaline secretion from PC12 cells induced by a high K(+) concentration was enhanced by forskolin or PMA. Stable depletion of SNAP-25 inhibited high-K(+)-induced noradrenaline secretion. Forced expression of WT SNAP-25 restored the secretory response of the SNAP-25-depleted cells to high-K(+), and this response was enhanced by forskolin or PMA. Expression of the nonphosphorylatable T138A or S187A mutants of SNAP-25 similarly rescued the secretory response to high-K(+), but the augmentation of this response by forskolin was more pronounced in the cells expressing SNAP-25 (T138A) than in those expressing SNAP-25 (WT), whereas that by PMA was less pronounced in those expressing SNAP-25 (S187A). Our results thus suggest that SNAP-25 phosphorylation by PKA or PKC contributes differentially to the control of exocytosis in PC12 cells by regulating SNARE complex formation.
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http://dx.doi.org/10.1016/j.cellsig.2015.12.014DOI Listing
May 2016

Time-Series Data Analysis of Long-Term Home Blood Pressure Measurements in Relation to Lifestyle.

Stud Health Technol Inform 2015 ;216:751-5

Department of Healthcare Informatics, Takasaki University of Health and Welfare, Takasaki, Gunma, Japan.

We conducted a long-term time-series analysis of an individual's home blood pressure measurements, stored on a personal healthcare system in cloud, relative to the individual's life-style. In addition to daily scattering, apparent seasonal variations were observed in both systolic and diastolic blood pressure measurements. We examined the effect of seasonal variations on the outcome of a healthcare data mining process that extracts rules between blood pressure measurements and life-style components such as exercise and diet, and found that the daily blood pressure data approached a normal distribution when adjusted for the seasonal variations. This implies that an adjustment is desirable in order to produce appropriate rules in the healthcare data mining process.
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December 2016

Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin.

Biochem Biophys Res Commun 2015 Apr 28;459(3):437-42. Epub 2015 Feb 28.

Division of Applied Pharmacology, Kyushu Dental University, Kitakyushu 803-8580, Japan. Electronic address:

A close relationship between the bone and systemic glucose metabolism has recently been the center of attention, since the uncarboxylated form of osteocalcin (GluOC), a bone-derived protein, but not the γ-carboxylated form, is involved in glucose metabolism. However, the analysis of GluOC effect using isolated organs and related cell lines are required to understand its roles in a whole systemic metabolic status. In the present study, we examined the effect of GluOC on cell lines derived from skeletal muscle to explore the mechanisms by which GluOC regulates glucose uptake. In the differentiated C2C12 myotubes, GluOC dose-dependently induced the phosphorylation of ERK without affecting intracellular cAMP and Ca(2+) levels. This effect was inhibited by U0126, an inhibitor of ERK kinase (MEK). Additionally, U73122, an inhibitor of phospholipase C tended to inhibit it as well. Furthermore, cell treatment with GluOC for a long period promoted insulin-induced Akt phosphorylation and glucose uptake in the myotubes, which was abolished by ERK signaling inhibition. These results indicate that GluOC does not triggered Akt phosphorylation and glucose uptake by itself but promotes insulin-induced glucose uptake in myotubes, probably by up-regulating Akt signaling through ERK activation.
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http://dx.doi.org/10.1016/j.bbrc.2015.02.123DOI Listing
April 2015

Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis.

Int J Biochem Cell Biol 2015 May 25;62:62-71. Epub 2015 Feb 25.

Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.

Insulin triggers glucose uptake into skeletal muscle and adipose tissues by gaining the available number of glucose transporter 4 (GLUT4) on the cell surface. GLUT4-loaded vesicles are targeted to plasma membrane from the intracellular reservoir through multiple trafficking and fusion processes that are mainly regulated by Akt. However, it is still largely unknown how GLUT4 expression in the cell surface is promoted by insulin. In the present study, we identified tomosyn at Ser-783 as a possible Akt-substrate motif and examined whether the phosphorylation at Ser-783 is involved in the regulation of GLUT4 expression. Both Akt1 and Akt2 phosphorylated the wild-type tomosyn, but not the mutant tomosyn in which Ser-783 was replaced with Ala. Phosphorylation of tomosyn at Ser-783 was also observed in the intact cells by insulin stimulation, which was blocked by PI3K inhibitor, LY294002. In vitro pull-down assay showed that phosphorylation of tomosyn at Ser-783 by Akt inhibited the interaction with syntaxin 4. Insulin stimulation increased GLUT4 in the cell surface of CHO-K1 cells to promote glucose uptake, however exogenous expression of the mutant tomosyn attenuated the increase by insulin. These results suggest that Ser-783 of tomosyn is a target of Akt and is implicated in the interaction with syntaxin 4.
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http://dx.doi.org/10.1016/j.biocel.2015.02.013DOI Listing
May 2015

Validity of association rules extracted by healthcare-data-mining.

Annu Int Conf IEEE Eng Med Biol Soc 2014 ;2014:4960-3

A personal healthcare system used with cloud computing has been developed. It enables a daily time-series of personal health and lifestyle data to be stored in the cloud through mobile devices. The cloud automatically extracts personally useful information, such as rules and patterns concerning the user's lifestyle and health condition embedded in their personal big data, by using healthcare-data-mining. This study has verified that the extracted rules on the basis of a daily time-series data stored during a half- year by volunteer users of this system are valid.
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http://dx.doi.org/10.1109/EMBC.2014.6944737DOI Listing
July 2016