Publications by authors named "Chika Miyoshi"

22 Publications

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Two novel mouse models mimicking minor deletions in 22q11.2 deletion syndrome revealed the contribution of each deleted region to psychiatric disorders.

Mol Brain 2021 04 12;14(1):68. Epub 2021 Apr 12.

Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

22q11.2 deletion syndrome (22q11.2DS) is a disorder caused by the segmental deletion of human chromosome 22. This chromosomal deletion is known as high genetic risk factors for various psychiatric disorders. The different deletion types are identified in 22q11.2DS patients, including the most common 3.0-Mb deletion, and the less-frequent 1.5-Mb and 1.4-Mb deletions. In previous animal studies of psychiatric disorders associated with 22q11.2DS mainly focused on the 1.5-Mb deletion and model mice mimicking the human 1.5-Mb deletion have been established with diverse genetic backgrounds, which resulted in the contradictory phenotypes. On the other hand, the contribution of the genes in 1.4-Mb region to psychiatric disorders is poorly understood. In this study, we generated two mouse lines that reproduced the 1.4-Mb and 1.5-Mb deletions of 22q11.2DS [Del(1.4 Mb)/+ and Del(1.5 Mb)/+] on the pure C57BL/6N genetic background. These mutant mice were analyzed comprehensively by behavioral tests, such as measurement of locomotor activity, sociability, prepulse inhibition and fear-conditioning memory. Del(1.4 Mb)/+ mice displayed decreased locomotor activity, but no abnormalities were observed in all other behavioral tests. Del(1.5 Mb)/+ mice showed reduction of prepulse inhibition and impairment of contextual- and cued-dependent fear memory, which is consistent with previous reports. Furthermore, apparently intact social recognition in Del(1.4 Mb)/+ and Del(1.5 Mb)/+ mice suggests that the impaired social recognition observed in Del(3.0 Mb)/+ mice mimicking the human 3.0-Mb deletion requires mutations both in 1.4-Mb and 1.5 Mb regions. Our previous study has shown that Del(3.0 Mb)/+ mice presented disturbance of behavioral circadian rhythm. Therefore, we further evaluated sleep/wakefulness cycles in Del(3.0 Mb)/+ mice by electroencephalogram (EEG) and electromyogram (EMG) recording. EEG/EMG analysis revealed the disturbed wakefulness and non-rapid eye moving sleep (NREMS) cycles in Del(3.0 Mb)/+ mice, suggesting that Del(3.0 Mb)/+ mice may be unable to maintain their wakefulness. Together, our mouse models deepen our understanding of genetic contributions to schizophrenic phenotypes related to 22q11.2DS.
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http://dx.doi.org/10.1186/s13041-021-00778-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042712PMC
April 2021

Induction of Mutant Allele in Neurons in Late Infancy Increases Sleep Need.

J Neurosci 2021 Mar 8;41(12):2733-2746. Epub 2021 Feb 8.

International Institute for Integrative Sleep Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function () mutation in the () gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in mice remain to be elucidated. Here, we generated two mouse lines, and mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep. The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.
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http://dx.doi.org/10.1523/JNEUROSCI.1004-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018738PMC
March 2021

Gut microbiota depletion by chronic antibiotic treatment alters the sleep/wake architecture and sleep EEG power spectra in mice.

Sci Rep 2020 11 11;10(1):19554. Epub 2020 Nov 11.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.

Dysbiosis of the gut microbiota affects physiological processes, including brain functions, by altering the intestinal metabolism. Here we examined the effects of the gut microbiota on sleep/wake regulation. C57BL/6 male mice were treated with broad-spectrum antibiotics for 4 weeks to deplete their gut microbiota. Metabolome profiling of cecal contents in antibiotic-induced microbiota-depleted (AIMD) and control mice showed significant variations in the metabolism of amino acids and vitamins related to neurotransmission, including depletion of serotonin and vitamin B6, in the AIMD mice. Sleep analysis based on electroencephalogram and electromyogram recordings revealed that AIMD mice spent significantly less time in non-rapid eye movement sleep (NREMS) during the light phase while spending more time in NREMS and rapid eye movement sleep (REMS) during the dark phase. The number of REMS episodes seen in AIMD mice increased during both light and dark phases, and this was accompanied by frequent transitions from NREMS to REMS. In addition, the theta power density during REMS was lower in AIMD mice during the light phase compared with that in the controls. Consequently, the gut microbiota is suggested to affect the sleep/wake architecture by altering the intestinal balance of neurotransmitters.
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http://dx.doi.org/10.1038/s41598-020-76562-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659342PMC
November 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Loss of the conserved PKA sites of SIK1 and SIK2 increases sleep need.

Sci Rep 2020 05 26;10(1):8676. Epub 2020 May 26.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, 305-8575, Japan.

Although sleep is one of the most conserved behaviors, the intracellular mechanism regulating sleep/wakefulness remains unknown. We recently identified a protein kinase, SIK3, as a sleep-regulating molecule. Mice that lack a well-conserved protein kinase A (PKA) phosphorylation site, S551, showed longer non-rapid eye movement (NREM) sleep and increased NREMS delta density. S551 of SIK3 is conserved in other members of the SIK family, such as SIK1 (S577) and SIK2 (S587). Here, we examined whether the PKA phosphorylation sites of SIK1 and SIK2 are involved in sleep regulation by generating Sik1 and Sik2 mice. The homozygous Sik1 mice showed a shorter wake time, longer NREMS time, and higher NREMS delta density than the wild-type mice. The heterozygous and homozygous Sik2 mice showed increased NREMS delta density. Both the Sik1 and Sik2 mice exhibited proper homeostatic regulation of sleep need after sleep deprivation. Despite abundant expression of Sik1 in the suprachiasmatic nucleus, the Sik1 mice showed normal circadian behavior. Although Sik2 is highly expressed in brown adipose tissue, the male and female Sik2 mice that were fed either a chow or high-fat diet showed similar weight gain as the wild-type littermates. These results suggest that PKA-SIK signaling is involved in the regulation of sleep need.
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http://dx.doi.org/10.1038/s41598-020-65647-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250853PMC
May 2020

Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice.

Proc Natl Acad Sci U S A 2019 08 23;116(32):16062-16067. Epub 2019 Jul 23.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, 305-8575 Ibaraki, Japan;

The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the gene is described as an example.
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http://dx.doi.org/10.1073/pnas.1906774116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689935PMC
August 2019

A single phosphorylation site of SIK3 regulates daily sleep amounts and sleep need in mice.

Proc Natl Acad Sci U S A 2018 10 25;115(41):10458-10463. Epub 2018 Sep 25.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 305-8575 Tsukuba, Japan;

Sleep is an evolutionally conserved behavior from vertebrates to invertebrates. The molecular mechanisms that determine daily sleep amounts and the neuronal substrates for homeostatic sleep need remain unknown. Through a large-scale forward genetic screen of sleep behaviors in mice, we previously demonstrated that the mutant allele of the protein kinase gene markedly increases daily nonrapid-eye movement sleep (NREMS) amounts and sleep need. The mutation deletes the in-frame exon 13 encoding a peptide stretch encompassing S551, a known PKA recognition site in SIK3. Here, we demonstrate that single amino acid changes at SIK3 S551 ( and ) reproduce the hypersomnia phenotype of the mutant mice. These mice exhibit increased NREMS amounts and inherently increased sleep need, the latter demonstrated by increased duration of individual NREMS episodes and higher EEG slow-wave activity during NREMS. At the molecular level, deletion or mutation at SIK3 S551 reduces PKA recognition and abolishes 14-3-3 binding. Our results suggest that the evolutionally conserved S551 of SIK3 mediates, together with PKA and 14-3-3, the intracellular signaling crucial for the regulation of daily sleep amounts and sleep need at the organismal level.
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http://dx.doi.org/10.1073/pnas.1810823115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187192PMC
October 2018

Ablation of Central Serotonergic Neurons Decreased REM Sleep and Attenuated Arousal Response.

Front Neurosci 2018 7;12:535. Epub 2018 Aug 7.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.

Sleep/wake behavior is regulated by distinct groups of neurons, such as dopaminergic, noradrenergic, and orexinergic neurons. Although monoaminergic neurons are usually considered to be wake-promoting, the role of serotonergic neurons in sleep/wake behavior remains inconclusive because of the effect of serotonin (5-HT)-deficiency on brain development and the compensation for inborn 5-HT deficiency by other sleep/wake-regulating neurons. Here, we performed selective ablation of central 5-HT neurons in the newly developed mouse line that was crossed with mice to examine the role of 5-HT neurons in the sleep/wake behavior of adult mice. Intracerebroventricular administration of diphtheria toxin completely ablated tdTomato-positive cells in mice. Electroencephalogram/electromyogram-based sleep/wake analysis demonstrated that central 5-HT neuron ablation in adult mice decreased the time spent in rapid eye movement (REM) sleep, which was associated with fewer transitions from non-REM (NREM) sleep to REM sleep than in control mice. Central 5-HT neuron-ablated mice showed attenuated wake response to a novel environment and increased theta power during wakefulness compared to control mice. The current findings indicated that adult 5-HT neurons work to support wakefulness and regulate REM sleep time through a biased transition from NREM sleep to REM sleep.
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http://dx.doi.org/10.3389/fnins.2018.00535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090062PMC
August 2018

Quantitative phosphoproteomic analysis of the molecular substrates of sleep need.

Nature 2018 06 13;558(7710):435-439. Epub 2018 Jun 13.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.

Sleep and wake have global effects on brain physiology, from molecular changes and neuronal activities to synaptic plasticity. Sleep-wake homeostasis is maintained by the generation of a sleep need that accumulates during waking and dissipates during sleep. Here we investigate the molecular basis of sleep need using quantitative phosphoproteomic analysis of the sleep-deprived and Sleepy mouse models of increased sleep need. Sleep deprivation induces cumulative phosphorylation of the brain proteome, which dissipates during sleep. Sleepy mice, owing to a gain-of-function mutation in the Sik3 gene , have a constitutively high sleep need despite increased sleep amount. The brain proteome of these mice exhibits hyperphosphorylation, similar to that seen in the brain of sleep-deprived mice. Comparison of the two models identifies 80 mostly synaptic sleep-need-index phosphoproteins (SNIPPs), in which phosphorylation states closely parallel changes of sleep need. SLEEPY, the mutant SIK3 protein, preferentially associates with and phosphorylates SNIPPs. Inhibition of SIK3 activity reduces phosphorylation of SNIPPs and slow wave activity during non-rapid-eye-movement sleep, the best known measurable index of sleep need, in both Sleepy mice and sleep-deprived wild-type mice. Our results suggest that phosphorylation of SNIPPs accumulates and dissipates in relation to sleep need, and therefore SNIPP phosphorylation is a molecular signature of sleep need. Whereas waking encodes memories by potentiating synapses, sleep consolidates memories and restores synaptic homeostasis by globally downscaling excitatory synapses. Thus, the phosphorylation-dephosphorylation cycle of SNIPPs may represent a major regulatory mechanism that underlies both synaptic homeostasis and sleep-wake homeostasis.
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http://dx.doi.org/10.1038/s41586-018-0218-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350790PMC
June 2018

Large-scale forward genetics screening identifies Trpa1 as a chemosensor for predator odor-evoked innate fear behaviors.

Nat Commun 2018 05 23;9(1):2041. Epub 2018 May 23.

Functional Neuroscience Lab, Kansai Medical University, Hirakata, Osaka, 573-1010, Japan.

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1 mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.
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http://dx.doi.org/10.1038/s41467-018-04324-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966455PMC
May 2018

Localization of orexin B and orexin-2 receptor in the rat epididymis.

Acta Histochem 2018 Apr 26;120(3):292-297. Epub 2018 Feb 26.

Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Via Delpino 1, 80137, Naples, Italy. Electronic address:

The peptides orexin A (OXA) and orexin B (OXB) derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin, were originally described in the rat hypothalamus. Successively, they have been found in many other brain regions as well as in peripheral organs of mammals and other less evolved animals. The widespread localization of orexins accounts for the multiple activities that they exert in the body, including the regulation of energy homeostasis, feeding, metabolism, sleep and arousal, stress, addiction, and cardiovascular and endocrine functions. Both OXA and OXB peptides bind to two G-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor, though with different binding affinity. Altered expression/activity of orexins and their receptors has been associated with a large number of human diseases. Though at present evidence highlighted a role for orexins and cognate receptors in mammalian reproduction, their central and/or local effects on gonadal functions remain poorly known. Here, we investigated the localization of OXB and OX2R in the rat epididymis. Immunohistochemical staining of sections from caput, corpus and cauda segments of the organ showed intense signals for both OXB and OX2R in the principal cells of the lining epithelium, while no staining was detected in the other cell types. Negative results were obtained from immunohistochemical analysis of hypothalamic and testicular tissues from OX2R knock-out mice (OX2R) and OX1R/OX2R double knock-out (OX1R; OX2R) mice, thus demonstrating the specificity of the rabbit polyclonal anti-OX2R antibody used in our study. On contrary, the same antibody clearly showed the presence of OX2R in sections from hypothalamus and testis of normal mice and rats which are well known to express the receptor. Thus, our results provide the first definite evidence for the immunohistochemical localization of OXB and OX2R in the principal cells of rat epididymis.
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http://dx.doi.org/10.1016/j.acthis.2018.02.011DOI Listing
April 2018

Sleep/Wake Behaviors in Mice During Pregnancy and Pregnancy-Associated Hypertensive Mice.

