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Tacrolimus, as one of cytochrome P450 (CYP) 4/5 substrates is known to have many drug–drug interactions. Metabolism of tacrolimus is inhibited by many drugs, such as the azole antifungal agents due to their effects on CYP 3A4/5. Implementation of therapeutic drug monitoring is commended during tacrolimus treatment for maintaining optimal plasma concentration levels, and to avoid its toxicity. We herein report a case of a 58-year-old female patient with lupus nephritis whose plasma concentration of orally ministered tacrolimus increased after a single administration of 300 mg voriconazole for suspected Aspergillus infection. Our observations were the result of therapeutic drug monitoring before the second administration of voriconazole. Although tacrolimus dose did not vary, the plasma tacrolimus concentration was markedly increased and concentration dose ratio was 2.1 fold more than prior to voriconazole treatment. This case suggests that even after a single intravenous administration of voriconazole, the plasma concentration of tacrolimus may increase. Dose optimization of tacrolimus by therapeutic drug monitoring soon after the first dose of voriconazole is therefore warranted to minimize the risk of tacrolimus toxicity.

Dihydrobiopterin (BH2), an oxidized form of tetrahydrobiopterin (BH4) which is an essential cofactor of nitric oxide synthase, has been reported to be elevated in association with oxidative stress-related cardiovascular disorders such as hypertension and diabetes. However, the role of BH2 in the regulation of endothelial nitric oxide synthase (eNOS) activity in vivo remains unknown. Thus, we aimed to clarify whether increasing intracellular BH2 levels causes eNOS dysfunction in rats. The BH2 precursor sepiapterin (SEP) was intravenously given after administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH2 to BH4. MTX/SEP treatment markedly augmented aortic BH2 levels by 8.7- fold but did not affect aortic BH4 levels compared with control (saline) treatment. MTX alone did not significantly alter BH4 or BH2 levels. MTX/SEP, but not MTX alone, impaired vasodilator responses induced by acetylcholine (ACh), an endotheliumdependent vasodilator, and also attenuated ACh-induced relaxations of isolated aortas, indicating impairment of endothelial function. Importantly, MTX/SEP treatment significantly enhanced NOS inhibitor-sensitive superoxide production, suggesting the involvement of eNOS uncoupling. MTX/SEP treatment modified the eNOS phosphorylation state into a reduced eNOS activity. The present data indicate that BH2, even in the absence of BH4 deficiency, causes impairment of eNOS function in vivo, and suggest a novel role of BH2 in endothelial dysfunction.

すでに知られたガジュツの健胃剤としての作用に加えて、Curvuma zadoaria Roscoeの主成分デヒドロクルジオンは、抗酸化性によると思われる抗炎症作用をもつことを明らかにした。, In addition to the well-known effect of zedoary as a stomachic, dehydrocurdione, the major component of Curcuma zedoaria Roscoe has antiinflammatory potency related to its antioxidant effect.

ジアシルグリセロールキナーゼ (DGK) はジアシルグリセロールをリン酸化し、ホスファチジン酸へと変換する酵素である。現在までに哺乳類において10 種類のアイソフォームが同定されており、脳において強く発現していることが知られている。DGKβ はI 型に分類されるDGK アイソフォームであり、マウス脳においては、嗅球、皮質、海馬、線条体において特に強く発現している。しかしながら、その機能については不明な点が多いため、DGKβ 欠損マウスを用いて表現型解析を行った。DGKβ 欠損マウスでは、躁病様の表現型が認められ、これら表現型は既存の躁病治療薬で改善されることが明らかとなった。また、DGKβ 欠損マウスは多動性に加えて注意欠損様の行動が認められ、注意欠陥多動性障害(ADHD) 治療薬メチルフェニデートは注意欠損様の行動を改善することが示唆された。さらに、DGKβ 欠損マウスに痙攣誘発薬を投与したところ、痙攣行動の増悪が認められ、その一因に海馬における抑制性神経細胞の減少が考えられる。以上の結果より、DGKβ の欠損が躁病をはじめとする様々な疾患に関与している可能性およびこれら疾患の病態解明・治療薬探索にDGKβ 欠損マウスが有用であると考えられる。, Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol to phosphatidic acid. To date, ten isoforms ofDGKs have been identified in mammals, and it is reported that DGKs is strongly expressed in the brain. DGKβ, a type of DGK, isexpressed in the olfactory bulb, cortex, hippocampus, and striatum. The roles of DGKβ are still unknown. We generated the DGKβknockout (KO) mice and investigated the phenotypes of DGKβ KO mice. DGKβ KO mice showed mania-like behaviors such ashyperactivity and reduced anxiety. These mania-like behaviors were ameliorated after commonly used medication for mania wereadministered. Furthermore, DGKβ KO mice showed attention-deficit behavior, which was ameliorated by treatment withmethylphenidate. Furthermore, DGKβ KO mice showed increased seizure sensitivity due to the decreased numbers of depressorneurons in the hippocampus. These results suggest that deficit of DGKβ is involved in various neurological diseases including mania,and DGKβ KO mice would be useful to elucidate the pathogenesis and find the therapeutic targets in these disorders of the centralnervous system.

