Diacylglycerol Kinase β Knockout Mice Exhibit Attention-Deficit Behavior and an Abnormal Response on Methylphenidate-Induced Hyperactivity

BACKGROUND: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKβ knockout (KO) mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density), hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKβ KO mice in order to investigate the function of DGKβ in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD). METHODOLOGY/PRINCIPAL FINDINGS: DGKβ KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.). In the open field test, DGKβ KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p.), but showed a similar response to an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.3 mg/kg, i.p.), when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK), which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKβ KO mice. CONCLUSIONS/SIGNIFICANCE: These findings suggest that DGKβ KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKβ KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKβ has a pivotal involvement in ERK regulation in the striatum

Restraint-Induced Expression of Endoplasmic Reticulum Stress-Related Genes in the Mouse Brain

ABSTRACT Depression is a significant public health concern but its pathology remains unclear. Previously, increases in an end

Laxative effects of agarwood on low-fiber diet-induced constipation in rats

<p>Abstract</p> <p>Background</p> <p>Agarwood (<it>Aquilaria sinensis</it>), well known as incense in Southeast Asia, has been used as a digestive in traditional medicine. We investigated the laxative effects of an ethanol extract of agarwood leaves (EEA) in a rat model of low-fiber diet-induced constipation.</p> <p>Methods</p> <p>A set of rats was bred on a normal diet while another set was placed on a low-fiber diet to induce constipation. The laxative effect of agarwood was then investigated on both sets of rats.</p> <p>Results</p> <p>Pretreatment of normal rats with single dose of EEA (600 mg/kg, p.o.) significantly increased frequency and weight of stools. Also, treatments with EEA (300 and 600 mg/kg, p.o.) for 14 days caused a significant increase in stool frequency and weight. Feeding of the animals with a low-fiber diet resulted in a decrease in stool weight, frequency, and water content and also delayed carmine egestion. A single treatment with EEA (600 mg/kg) or senna (150 and 300 mg/kg) significantly increased stool frequency, weight, and water content and also accelerated carmine egestion in the model rats. Once daily administrations of EEA (150 mg/kg), for 14 days, caused a significant increase in water content of stools. The higher doses of EEA (300 and 600 mg/kg) significantly increased frequency, weight, and water content of the stools while accelerating carmine egestion in the constipated rats. Senna (150 and 300 mg/kg) produced similar effect as the higher doses of EEA but, in addition, induced severe diarrhea.</p> <p>Conclusion</p> <p>These findings indicate that EEA has a laxative effect, without causing diarrhea, in a rat model of low-fiber diet-induced constipation. These findings suggest that EEA may be highly effective on constipation as a complementary medicine in humans suffering from life style-induced constipation.</p

カリジノゲナーゼの血管新生抑制作用

現在、異常な網膜血管新生によって、不可逆的な視野欠損あるいは失明に至る患者が増加の一途をたどっている。網膜中の異常な血管新生は血管内皮細胞増殖因子 (VEGF) などの特定のサイトカインによって誘導される。抗VEGF 治療薬が眼内血管新生疾患をターゲットにした治療薬として硝子体内投与によって使用されている。しかし、硝子体内に対する連続投与は硝子体出血、網膜はく離の危険性があり、さらにコンプライアンスの低下が懸念されている。したがって、末梢投与のような非侵襲的な薬物送達が求められている。近年、カリジノゲナーゼが末梢経路を介して網脈絡膜の循環を改善し、さらには網膜血管透過性亢進を抑制することが報告された。網膜血管新生におけるカリジノゲナーゼの役割を明らかにするために、増殖糖尿病網膜症患者 (PDR) の硝子体液を用いてその濃度を測定し、in vitro およびin vivo 血管新生モデルを用いて抗血管新生作用について検討を行った。硝子体中カリジノゲナーゼおよびVEGF 濃度は、黄斑円孔および黄班上膜患者に比べPDR 患者で高値を示した。カリジノゲナーゼはVEGF165 の切断を介して、in vitro 血管新生モデルにおけるVEGF165 誘発管腔形成、増殖、遊走を抑制した。また、カリジノゲナーゼは皮下投与によってマウス高酸素負荷網膜血管新生モデルにおける病的な血管新生を抑制した。これらの知見はカリジノゲナーゼが増殖糖尿病網膜症の病態に一部関与し、さらに末梢経路によってVEGF165 自体を切断する有望な治療薬になり得ることを示唆している。Irreversible vision loss and blindness due to abnormal retinal neovascularization has been increasing. An abnormalproliferation of new blood vessels in the retina is induced by a specific cytokine, vascular endothelial growth factor (VEGF). Theintravitreal injection of anti-VEGF therapeutic agents has been used in the treatment of ocular neovascular diseases. However,repeated injections are associated with potential risks of vitreous hemorrhage, retinal detachment, and decrease in compliance.Therefore, noninvasive delivery systems, such as peripheral administration, are required. Recently, it has been reported thatkallidinogenase improved choroidal and retinal circulation, and prevented the retinal vascular hyperpermeability by the peripheralroute. To identify the role of kallidinogenase in retinal neovascularization, we measured the concentrations in vitreous fluid frompatients with proliferative diabetic retinopathy, and investigated the anti-angiogenic effect by using in vitro and in vivo angiogenesismodels. Kallidinogenase in vitreous fluid was markedly elevated in proliferative diabetic retinopathy patients compared with that incontrol patients with macular holes and epiretinal membranes. Kallidinogenase inhibited VEGF165-induced tube formation,proliferation, and migration in an in vitro angiogenesis model via the cleavage of VEGF165. When administered subcutaneously,kallidinogenase reduced the pathologic neovascularization in the murine oxygen-induced retinopathy model. These findings indicatethat kallidinogenase is partly involved in the pathogenesis of proliferative diabetic retinopathy and may be a promising therapeuticagent that could cleave VEGF165 itself when administered by a peripheral route