Sleep 2018 03;41(3)

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Ibaraki, Japan.

Study Objectives: In humans and other mammals, sleep is altered during pregnancy. However, no studies have been conducted on sleep/wakefulness during pregnancy in mice. In this study, we examined sleep/wakefulness in female C57BL/6 mice during pregnancy. We also examined sleep/wake behaviors in an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice, in which increased angiotensin causes hypertension.

Methods: Sleep/wake behaviors of female C57BL/6 and PAH mice were examined based on electroencephalogram (EEG) or electromyogram recordings before, during, and after pregnancy. To examine whether high blood pressure disrupts the integrity of the blood-brain barrier in PAH mice, Evans blue dye was injected intravenously. Angiotensin II receptor blocker (olmesartan)-administered PAH mice and female Tsukuba hypertensive mice were also examined.

Results: C57BL/6 mice showed a decreased total wake time and increased nonrapid eye movement (NREM) sleep time during late pregnancy. Rapid eye movement (REM) sleep time did not change during the course of pregnancy. PAH mice exhibited a general slowing of EEG during late pregnancy and subsequently returned to apparently normal sleep/wakefulness after delivery. All PAH mice exhibited multiple focal leakages of Evans blue dye in the brain. Spike-and-wave discharges were observed in 50% of PAH mice. Olmesartan-administered PAH mice did not show general slowing of EEG. Tsukuba hypertensive mice showed a normal time spent in wakefulness and NREM sleep and a decreased total REM sleep time.

Conclusions: This study showed pregnant-stage-specific changes in sleep/wakefulness in C57BL/6 mice. Furthermore, PAH mice may be useful as an animal model for eclampsia.
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http://dx.doi.org/10.1093/sleep/zsx209DOI Listing
March 2018

Forward-genetics analysis of sleep in randomly mutagenized mice.

Nature 2016 11 2;539(7629):378-383. Epub 2016 Nov 2.

Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.
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http://dx.doi.org/10.1038/nature20142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6076225PMC
November 2016

Phase I trial of GBS-01 for advanced pancreatic cancer refractory to gemcitabine.

Cancer Sci 2016 Dec 19;107(12):1818-1824. Epub 2016 Dec 19.

Division of Translational Research, Exploratory Oncology and Clinical Trial Center, National Cancer Center, Kashiwa, Japan.

GBS-01, an extract from the fruit of Arctium lappa L. is an orally administered drug rich in arctigenin, which has been reported to exert antitumor activity by attenuating the tolerance of cancer cells to glucose deprivation. We investigated the maximum tolerated dose of GBS-01 based on the frequency of the dose-limiting toxicities (DLTs) and pharmacokinetics in patients with advanced pancreatic cancer refractory to gemcitabine. GBS-01 was given orally at escalating doses from 3.0 g (containing 1.0 g burdock fruit extract) to 12.0 g q.d. A DLT was defined as a grade 4 hematological toxicity and grade 3 or 4 non-hematological toxicity appearing during the first 28 days of treatment. Fifteen patients (GBS-01 dose level 1 [3.0 g], three patients; dose level 2 [7.5 g], three patients; and dose level 3 [12.0 g], nine patients) were enrolled. None of the patients at any of the three dose levels showed any sign of DLTs. The main adverse events were increased serum γ-glutamyl transpeptidase, hyperglycemia, and increased serum total bilirubin; however, all the toxicities were mild. Of the 15 patients, 1 showed confirmed partial response and 4 patients had stable disease. The median progression-free and overall survival of the patients were 1.1 and 5.7 months, respectively. The pharmacokinetic study revealed a high bioavailability of arctigenin and rapid conjugation of the drug with glucuronic acid. The recommended dose of GBS-01 was 12.0 g q.d, and favorable clinical responses were obtained. This trial was registered at UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number UMIN000005787.
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http://dx.doi.org/10.1111/cas.13086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198948PMC
December 2016

Identification of mutations through dominant screening for obesity using C57BL/6 substrains.

Sci Rep 2016 09 2;6:32453. Epub 2016 Sep 2.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.
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http://dx.doi.org/10.1038/srep32453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009433PMC
September 2016

Antiausterity activity of arctigenin enantiomers: importance of (2R,3R)-absolute configuration.