わたし達の目は、日常的に太陽の紫外線や酸素などの影響を受けている。加えて、スマートフォンやパソコンなど電子端末機器の普及が進む現代社会において、目に関するトラブルの増加が懸念される。視機能の維持は我々のQOLに大きく関わるため、薬剤による治療や進行の抑制とともに、食品の機能性による予防も重要である。健康食品素材として広く利用されるビルベリーは、ブルーベリーの近縁種にあたる果実であり、果皮および果実内部にポリフェノールの一種であるアントシアニン色素を多く含んでいる。ビルベリー由来アントシアニンが目に与える機能性としては、in vitroおよびin vivo 試験において、網膜神経節細胞保護作用や光刺激に対する網膜視細胞保護作用、血管新生抑制作用、網膜炎症の軽減による視機能低下抑制作用などが報告されている。一方で、ヒト臨床試験の報告が少ないことが課題とされていたが、2015 年4 月より新たな食品表示基準として機能性表示食品制度が施行されたことにより、ヒト臨床試験の報告が増えてきている。ビルベリーエキスに含まれるアントシアニン（以下、ビルベリー由来アントシアニン）においても新たな報告がなされている。ビルベリー由来アントシアニンを健常人が接種することで得られる機能性として、物の遠近を見る近見視力や毛様体筋の緊張状態、眼精疲労の客観的指標であるフリッカー値、および目の疲労感の主観的指標であるvisual analogue scale（VAS）スコアや自覚症状アンケートにおいて、プラセボ接種群と比較して有意な改善が示されている。そのため、機能性表示食品として、目のピント調節力を改善すること、目の疲労感を和らげることが表示されている。今後もさらにヒト臨床試験が実施されることで、新たな機能性が明らかになることが予想される。, Eyes are organs, which protrude outside the body, exposed to ultraviolet rays from the sun and oxygen. Furthermore, thedisorders of the eyes have increased due to the widespread use of electronic terminal equipment, such as smartphones and personalcomputers. Visual function is very important for our quality of life. Prevention of diseases by using drugs and beneficial food-derivedsubstances is important. Bilberry, which is a species related to blueberry, contains large amounts of anthocyanin in the peel and fruit.The beneficial effects of anthocyanins derived from bilberry on eye health, such as their protective effects on retinal ganglion cellsagainst oxidative stress and on photoreceptor cells against light-induced damage, inhibitory effect against angiogenesis, andinhibitory effect on visual function decline by reducing inflammation in the retina, have been reported from in vitro and in vivostudies. However, few human clinical trials on the intake of anthocyanins derived from bilberry are present. In Japan, the new systemof functional display of foods started from April 2015, thereby resulting in some new results from human clinical trials with asupplement containing anthocyanins derived from bilberry. The beneficial effects of anthocyanins derived from bilberry in humanshave been reported including improvement of factors such as near visual acuity, tension of the ciliary body, flicker fusion frequencyindicative of eye strain, and visual analogue scale (VAS) score or subjective symptoms evaluation using questionnaires compared toplacebo. The beneficial effects of anthocyanins derived from bilberry have also been indicated in the improvement of eye focusingand reduction of eye fatigue. In the future, new beneficial functions related to eye health may emerge by conducting further humanclinical trials with bilberry.

キンミズヒキのタンニンの1種であるagrimoniinの抗腫瘍作用を検討した。AgrimoniinはC57BL/6マウスに移植したE.L.4およびBALB/cマウスに移植したMethA腫瘍の増殖を有意に抑制し, MethAを移植したマウスの生存期間の延長傾向を示した。また, agrimoniinはE.L.4,MethAおよびL-929細胞に対してin vitroで用量依存的な障害作用を示し, その細胞障害性は4時間以内のincubationによってみられた。一方, agrimoniinはヒツジ赤血球で免疫したhemolytic plaque forming cellおよびhemolysinの産生に影響を及ぼさず, また, picrylchlorideによる細胞性免疫の成立に対しても影響を及ぼさなかった。以上のことから, agrimoniinはキンミズヒキの抗腫瘍成分の一つであり, その作用機序は直接的な細胞障害作用によるものと思われる。, Studies were carried out to examine the antitumor activity of agrimoniin, one of tannins isolated from Agrimonia pilosa Ledeb., using syngeneic systems including C57BL/6 mice-E.L.4 lymphosarcoma and BALB/C mice-Meth A fibrosarcoma, and the effect on immune responses. Agrimoniin significantly inhibited the growth of both the tumors and showed a tendency to prolong the survival period of BALB/C mice bearing Meth A fibrosarcoma. In the in vitro study the tannin also exhibited a cytotoxic activity against E. L. 4 lymphosarcoma, Meth A fibrosarcoma and L-929 cells in a dosedependent fashion, and the activity was seen within 4 hour-incubation. Agrimoniin, however, neither affected the production of humoral antibodies such as hemolytic plaque forming cell and hemolysin against sheep red blood cells nor the formation of cellular immune response induced by picryl chloride These results suggest that agrimoniin may be one of the antitumor constituents of A. pilosa and display the activity through the direct cytotoxicity rather than host-mediated mechanism.

Following experiments were made mainly about the effects of VB_1 and its related compounds on the action and synthesis of ACh. 1) The ACh-induced contraction of isolated ileum of guinea pigs was increased by the pretreatment of these examined substances. 2) Inhibitory effect of examined substances on ChE of horse's serum was found only in a high concentration. 3) The formation of free and total ACh in chopped tissue of frog's brain was increased with a low concentration of examined substances. The order of intensity was as follows : TPD>TPP> VB_1. 4) In the contraction of rat's phrenic nerve-diaphram preparation by indirect stimulation, these substances showed a decrease seems to be a curare-like action in a concentration of 5×l0^M, but an increase in l0^M. Their potentiation for the contraction was more remarkable by the pretreatment of DFP, one of potent cholinesterase inhibitors, or in the fatigued preparation. On the other hand, the effect of these substances was somewhat or little in the contraction by direct stimulation. 5) Analgesic activity of these substances was found somewhat or little in rabbits subjected to dental pulp-stimulation method. 6) These substances didn't affect the ED_ value of morphine in mice.

緑内障は、自覚症状がなく視力が徐々に低下し、失明に至る危険性の高い慢性疾患の一つである。緑内障発症の原因の一つは眼圧が亢進し、眼底部にある視神経が物理的障害を受けて、視力が低下すると考えられている。そのため、眼圧を下げることが緑内障治療の第一選択肢となる。眼圧は房水の産生量と流出量のバランスで制御されており、線維柱帯細胞の収縮弛緩の変化でこのバランスが調節されている。仮に房水流出量が減少すれば、眼圧は徐々に高まり、緑内障の発症危険率が高まる。現在のところ、主経路からの房水流出を促進させる薬剤が少なく、新しい作用機序を有する治療薬の開発、販売が望まれている。本研究では、主経路からの房水流出促進を評価するためブタ由来線維柱帯細胞を用いた3次元コラーゲンゲル培養方法をin vitro で簡便に評価する方法を構築し、多数の薬剤を評価した結果、cyclin 依存性kinase（CDK）阻害薬であるroscovtine が強いコラーゲンゲル収縮抑制作用を示した。Roscovitine にはR 体とS 体の2 つの異性体が存在するため、両異性体をウサギに点眼した後の眼圧下降作用を検討したところ、いずれも眼圧下降作用を示した。さらに培養神経節細胞を用いたストレス負荷試験でroscovitine 両異性体の抗ストレス作用を検討したところ、S 体の異性体のみに保護作用が認められた。, Glaucoma comprises group of eye diseases that gradually diminish eyesight, often without warning or symptoms. Loss ofvision results from optic nerve damage, which was once attributed primarily to high intraocular pressure (IOP). Therefore, loweringIOP is the first choice for glaucoma therapy. The IOP is regulated by a balance between aqueous inflow and outflow, and ismodulated by changes in the contractile state of the TM. If aqueous outflow decreases, IOP may gradually increase, resulting inhypertensive glaucoma. At present, only a limited number of agents that have the capability of modulating outflow through theconventional route are available. Identification of new and improved agents that can improve outflow is therefore important.We developed a 3-D collagen gel assay using primary porcine trabecular meshwork (pTM) cells and evaluated the inhibitory effectsof various agents on the contractility of collagen gel. Roscovitine, a cell cyclin-dependent kinase (CDK) inhibitor, strongly inducedrelaxation of pTM cells. Additionally, we investigated the effects of both the R- and S-isomer on the IOP of rabbits and on the deathof cultured retinal ganglion cells. In the in vivo rabbit experiment, instillation of both isomers significantly lowered the IOP. In the invitro cell experiment, the S-isomer alone inhibited tunicamycin- and glucose deprivation-induced cell damage.

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.

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.

ヘパリン結合性上皮成長因子(heparin-binding epidermal growth factor-like growth factor: HB-EGF)はEGF ファミリーの主要な増殖因子の一つである。これまでの研究で、HB-EGF は生体内で細胞の癌化、正常な心臓機能の発現、表皮の創傷治癒、目蓋の形成などに関与していることが報告されている。一方、HB-EGF は海馬や大脳皮質、小脳など中枢神経系で高い発現を示すことから、神経細胞のネットワーク回路の構築や高次脳機能への関与が示唆される。そこで、前脳選択的にHB-EGF 欠損させたマウスを作製し、中枢神経系の高次脳機能におけるHB-EGF の役割について検討した。前脳選択的HB-EGF 欠損マウスは自発運動量の増加や社会性行動の低下、プレパルスインヒビションの低下、記憶の低下などの諸種の行動変化を示した。また、HB-EGF KO マウスではドパミンやセロトニンなどの脳内モノアミン含量の変化や、大脳皮質第三層のスパイン密度の低下、海馬における長期増強(LTP)の低下などの機能的・器質的変化を示した。以上よりHB-EGF は、精神行動や記憶などの中枢神経系の高次脳機能の調節に重要な役割を果たしていることが示唆された。さらにHB-EGF シグナルの制御が複雑な神経疾患の病態機序解明、新規治療法並びに治療薬の開発の糸口になることが期待できる。, Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors.Previously, HB-EGF has been reported to be involved in diverse biological processes, including tumor formation, heart function,wound healing, and eyelid formation. On the other hand, HB-EGF is widely expressed in the central nervous system, including thehippocampus, cerebral cortex and cerebellum, and is considered to play pivotal roles in the development of the adult nervous systemand higher brain function. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain andinvestigated the roles of HB-EGF in higher brain function. These knockout mice showed behavioral abnormalities such as anincrease in locomotor activity, decreased social interaction, a deficit of prepulse inhibition, and memory impairment. HB-EGF KOmice also showed altered monoamine factors such as dopamine and serotonin, decreased spine density in neurons of the prefrontalcortex, and impaired long-term potentiation (LTP) in the hippocampus. These results suggest that HB-EGF exerts significantinfluence in higher brain functions, such as psychomotor behavior and memory formation and careful regulation of its activity will bean important goal for treating a number of neurological diseases of the central nervous system.

近年、細胞死の原因として、従来知られていたミトコンドリア障害やDNA障害によるものとは異なる、小胞体機能の破綻（小胞体ストレス）が関与することが明らかになってきた。小胞体ストレスは、アルツハイマー病などの神経変性疾患の神経細胞死メカニズムに関与していることが示唆され、アポトーシスシグナルの新たな発信地として注目を集めている。そこで、脳虚血後神経細胞障害の病態・機序を解明するための一環として、動物脳虚血モデルにおける小胞体ストレスの役割について検討した。砂ネズミ前脳虚血モデルにおける一過性脳虚血誘発海馬CA1野選択的神経細胞死およびマウス中大脳動脈閉塞モデルにおける永久閉塞誘発神経細胞死において小胞体ストレスの関与が認められた。そこで、新規脳卒中治療薬探索のため、小胞体ストレスにより誘導される分子シャペロンBiPの選択的誘導薬（BiP inducer X: BIX）の脳保護作用について検討した。BIXは、砂ネズミ前脳虚血誘発海馬CA1野選択的神経細胞死およびマウス中大脳動脈永久閉塞誘発神経細胞死を抑制した。以上より、脳虚血後神経細胞障害の機序に小胞体ストレス誘導性アポトーシスの関与が示唆され、BiP選択的誘導薬は神経細胞保護作用を有する新規な脳卒中治療薬の候補となる可能性が示唆された。, Recent studies have revealed that perturbation of endoplasmic reticulum (ER) functions, which is called ER stress, induces apoptosis. ER stress, which is caused by an accumulation of unfolded proteins in the ER lumen, is associated with neurodegenerative diseases such as Alzheimer disease. The purpose of this study was to provide new insights into the pathogenesis of brain ischemia for development of new therapeutic approaches to ischemic brain diseases. We suggest that ER stress is involved in the CA1-selective neuronal cell death and permanent middle cerebral artery occlusion (MCAO) induced cell death. Furthermore, to investigate a possible role of a selective inducer of BiP (BIX), we evaluated the neuroprotective effects of BIX against acute ischemic neuronal damage. BIX provided significant protection against CA1-selective neuronal cell death and permanent MCAO-induced cell death. In conclusion, ER stress plays an important causal role both in transient and permanent ischemic damage, and drugs which selectively induce BiP may exert a neuroprotective effect and may be a candidate of new therapeutic treatments of stroke.

本実験では,下顎切歯の歯間離開処置がマウスの情動行動に及ぼす影響を検討した.40匹のマウスを用い,対照群20匹と歯間離開処置を行った処置群20匹に分け,情動行動の評価として強制水泳法に基づくswimming behavior,climbing behavior,immobility behaviorを測定した.測定は,歯間離開処置後1日目,2日目,7日目に行った.その結果,処置群では対照群と比較してclimbing behaviorが1日目と2日目で有意に増加し,immobility behaviorが1日目と2日目で有意に減少していた.以上の結果から,歯間離開処置はマウスの情動機能に一時的な影響を及ぼす可能性が示唆される., In the present study, the effect of experimental separation of the lower incisors on emotional behavior was examined in mice at day 1, 2 and 7 after separation treatment. Emotional behaviors were swimming behavior, climbing behavior and immobility behavior based on the modified forced swim test. The experimental incisor separation increased climbing behavior and decreased immobility behavior on day 1 and 2 after treatment. It is reported that an incisor separation in the mouse increases the turnover of the central noradrenergic system. Thus, from these results, it is suggested that incisor separation in mice temporarily influences emotional functions.

血管新生（既存の血管から新しい血管ネットワークが形成される現象）は通常、厳密に制御されているが、一旦この制御機構が崩壊すると、調節不能な血管新生（病的血管新生）が発生する。糖尿病網膜症における不可逆的な視野欠損や失明の原因は網膜に生じた病的血管新生である。そこで、糖尿病網膜症の病態・機序を解明するための一環として、増殖糖尿病網膜症（PDR）における硝子体中細胞外スーパーオキシドジスムターゼ（EC-SOD）の役割について検討した。硝子体中EC-SODおよび血管新生の主要因子である血管内皮細胞増殖因子（VEGF）濃度は、黄斑円孔患者に比べPDR患者で高値を示し、全患者において両者間に強い正の相関関係が認められた。そこで、EC-SODの役割を探索するため、in vitro血管新生モデルを用いてEC-SODの抗血管新生作用を検討した。EC-SODはVEGF誘発ヒト臍帯静脈血管内皮細胞（HUVEC）およびヒト網膜毛細血管内皮細胞増殖、およびHUVEC管腔形成を抑制した。以上より、EC-SODはPDR患者の硝子体内で上昇し、in vitroにおいて抗血管新生作用を有したことから、血管新生の病態において中心的な役割を担う可能性が示唆された。, Angiogenesis, in which new vessels are formed from existing vessels, is usually regulated strictly. However, once the regulated mechanism is ruptured, dysregulated angiogenesis (pathological neovascularization) is generated. In diabetic retinopathy (DR), retinal pathological neovascularization is leading causes of irreversible failing vision and blindness. Therefore, as part to clarify the mechanism of pathogenesis in DR, we investigated the role of intravitreal EC-SOD in proliferative diabetic retinopathy (PDR). The intravitreal concentrations of EC-SOD and vascular endothelial growth factor (VEGF), which is a major factor in angiogenesis, were significantly higher in PDR patients than in macular hole patients, and showed a positive correlation with each other (in the whole patients). Furthermore, to investigate possible roles of EC-SOD, we evaluated the angiostatic effect of EC-SOD using an in vitro angiogenesis model. EC-SOD significantly suppressed VEGF-induced cell proliferation in human umbilical vein endothelial cells (HUVECs) and human retinal microvascular endothelial cells, and in vitro tube formation in HUVECs. In conclusion, EC-SOD was increased in the vitreous body from PDR patients and had the angiostatic effects in vitro. Therefore, these results suggest that EC-SOD may play a pivotal role in the pathogenesis of angiogenesis.