膜貫通糖タンパク質GPNMB のALS 病態に対する神経保護因子としての可能性

筋萎縮性側索硬化症(ALS) は上位および下位運動ニューロンが選択的かつ進行性に変性、脱落する重篤な指定神経難病である。長年、ALS に対する精力的な研究が行われてきたが、現時点でALS 病態の原因は不明である。本試験において、変異Cu/Zn superoxide dismutase (SOD1G93A) 発現ALS モデルマウスを用いたDNA マイクロアレイ解析を実施したところ、glycoprotein nonmetastatic melanoma protein B (GPNMB) が新規 ALS 病態関連因子として同定された。ALS 患者およびALS モデルマウス脊髄において病態の進行に伴うGPNMB の顕著な増加が認められた。また、GPNMB の発現は、運動ニューロンおよびアストロサイトにおいて認められた。さらに、運動ニューロン様細胞であるNSC34 細胞株を用いた検討により、SOD1G93AがGPNMBと結合しGPNMBの糖鎖修飾を阻害することで運動ニューロンの脆弱性が亢進した。一方、活性化したアストロサイトでは、GPNMB細胞外断片の分泌が認められ、その結果運動ニューロンに対するSOD1G93A毒性を減弱させた。さらに、ALS 患者血清中のGPNMB 量は、コントロール群および他の中枢神経変性疾患群に比べ高値を示した。以上より、GPNMB は、ALS の有用な治療標的となる可能性が示唆された。Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by the loss of motorneurons. Despite substantial research, the causes of ALS remain unclear. Glycoprotein nonmetastatic melanoma protein B (GPNMB)was identified as an ALS-related factor using DNA microarray analysis with mutant superoxide dismutase (SOD1G93A) mice.GPNMB was greatly induced in the spinal cords of ALS patients and a mouse model as the disease progressed. It was especiallyexpressed in motor neurons and astrocytes. In a NSC34 cell line, glycosylation of GPNMB was inhibited by interaction withSOD1G93A, increasing motor neuron vulnerability, whereas extracellular fragments of GPNMB secreted from activated astrocytesattenuated the neurotoxicity of SOD1G93A in neural cells. Furthermore, GPNMB expression was more substantial in the sera ofsporadic ALS patients than in other diseased patients. This study suggests that GPNMB can be a target for therapeutic interventionfor suppressing motor neuron degeneration in ALS

Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

<p>Abstract</p> <p>Background</p> <p>Vascular endothelial growth factor (VEGF) is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ), bee pollen, and Chinese red propolis] from the honeybee, <it>Apis mellifera</it>, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs).</p> <p>Methods</p> <p>In an <it>in vitro </it>tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE)]. To clarify the mechanism of <it>in vitro </it>angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE.</p> <p>Results</p> <p>RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced <it>in vitro </it>tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation.</p> <p>Conclusion</p> <p>Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.</p

Efficacy of Prednisolone in Generated Myotubes Derived From Fibroblasts of Duchenne Muscular Dystrophy Patients

Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy characterized by progressive muscle degeneration. This disease is caused by the mutation or deletion of the dystrophin gene. Currently, there are no effective treatments and glucocorticoid administration is a standard care for DMD. However, the mechanism underlying prednisolone effects, which leads to increased walking, as well as decreased muscle wastage, is poorly understood. Our purpose in this study is to investigate the mechanisms of the efficacy of prednisolone for this disease. We converted fibroblasts of normal human cell line and a DMD patient sample to myotubes by MyoD transduction using a retroviral vector. In myotubes from the MyoD-transduced fibroblasts of the DMD patient, the myotube area was decreased and its apoptosis was increased. Furthermore, we confirmed that prednisolone could rescue these pathologies. Prednisolone increased the expression of not utrophin but laminin by down-regulation of MMP-2 mRNA. These results suggest that the up-regulation of laminin may be one of the mechanisms of the efficacy of prednisolone for DMD

Phosphodiesterase-III Inhibitor Prevents Hemorrhagic Transformation Induced by Focal Cerebral Ischemia in Mice Treated with tPA

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA