α1-酸性糖タンパク質の甲状腺ホルモンによる誘導機構の解明

金沢大学自然科学研究科α1-酸性糖タンパク質(AGP)遺伝子の甲状腺ホルモンT3やグルココルチコイドによる転写活性化に伴い、遺伝子5′上流のDNA領域にクロマチンの構造変化が起き、それはDNasel感受性の変化として検出される。転写開始点に近い二つの高感受性部位HS1,HS2についてその近辺のDNA領域がどのように転写活性化に関わっているかをラット肝臓とラット肝臓由来の培養細胞を用いて調べた。1)HS2近辺のDNA領域に結合するタンパク質のフットプリントを行った。3カ所のサイトが検出され、各々のサイトには複数のタンパク質が結合するようである。現在各タンパク質の精製を行っている。2)T3による誘導にはHS1やHS2を含む領域が必要であったが、グルココルチコイドによる誘導にも必要であることがわかった。HS1とHS2の効果はアディティブで両方あると最大の効果を示した。3)HS2断片に結合するタンパク質を調べたところ、グルココルチコイド処理後、結合活性の大きな上昇がみられ、このタンパク質の結合活性上昇とクロマチンの構造変化には関連があると思われる。4)培養細胞RLN‐10において、グルココルチコイドによるAGPの誘導をT3やレチノイン酸が押さえることが示された。この抑制機構についても検討したところ、抑制にはTRE、HS1やHS2DNA領域は関与していないことがわかった。この抑制機構はT3やレチノイン酸の今までに知られていない新しい作用機構を示していると思われ、現在詳細を解析中である。研究課題/領域番号:06672178, 研究期間(年度):1994出典：研究課題「α1-酸性糖タンパク質の甲状腺ホルモンによる誘導機構の解明」課題番号06672178（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-06672178/）を加工して作

新しいトランスジェニック魚の開発

金沢大学医薬保健研究域医学系我々は組織あるいは細胞特異的に、かつ必要な時にだけ遺伝子のノックアウトができるようなノックアウト魚の開発を目指した。ノックアウトはその遺伝子のアンチセンス鎖を発現させることにより行うことにし、最初テラピアを用いたトランスジェニック魚の開発を計画した。しかしテラピアはなわばりを持つ性質があり、集団飼育がきわめて困難で、いろいろ工夫したが、よほど大きな水槽がないと飼育が難しいことが分かった。使いにくい魚であることが分かったので、我々はキンギョやゼブラフィッシュを用いた系や、アンチセンスオリゴヌクレオチドを用いたノックアウト法を開発することにした。キンギョ程度の大きさが有れば網膜なども簡単に単離でき、いろいろな実験に使用するには向いている。しかし、卵を年一回しか産まない。実験的には熱帯魚のように一年中産卵可能な魚が望ましい。キンギョの飼育温度を変えるなどして年中産卵できないかと試したが、あまりうまくいかなかった。アンチセンスオリゴヌクレオチドをキンギョ網膜に注入し特定のタンパク質の生成を押さえるのはうまくいったので、キンギョ網膜で特定の遺伝子の発現を押さえるにはアンチセンスオリゴヌクレオチドを使用するのが現実的であると思われる。トランスジェニックフィッシュに関しては結局ゼブラフィッシュを用いることとし、ノックアウトフィッシュ作成に必要なゼブラフィッシュの遺伝子のクローニングを行っている。ゼブラフィッシュゲノミックライブラリーからスクリーニングし現在塩基配列を解析し、これら遺伝子の発現制御領域を調べているところである。今後はこの発現制御領域に誘導剤(エクダイソン)リセプターの結合塩基配列を組み込み、発現ヴェクターとして使用する予定である。研究課題/領域番号:12878134, 研究期間(年度):2001 – 2002出典：「新しいトランスジェニック魚の開発」研究成果報告書　課題番号12878134（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （ https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-12878134/ ）を加工して作

急性期たん白質の甲状腺ホルモンによる誘導の効果

金沢大学薬学部本年度において我々は以下のことを明らかにした。1)グルココルチコイドによりAGPを発現するラット肝臓由来の培養細胞を数株得た。グルココルチコイドによる遺伝子発現の機構について検討を行っている。2)甲状腺ホルモンリセプター(TR)の結合サイトを決定した。TRは第一イントロン中のパリンドローム様構造に結合し、我々の予想と一致した。3)AGP遺伝子の発現に関与していると思われる5\u27上流領域のDNaseI高感受性部位(HS)1および2について、各々の領域をサブクローニングした。サブクローニングしたDNA断片について、ゲルシフトアッセイにより結合するタンパク質有無を調べたところ、各々の断片に特異的に結合するタンパク質が存在した。4)上記断片のうちHS2を更に2つの断片に分け(HS2a,HS2b)各々について解析したところ、HS2a、b断片共に結合タンパク質が存在した。5)HS2a、HS2b各断片とタンパク質の結合は互いに阻害し合うことから同じものであると思われた。6)HS2に結合するタンパク質の部分精製を行った。サウスウェスタン法により分子量を求めたところ、HS2a,b共に分子量120k,77k,55kのタンパク質が検出された。このことから各断片には同一のタンパク質が結合していることが判った。7)HS2断片を用いて、上記タンパク質の結合サイトを決定したところ、2カ所のサイトが検出された。しかし、その2カ所で塩基配列は異なっていた。We found someresults as follows ;1) Some cultured cell lines, which were established from rat liver and expressed AGP by glucocorticoid stimulation, were obtained. We are now investigating the expression mechanism by glucocorticoid.2) The palindromic region in the 1st intron of rat AGP gene was thyroid hormone responsive element and postulated thyroid hormone receptorTTR) binding region. It was determined that TR bound this region.3) Two DNaseI hypersensitive sites(HS-1 and 2) were found in 5\u27franking region of rat AGP gene. These regions were postulated to play an important role for gene expression induced by T3. These regions were subcloned. Binding protein(s) to these regions was studied by gel shift analysi, and the protein which specifically bind to these regions was observed.4) The HS-2 fragments, one of two DNA fragments above, was further subcloned into two fragments, HS2a, and HS2b. There found specific binding protein(s) toboth of HS2a and HS2b.5) The same protein(s) could bind to HS2a and HS2b, we found.6) The protein(s) was partially purified and characterized by South-Western method. The MWs of proteins that bound to these fragments were 120k, 77k, and 55k, for both fragments. It was further confirmed that the same protein(s) bound to two different fragments.The binding sites were determined by Methylation-interference Assay, and was different in the two fragments.研究課題/領域番号:04671347, 研究期間(年度):1992 – 1993出典：研究課題「急性期たん白質の甲状腺ホルモンによる誘導の効果」課題番号04671347（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-04671347/046713471993kenkyu_seika_hokoku_gaiyo/）を加工して作

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

金沢大学医薬保健研究域　医学系Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury. © 2007 Elsevier Ltd. All rights reserved

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

金沢大学医薬保健研究域　医学系Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish (Carassius auratus). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5-40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5-40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo. None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning. © 2009 International Society for Neurochemistry.出版社許諾要件により、2010年9月より全文公開

Neuritogenic activity of a genipin derivative in retinal ganglion cells is mediated by retinoic acid receptor β expression through nitric oxide/S-nitrosylation signaling

Genipin, a herbal iridoid, is known to have both neuroprotective and neuritogenic activity in neuronal cell lines. As it is structurally similar to tetrahydrobiopterin, its activity is believed to be nitric oxide (NO)-dependent. We previously proposed a novel neuroprotective activity of a genipin derivative, (1R)-isoPropyloxygenipin (IPRG001), whereby it reduces oxidative stress in RGC-5, a neuronal precursor cell line of retinal origin through protein S-nitrosylation. In the present study, we investigated another neuritogenic property of IPRG001 in RGC-5 cells and retinal explant culture where in we focused on the NO-cGMP-dependent and protein S-nitrosylation pathways. IPRG001 stimulated neurite outgrowth in RGC-5 cells and retinal explant culture through NO-dependent signaling, but not NO-dependent cGMP signaling. Neurite outgrowth with IPRG001 requires retinoic acid receptor β (RARβ) expression, which is suppressed by an RAR blocking agent and siRNA inhibition. Thereby, we hypothesized that RARβ expression is mediated by protein S-nitrosylation. S-nitrosylation of histone deacetylase 2 is a key mechanism in chromatin remodeling leading to transcriptional gene activation. We found a parallelism between S-nitrosylation of histone diacetylase 2 and the induction of RARβ expression with IPRG001 treatment. The both neuroprotective and neuritogenic activities of genipin could be a new target for the regeneration of retinal ganglion cells after glaucomatous conditions. © 2011 International Society for Neurochemistry

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG R) mRNA increased in RGCs 1-6 weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1 h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40 days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10 days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons. © 2012 Elsevier Ltd. All rights reserved

A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness

Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362