Nat Prod Commun 2014 Jan;9(1):79-82

From a MeOH extract of powdered roots of Wikstroemia indica, six dibenzyl-gamma-butyrolactone-type lignans with (2S,3S)-absolute configuration [(+)-arctigenin (1), (+)-matairesinol (2), (+)-trachelogenin (3), (+)-nortrachelogenin (4), (+)-hinokinin (5), and (+)-kusunokinin (6)] were isolated, whereas three dibenzyl-gamma-butyrolactone-type lignans with (2R,3R)-absolute configuration [(-)-arctigenin (1*), (-)-matairesinol (2*), (-)-trachelogenin (3*)] were isolated from Trachelospermum asiaticum. The in vitro preferential cytotoxic activity of the nine compounds was evaluated against human pancreatic PANC-1 cancer cells in nutrient-deprived medium (NDM), but none of the six lignans (1-6) with (2S,3S)-absolute configuration showed preferential cytotoxicity. On the other hand, three lignans (1*-3*) with (2R,3R)-absolute configuration exhibited preferential cytotoxicity in a concentration-dependent manner with PC50 values of 0.54, 6.82, and 5.85 microM, respectively. Furthermore, the effect of (-)- and (+)-arctigenin was evaluated against the activation of Akt, which is a key process in the tolerance to nutrition starvation. Interestingly, only (-)-arctigenin (1*) strongly suppressed the activation of Akt. These results indicate that the (2R,3R)-absolute configuration of (-)-enantiomers should be required for the preferential cytotoxicity through the inhibition of Akt activation.
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January 2014

Synthesis and antitumor evaluation of arctigenin derivatives based on antiausterity strategy.

Eur J Med Chem 2013 Feb 28;60:76-88. Epub 2012 Nov 28.

Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.

A series of new (-)-arctigenin derivatives with variably modified O-alkyl groups were synthesized and their preferential cytotoxicity was evaluated against human pancreatic cancer cell line PANC-1 under nutrient-deprived conditions. The results showed that monoethoxy derivative 4i (PC(50), 0.49 μM), diethoxy derivative 4h (PC(50), 0.66 μM), and triethoxy derivative 4m (PC(50), 0.78 μM) showed the preferential cytotoxicities under nutrient-deprived conditions, which were identical to or more potent than (-)-arctigenin (1) (PC(50), 0.80 μM). Among them, we selected the triethoxy derivative 4m and examined its in vivo antitumor activity using a mouse xenograft model. Triethoxy derivative 4m exhibited also in vivo antitumor activity with the potency identical to or slightly more than (-)-arctigenin (1). These results would suggest that a modification of (-)-arctigenin structure could lead to a new drug based on the antiausterity strategy.
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http://dx.doi.org/10.1016/j.ejmech.2012.11.031DOI Listing
February 2013

Hypoxia-inducible transcription factor-2alpha in endothelial cells regulates tumor neovascularization through activation of ephrin A1.

J Biol Chem 2008 Jul 23;283(27):18926-36. Epub 2008 Apr 23.

Department of Regenerative Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan.

The hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha mediate responses to hypoxia, such as tumor neovascularization. To determine the function of HIF-2alpha in vascular endothelial cells (ECs), we examined vascular formation in HIF-2alpha knockdown (kd/kd) mice transplanted with tumors. We observed that both the tumor size and the number of large vessels growing within transplanted melanomas were significantly reduced in kd/kd recipients compared with wild-type (WT) mice. In contrast, we observed a similar extent of vascular formation within fibrosarcomas transplanted from either kd/kd or WT mice into WT recipients. Thus, HIF-2alpha expression in host animal ECs, but not in the tumor cells, is crucial for tumor neovascularization. HIF-2alpha may function through ephrin A1 as the expression of ephrin A1 and related genes was markedly reduced in kd/kd ECs, and HIF-2alpha specifically bound a hypoxia-response element sequence in the ephrin A1 promoter. Treatment of WT ECs with an ephrin A1 inhibitor (ephrin A1-Fc) also impaired neovascularization. We conclude that in ECs, HIF-2alpha plays an essential role in vascular remodeling during tumor vascularization through activation of at least ephrin A1.
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http://dx.doi.org/10.1074/jbc.M709133200DOI Listing
July 2008

Comparison of individual perceptions of medication costs and benefits between intentional and unintentional medication non-adherence among Japanese patients.