モルヒネをはじめとするいくつかのオピオイド鎮痛薬、合成したオピオイド様化合物の腫瘍細胞増殖抑制作用について調べた。腫瘍細胞として、A549(扁平上皮肺がん細胞)、N417(小細胞肺がん細胞)、MCF-7(乳がん細胞)およびKATO III(胃がん細胞)を試験に用いた。市販のオピオイド鎮痛薬ブプレノルフィンは、A549細胞に対してアポトーシス小体、DNAの断片化とDNAラダーの発現を伴うアポトーシス死を引き起こした。合成したオピオイド様化合物(100μM)について、A549細胞に対する細胞増殖抑制作用をalamar blue染色法により検討した。ブプレノルフィンと同様のエテノモルヒネ構造を有するPEO(8c)およびH_2-PEO (14c)は強い細胞増殖抑制効果(100%近い)を示した。一方、ピペリジン骨格のfentanylおよびmeperidineはむしろ細胞増殖を活性化した。A549細胞の生存活性に対する主な試験化合物のIC_値はそれぞれ、buprenorphine = 38.0μM, PEG (8c) = 73.8μMならびにloperamide = 3,7μMであった。, We examined the effects of opioid analgesics and synthetic opioid analogues on the proliferation of human cancer cells. Cell lines used were A549 (squamous epithelial cells of lung cancer), N417 (small cells of lung cancer), MCF-7 (breast cancer) and KATO III (stomach cancer). We found buprenorphine, a commercially available analgesic, induced apoptotic cell death on A549 cells in the appearance of the apoptotic body, nuclear fragmentation and DNA laddering. Cytostatic activity of synthetic opioid analogues was also evaluated at 100μM by alamar blue assay on the A549 cell line. PEO (8c) and H_2-PEO (14c) which had an ethenomorphine structure the same as buprenorhine, showed potent inhibition (by 100%) of the cell growth, however fentanyl and meperidine which had a piperidine molecular frame, enhanced the cell proliferation. The IC_ values of the compounds on the survival activity of A549 cells were 38.0μM (buprenorphine), 73.8μM (PEG, 8c), and 3.7μM (loperamide).

Calcium ion (Ca^) plays an essential role as a second messenger in secretion of water and electrolytes in salivary glands. Many studies using fluorescent Ca^ indicators such as quin-2 and fura-2 have shown that stimulation of muscarinic, α-adrenergic, or substance P receptors cause a rapid increase in [Ca^]_i in salivary gland cells resulting from the production of inositol 1,4,5-trisphosphate (IP_3). The [ra～]_i response is attributed to Ca^ release from intracellular Ca^ store followed by Ca^ entry across the plasma membrane. The increase in [Ca^]_i is initiated in the apical pole of acinar cells and then rapidly spreads as a Ca^ wave toward the basolateral region. The apical-to-basal pattern of Ca^ signaling is probably important to activate the ion transport systems in salivary glands. In this review, I discuss mechanisms and physiological significance of the polarized Ca^ signaling in salivary gland cells.

The present study clearly demonstrated the effects of complete cerebral ischemia and selective ischemia on brain choline metabolism in ddY mice The complete cerebral ischemia mouse was produced by cervical dislocation, and the selective ischemia mouse was produced by bilateral occlusion of the common carotid arteries. The following results were obtained 1 Complete cerebral ischemia increased brain choline contents rapidly and remarkably for 10 minutes After 10 minutes, the rate of increase of brain choline contents suddenly fell. 2 The increased level of choline contents by complete cerebral ischemia differed among the brain regions (cerebral cortex, hippocampus, medulla oblongata, cerebellum) tested 3 Selective ischemia increased choline contents in the cerebral cortex and hippocampus but not in the medulla oblongata or cerebella. 4. As with complete ischemia, hypoxia induced by N_2 gas inhalation increased choline contents in all brain regions. 5. Hypoglycemia induced by insulin administration did not increase brain choline contents However, the ischemia under a hypoglycemia state produced a greater choline increase than ischemia alone 6 Complete cerebral ischemia decreased the contents of both phosphatidylcholme (PtdCh) and glycerophosphocholine (GlyCh) in the mouse brain. The present results suggest that a main cause for the ischemic increase of brain choline contents is hypoxia by cessation of blood flow, and a low energy state caused by hypoglycemia partly contributes to this increase Furthermore, it is suggested that the ischemic increase of brain choline depends on the choline accumulation that results from decomposition of both PtdCh and GlyCh

The mechanisms for cross-desensitization between protease-activated receptor (PAR)-1 and PAR-2 in human parotid cell line, HSY-EA1 cells were examined. The HSY-EA1 cells strongly expressed mRNAs for PAR-1 and PAR-2, whereas the expression of PAR-3 and PAR-4 was very limited. Stimulation of HSY-EA1 cells with various concentrations of thrombin or trypsin produced a striking increase in [Ca^]_i followed by a slow return to the basal level within 2-3 min. When cells were pretreated with various concentrations of trypsin, the subsequent Ca^ response with thrombin diminished in a dose-dependent manner over the trypsin concentration, indicating that trypsin desensitized PAR-1. The desensitization of the thrombin-induced Ca^ response was also induced by the pretreatment with PAR-2 agonist peptide, SLIGKV. These results suggest that trypsin-induced inactivation of PAR-1 is due to the heterologous desensitization of PAR-1 through the activation of PAR-2 in addition to a proteolytic degradation of PAR-1.