Patient Educ Couns 2008 Feb;70(2):292-9

Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki-City, Kagawa 769-2193, Japan.

Objective: To identify Japanese patients' perceptions of the costs and benefits of their medications by administering a questionnaire validated in Western patients and to compare the association between the perception levels and non-adherence to medication in the two non-adherent patient types, intentional, and unintentional.

Methods: Japanese patients with chronic diseases were given a questionnaire and interviewed, and the validity and reliability of the scales generated were assessed. Logistic regression was used to analyse the association between individual perception levels and non-adherence to the medication regimen.

Results: From 151 responses, two kinds of scales were generated following a report of Western patients; the necessity scale showed satisfactory reliability (Cronbach's alpha 0.79) but the concerns scale did not. Individual levels of perception of the necessity of medications were associated with unintentional non-adherence (the higher the level, the lower the odds ratio 1.0, 0.56, 0.40, and 0.15), while they were not associated with intentional non-adherence.

Conclusion: Japanese patients' perceptions of the benefits of medications, but not the costs were similar to those of Western patients, and these perceptions were likely to be different between intentionally and unintentionally non-adherent patients.

Practice Implications: Strategies to improve non-adherence should be designed according to the non-adherent type.
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http://dx.doi.org/10.1016/j.pec.2007.10.016DOI Listing
February 2008

Changes in attitudes among Japanese patients after Pharmacist Law revision.

Pharm World Sci 2008 Jun 25;30(3):258-64. Epub 2007 Oct 25.

Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Shido, Sanuki-city, Kagawa, Japan.

Objective: To assess changes in patients' perception of their medications and their adherence to regimens after enactment of the Japanese Pharmacist Law revision of 1997, which stipulated provision of drug information to patients as one of the principal duties of pharmacists. Setting A university hospital in Japan.

Method: Comparison of cross sectional analyses between two time periods: shortly after enactment of the Pharmacist Law revision and about a half-decade later.

Main Outcome Measure: Patient's knowledge of the medications, anxiety level, individual beliefs regarding taking medications without anxiety, and adherence to the medication regimens.

Results: There were 141 and 151 patients who participated during each period, respectively. The proportion of non-adherent patients significantly decreased from 68.8 to 53.6% (P = 0.008). Multiple logistic regression analysis indicated that the features of the intentionally non-adherent patients have changed; they have switched from persons who lack general comprehension about the medications (P = 0.01), ones who place an importance on knowing the side effects (P = 0.04), or who place no value on mutual reliance on their doctors (P = 0.03) into persons who place no value on understanding the purpose of taking their medications (P = 0.04) or who place value on multiple items to take medications without anxiety (P = 0.03), i.e., supposedly people who prefer thinking about their drug therapy from their own point of view based on comprehension of their disease and medications.

Conclusions: The rapid progression of drug information disclosure after enactment of the Pharmacist Law revision has likely resulted in drastic changes in patients' perception of their medications and led to improvements in medication adherence.
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http://dx.doi.org/10.1007/s11096-007-9171-6DOI Listing
June 2008

MafG sumoylation is required for active transcriptional repression.

Mol Cell Biol 2006 Jun;26(12):4652-63

Graduate School of Comprehensive Human Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.
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http://dx.doi.org/10.1128/MCB.02193-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489127PMC
June 2006

Leukocyte behavior in angiogenic vessels is affected by tumor-derived nitric oxide.

Jpn J Physiol 2003 Oct;53(5):343-50

Department of Biomedical Engineering, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305-8575 Japan.

Recent studies indicate a possible role of nitric oxide (NO) in regulating leukocyte-endothelial cell interactions, which plays a key role in the tumor immunity. The purpose of the present study is aimed to observe the tumor hemodynamics intravitally and to clarify the effect of NO on tumor microcirculation by means of a real-time confocal laser-scanning microscope using NO-reactive indicators. Visualization of localization of NO and the leukocyte behavior was made in the mesenteric microvessels of an experimental tumor model rat. Production of NO was clearly visualized along the endothelium of the tumor-free rats, but scarcely found in the newly formed tumor microvessels. A higher level of NO production was observed in a solid tumor region, where a more marked decrease in the leukocyte-endothelial cell interactions was observed. Local administration of a NO synthase (NOS) inhibitor increased leukocyte adhesion. This indicates that tumor-derived NOS creates the tumor specific microenvironment of the immature angiogenic tumor vessels, thereby modulating leukocyte behavior.
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http://dx.doi.org/10.2170/jjphysiol.53.343DOI Listing
October 2003