Antigen I/II protein is a cell surface protein antigen of Streptococcus mutans and is implicated in the adhesion mechanism to tooth surfaces. We have identified the antigen I/II homologous gene (paaA) from Streptococcus cricetus, a member of mutans streptococci. To clarify the mechanism of paaA expression, we sequenced the flanking regions of the paaA gene utilizing the gene-walking method in this study. In the upstream region of paaA, three homologous genes found in the corresponding region of Streptococcus sobrinus were identified. One of the corresponding genes in S. sobrinus was par, which has been characterized as a transcription represser of the antigen I/II (spaA) gene. Therefore, the homologous gene in S. cricetus could have a role in transcription of paaA. In the downstream region of paaA, the paaA homologous gene was found and designated as paaB. The paaB gene was interrupted by a novel insertion sequence element (ISScr1), a member of the IS982 family. Southern hybridization analysis of S. cricetus par indicated that no gene homologous to S. cricetus par was found in the oral streptococci used in this study. Furthermore, the downstream region of antigen I/II (spaA) in S. sobrinus was sequenced using the gene-walking method. No similar gene to antigen I/II gene was found in the region. It was suggested that the paaA might be regulated by the par in S. cricetus.

脳神経系の形成過程を分子レベルで解明し,神経疾患の病態の理解,治療法の開発に生かすことは神経科学研究の大きな目標のひとつである.神経栄養因子は脳の形成過程で重要な役割を果たすと推定されているが,実際には培養系を用いた断片的な知見しか得られていないのが現状である.成熟脳にも神経幹細胞が見いだされ,その臨床応用が視野に入った現在,神経幹細胞から神経細胞へと至る道筋に神経栄養因子がどのような役割を果たすのかを解明することは大きな意義があると考えている.本研究では大脳皮質形成過程に着目し,特に生体系での実験を中心に脳の形成過程に及ぼす神経栄養因子の役割について検討した.本研究結果から水頭症をはじめとする先天性の脳形成異常疾患に神経栄養因子の発現異常が関与している可能性が示唆された.ならびに正常な大脳皮質の発達には秩序だった神経栄養因子の発現が密接に関わっていることも明らかにした., Elucidation of molecular mechanisms and cellular processes during development of the nervous system is one of the major targets of neuroscience, because of merit for understanding or treatment of neurological diseases. Neurotrophic factors are thought to play a vital role during the course of constructing the cerebral cortex, but their actual functions in vivo remain unknown except for patchy findings obtained from in vitro experiments. Recently, neural stem cells have been found even in the mature brain, and their activation or proper differentiation is now expected to be useful for the clinical treatment of neurological diseases with neuronal loss such as Parkinson's disease or Alzheimer's disease. This encouraged us to investigate the effects of neurotrophic factors on neural stem cells. Ln the present study, we focused on the effects of neurotrophic factors on the developing rodent cerebral cortex, especially using in vivo experimental techniques. The obtained results suggest that abnormal expression of particular neurotrophic factors is responsible for brain malformation including hydrocephalus, and that timely- and spatially-regulated expression of neurotrophic factors is important in normal development of the cerebral cortex.

レチノイドは細胞内の特異的レセプターと結合し,各種遺伝子の発現を調節することにより,細胞の分化,増殖および抗炎症作用など,種々の生理活性を示すことが多数報告されている。本研究では,これらの欠点を補うべく新しく合成されたレチノイド誘導体である,4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid(Am-80)の免疫薬理学的作用について検討し,次いでその作用機序についても合わせて検討した。Am-80は,マウスの2,4-dinitrofluorobenzene(DNFB)誘発接触性皮膚炎およびハプテン特異的1gE産生が強く見られるDNFB反復塗布によるマウス接触性皮膚炎において,耳介の腫脹を用量依存的に強く抑制した。また,Am-80はin vitroでの単核球からの抗原によるinterleukin-6(IL-6)ならびにinterferon-γ(IFN一γ)産生を抑制し,その抑制は転写レベルにおいて観察された。Am-80は,マウスのtypeIIコラーゲン誘発関節炎の発症を抑制し,C.Paruvum 前処置マウスのLipopolysaccharide(LPS)によるIL-6産生を抑制したが,ConcanavarinA(ConA)刺激したT細胞からのIFN-γおよびinteleukin-4(IL-4)産生には影響を及ぼさなかった。さらにAm-80は,ラットの実験的アレルギー性脳脊髄炎(EAE)の臨床症状および脊髄への細胞浸潤を用量依存的に抑制したが,投与中止によりEAEの再発が観察された。また,脊髄におけるIL-6mRNAの発現レベルにおいても同様に,投与中止後のIL-6mRNAの発現上昇が観察された。以上の結果から,Am-80は,リンパ球以外の細胞からのIL-6およびIFN-γの産生を選択的に抑制することにより,マウスおよびラットの接触性皮膚炎ならびに自己免疫疾患発症を抑制することが示唆された。これらの知見は,免疫性疾患における炎症性サイトカインの重要性ならびにレチノィド誘導体の治療薬としての有用性を示唆するものである。, Retinoids have been reported to elicit a variety of biological activities such as cell differentiation, cell growth and anti-inflammatory effect, through the binding with specific intracellular receptors. In this study, we examined immunopharmacological effects of 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) carbamoyl]benzoic acid (Am-80), a new retinoid-derivative, and its mechanism of actions. Am-80 inhibited ear swelling in a dose-dependent manner on 2,4-dinitrofluorobenzene (DNFB)-induced contact dermatitis and on contact dermatitis by the repeated applications of DNFB in mice. In addition, Am-80 inhibited production of inteleukin-6 (IL-6) and interferon-γ (IFN-γ) on mononuclear cells in vitro, and its inhibition was observed on a transcriptional level. Am-80 inhibited the elicitation of type II collagen-induced arthritis in mice. Am-80 inhibited lipopolysaccharide (LPS)-induced IL-6 production in C. Parvum-pretreated mice, but not the production of IFN-γ or IL-4 in T cells stimulated with concanavarin A (Con A) in vitro. Am-80 diminished clinical symptoms and infiltration of inflammatory cells into the spinal code on experimental allergic encephalomyelitis (EAE)-induced rats. However, after stopping administration, EAE recurred in DA rats treated with AM-80. Furthermore, the expressional level of IL-6 mRNA was diminished during the administration of Am-80,but provoked as soon as it was stopped. Therefore, Am-80 cured mice contact dermatitis, arthritis and rat EAE through the selective inhibition of IL-6 and IFN-γ secreted from inflammatory cells except for T cells. This evidence may suggest the importance of inflammatory cytokines and the usefulness of retinoid derivatives in immune disorders.

Ductal cells were purified by Percoll centrifugation after collagenase digestion of rat submandibular glands. After centrifugation, the cells were separated into two distinct fractions. A cell fraction obtained from the interphase of the centrifuge tube contained predominantly duct-like tubular structures, while the acini sedimented to the bottom. The ductal fraction was highly enriched in kallikrein, a ductal maker. To test the functional integrity of the prepared ductal cells, the effects of the membrane receptor agonists on intracellular free calcium concentration ([Ca^]i) were examined using the Ca^ fluorescent indicator fura-2. Stimulation with carbachol, phenylephrine or isoproterenol increased significantly [Ca^]i in the ductal cells. These results indicate that the Percoll centrifugation may be used to isolate ductal cellsafter digestion of submandibular glands.

各種無機塩類による実験う歯の発生に関する一連の研究として,6属元素のテルル塩投与による歯牙への残留性について主として検討した。テルルは,化学的にはセレニウムに似た6属元素である。金属光沢を有する銀白色のmetalloidで遊離して存在するものもあるが,sylvinite,black telluriumなどの鉱石に含まれるものが多い。製品としては,bismuth鉱石の残渣から抽出される。工業的には,鋳鉄,合金,化学工業の触媒,ゴム製品,ガラスの色彩,冷凍装置の熱電材料などに広く使用されている。テルルの代謝については,Gmelinが1824年に発表して以来,吸収,排泄,蓄積など多くの研究が報告されている。動物に対する急性中毒,慢性中毒,ヒトに対する中毒では殊に産業中毒などに多くの研究がある。テルルは皮膚からも吸収され,これに接したヒトは特有なgallic呼気を排泄するという。またテルルは梅毒,肺結核の盗汗予防剤としても用いられたこともあるが,これによる種々の有害な症状もまた報告されている。このようにテルルは,社会的に広く存在する元素であるが,歯科学的分野における研究は少なく,これが実験的う歯の発生を増加させることは,興味あるところである。また,これに対する解毒剤としてBAL,ascorbic acidがあげられている。テルル単独ならびにglutathioneとの併用効果については,松本らの報告が,また,口腔内レンサ球菌叢の変動に関しては,馬場らが報告している。今回の研究の目的は,テルルが実験的う歯発生を増加させることを確認し,亜テルル酸ナトリウムを添加した飼料を与えたラットの歯牙への貯留性についてPIXE(Proton Induced X-ray Emission)法にて検討した。, Rats were fed a diet containing 0.1% sodium tellurite and 0.123% sodium tellurate for 12 weeks. The growth of the rats was markedly inhibited by the tellurium. The incidence of experimental dental caries was accelerated by the tellurium. A PIXE analysis did not detect tellurium in the teeth but tellurium was detected in the liver, kidney and spleen. These results suggest that the acceleration of dental caries by tellurium may be due to tropic disturbances.

グラム陰性菌の細胞壁の構成成分であるエンドトキシンは,アジュバント活性,抗腫瘍活性やマクロファージ活性化など免疫機能を亢進ずる。また,エンドトキシンは腎機能や肝機能を低下させることも知られている。特に,エンドトキシンによって誘発される急性腎不全は,薬物の体内動態ならびに腎排泄挙動を変化させると考えられる。エンドトキシンを静注することにより作製したエンドトキシン血症モデルラットにおいて,糸球体濾過速度の低下に伴ってアミノ配糖体系抗生物質の腎排泄が低下したが,尿細管からの再吸収は増加した。一方,有機アニオン性薬物の尿細管分泌は低下したが,有機カチオン輸送系による尿細管分泌は変化しなかった。エンドトキシンの活性本体であるリピドA部分を投与したところ,エンドトキシン血症時と同様の腎機能低下と薬物排泄能の低下が認められ,リピドAが急性腎不全の発現に関与すると考えられた。また,エンドトキシンによる薬物腎排泄の低下は時間依存的に回復した。さらに,アデノシン拮抗薬であるテオフィリンを前投与した場合にはエンドトキシンによる腎機能の低下が防御されたことから,エンドトキシンによる急性腎不全発現にはアデノシンが関与していることが明らかとなった。以上の知見は,エンドトキシン血症時の薬物腎排泄挙動ならびにグラム陰性菌感染症患者の治療に対して有益な情報を与えるものである。, Endotoxin, a Gram-negative bacterial cell-wall component, has various biological and immunological activities, including adjuvant effects, antitumour activity and macropharge activation. Endotoxin induces nephrotoxicity and hepatotoxicity. Acute renal failure induced by endotoxin may alter the phamacokinetics and renal handling of drugs. Renal excretion of aminoglycoside antibiotics decreases with glomerular filtration rate in endotoxemic rats made by intravenous administration of endotoxin at 250 μg/kg, although tubular reabsorption increases. The tubular secretion of anionic drug decreases, but organic cation transport system does not change in endotoxemic rats. Lipid A, an active component of endotoxin, decreases renal function and renal excretion of drugs as seen in cndotoxemic rats, indicating that lipid A contributes to the action of endotoxin. Recovery from decrease in renal excretion of drug after endotoxin treatment occurs time-dependently. Protective effect of theophylline, adenosine antagonist, against endotoxin-induced reduction in renal function suggests adenosine to be involve in the induction of acute renal failure by endotoxin. These findings provide clarification of the renal excretion of drugs in endotoxemia as well as information for treating patients with Gram-negative bacterial infection.

Intracellular free chloride concentrations ([Cl^-]_i) in rat parotid acinar cells were determined with the Cl^- sensitive fluorescent indicator SPQ. The [Cl^-]_i was calculated from the SPQ fluorescence based on Englom's method. The resulting level of [Cl^-]_i of the rat parotid acinar cells was estimated to be 73.4±1.9mM (mean ± S.E., n=31). Stimulation with 10μM carba-chol, a muscarinic agonist, decreased the [Cl^-]_i to 50.1±3.1mM (n=12). The ED_ of carbachol necessary to change the [Cl^-]_i was 1.0μM. A similar decrease in [Cl^-]_i was elicited with 10nM of substance P or 10μM of phenylephrine, an α-adrenergic agonist. When the intracelular Ca^ stores were previously depleted, no agonist-induced decrease in [Cl^-]_i was observed, suggesting that the decrease in [Cl^-]_i is induced by the increase in cytosolic Ca^ concentration. No detectable change in [Cl^-]_i was observed when cells were stimulated with 1μM isoproterenol, a β-adrenergic agonist which induces the production of cyclic AMP. This suggests that the [Cl^-]_i in rat parotid acinar cells is not regulated by the cyclic AMP pathway.

喘息, アレルギー性鼻炎, アトピー性皮膚炎などのアトピー性疾患はCoombs and Gellの分類によるI型アレルギー反応によってひき起こされる。I型アレルギー反応は以下の3 stageに分類される。1st stageではマクロファージ, T cell, B cellなどの免疫担当細胞によって抗原に対するIgE抗体が産生され, IgE抗体はmast cellやbasophilに結合する。2nd stageは抗原抗体反応によるmast cellやbasophilの脱顆粒やchemical mediatorの遊離である。最終の3rd stageは遊離mediatorによるアレルギー症状の発現である。アトピー性疾患に対する薬物は1940年頃から3rd stageに作用するものが用いられるようになり, 現在では2nd stageに作用する薬物が効果的に用いられている。1st stageに作用する薬物もやがて用いられるようになるものと思われる。我々は各stageに作用するすぐれた薬物を見出すための研究を行ってきた。ここでは2nd stageに作用する薬物(baicaleinとtranilast)および1st stageに作用する薬物(alkyl glycosideとIPD-1151T)について述べた。これらのうちのいくつかはアトピー性疾患の治療に寄与している。, Atopic diseases such as asthma, rhinitis and atopic dermatitis are caused by Type I allergic reaction classified by Coombs and Gell. Type I allergic reaction is divided into 3 stages. The 1st stage is IgE antibody formation by immunocompetent cells including macrophage, T cell and B cell against antigens, followed by fixation of the IgE antibody on mast cells or basophils. The 2nd stage is degranulation and chemical mediator release from mast cells or basophils induced by antigen-antibody reaction. Finally the 3rd stage is the expression of allergic symptoms caused by the mediators released. The drugs for atopic diseases has been applied on the 3rd stage since about 1940,and nowadays those acting on the 2nd stage are effectively used. The drugs on the 1st stage will soon be available.The author has studied to seek excellent remedies for each stage. In the present paper, the drugs on the 2nd stage (baicalein and tranilast) and on the 1st stage (alkyl glycoside and IPD-1151T) are discussed, and some of which have contributed the therapy of atopic diseases.

The toxic activities of Ga～Sn alloy (Adlloy-OH) in experimental rats, its relations to the nutritional condition and dental caries development, were studied for three months. Adlloy, 0.3g or 3g per body weight (kg), was fed orally with the basal diet consisting of casein, sucrose, bean oil, mineral, and vitamin mixtures. Biochemical assays of serum was carried out for total protein, albumin, calcium, inorganic phosphate, glucose, urea, creatine, alkaline phosphatase, GOT, and GPT. There was no convicing evidence of toxic effects on growth and biochemical data by the oral feeding of Addloy-OH.