ミトコンドリア ADP/ATP トウカ タンタイ ニオケル シンスイセイ ループ コウゾウ ト キノウテキ ヤクワリ

ミトコンドリア内膜に存在するADP/ATP透過担体(AAC)は、酸化的リン酸化の基質である ADPとその産物であるATPの交換輸送を行う酸化的リン酸化反応に必須の膜タンパク質である。 AACは種間を通じて約300のアミノ酸残基からなり、類似性の高い約100アミノ酸残基単位が3 回繰り返した配列をしている。各繰り返し配列は、2カ所の疎水性領域とそれを連結する約40残基 程度の長い親水性領域からなる。この親水性領域はミトコンドリア内腔(マトリックス)側に存在 するループを形成している。AACのトポロジーは、N，C両末端が細胞質側に突出した6回膜貫通 構造であることが明らかとなっている。またAACは、基質結合部位を細胞質側に向けたc-stateと マトリックス側に向けたm-stateの2種類の立体構造をとり、これらの立体構造間の転移によって 透過機能が発現すると考えられている。ウシ心筋AAC(bhAAC)の3つマトリックス側の親水性ル ープ(M1，M2，M3)にそれぞれ存在するシステイン残基(Cys56，cys，159，Cys256)のSH試薬に対する 反応性の違いから、各ループの立体配置がそれぞれ異なっていること、及びAACの機能発現に伴 った構造転移に際してこれらループは立体配置を変化させることが見いだされている。しかし、こ れら親水性ループと基質輸送との関連はいまだ不明である。そこで、本研究ではAACの基質透過 機構の解明を目的として、特に親水性ループの構造と機能的役割について検討した。 1.化学架橋試薬を用いたADP/ATP透過担体のループの空間的配置の解析 SS架橋試薬銅オルトフェナンスロリン(Cu(OP)₂)をウシ心筋ミトコンドリアの反転膜小胞 (SMP)に作用させSDS-PAGEに供したところ、30kDaのAACバンドの減少に伴って、約60kDa のバンドの出現し、このバンド強度はCu(OP)₂作用濃度に依存して増大した。アミノ酸配列分析の 結果、この60kDaのタンパク質はループM1中のCys56同士の分子間架橋により生じたbhAACの2 量体であることが明らかとなった。従ってAACは、膜中で2つのAAC分子が対称に対向した12 回膜貫通型の2量体として機能することが明らかとなった。次に、架橋距離が7.7～16.8Åと異な る架橋試薬である6種のジマレイミド誘導体をSMPに作用させたところ、全ての架橋試薬が Cu(OP)₂同様にCys56同士の分子間架橋を特異的に形成した.この結果から、Cys56を含むループM1 のみがマトリックス側に露出し、他のループは膜内に貫入していることが明らかとなった。更に、 この架橋反応を解析した結果、架橋距離が12Åのジマレイミドが最もCys56間の架橋を形成し易い ことを見いだした。また、全ての架橋試薬によるCys56間の架橋は、m-stateの状態で形成されたの に対し、c-stateの状態では全く形成されなかった。以上の結果から、ループM1はAACがm-state ではマトリックス側に露出し、Cys56間の距離が12Åの状態を中心とする広範囲な距離で揺動して いるのに対し、c-stateでは一転して膜中に貫入していると解釈することができる。即ち、基質輸送 に伴ってAACのループは立体配置を大幅に変化させることになる。また、架橋形成に伴いAACの 透過活性が阻害されたことから、ループM1の立体配置変化がAACの透過機能に必須であると考 えられ、ループM1は構造転移に伴ったこの‘開閉'的な立体配置変化により、基質輸送に際し、 マトリックス側のゲートとしての役目を果たしていることが示唆された。 2. bhAACの酵母細胞における機能的発現系の構築 bhAACは、現在までのAAC研究のほとんどで標品とされてきた。そのbhAACを遺伝子工学的 手法により機能解析するための実験系を確立することを目的として、固有のAAC遺伝子(yAAC1， yAAC2)が破壊された酵母細胞WB-12を宿主として、bhAACの機能的発現系の構築を試みた。し かし、bhAACは酵母細胞のミトコンドリアにほとんど発現しなかった.そこで、bhAAC と酵母 AACのアミノ酸配列の比較を行い、推定2次構造上で細胞側に突出しているN末端領域のアミノ 酸残基数及び配列が、bhAACと酵母AACで全く異なることに着目し、bhAACのN末端領域11残 基をyAAC2(26残基)またはyAAC1(16残基)の相当領域と置換したキメラbhAACのWB-12細胞 での発現を試みた。その結果、キメラbhAAC は顕著にミトコンドリアに発現した。更に、これら キメラbhAACがnativeなbhAACと同等の透過機能を有していた。以上の結果から、bhAACを機 能を保持したキメラ体として酵母細胞にて機能的に発現させることに成功した。 3.Cys56の機能的役割とループM1の立体配置変化に関与するアミノ酸残基の同定 膜透過性のSH試薬であるN-エチルマレイミド(NEM)が、Cys56を標識することでbhAACの透 過活性が阻害されることが知られている。そこで、Cys56の機能的役割を検討する目的でNEMの AACの透過機能への影響を検討した。その結果、NEMはCys56を修飾することで、マトリックス 側からの基質結合を阻害することなく、m-stateからc-stateへの構造転移を阻害した。従って、基質 結合後の構造転移に際したループM1の立体配置変化が、AACの透過機能に必須であることが明ら かとなった。更に、キメラbhAAC及びyAAC2のCys56に相当するシステイン残基を部位特異的 変異解析した結果、置換したアミノ酸残基の側鎖体積の増大に依存して、変異AACの基質透 過活性は低下した。従って、Cys56はAACの透過機能に必須のアミノ酸残基ではなく、NEMに よる活性阻害は、AACの構造転移の際Cys56周辺で生じる何らかの残基間相互作用に対して、立 体障害となっていると考えられた。そこで、Cys56のN末端側に隣接するアスパラギン酸残基が、 種間で保存されていることに着目し、この残基の部位特異的変異体を調製し、その基質透過活性を 検討したところ、アスパラギンやアラニンに置換することで、透過活性は完全に消失した。この結 果から、アスパラギン酸残基のカルポキシル基がAACの構造転移の際に他のアミノ酸残基と塩橋 を形成するなどの相互作用を行っていると思われる。 4. ループM2における基質認識機構の解明 過去の報告から、bhAACにおいて基質結合部位であることが示唆されているループM2領域の3 次元構造とATPとの相互作用の様式を分子力学計算及び分子動力学計算を用いたCGシュミレーシ ョンにより解析したところ、Arg151とAsp167が塩橋を形成し、これがATPのポリリン酸エステルを 静電的に吸引することと、この塩橋により形成されるループ内ループ中の疎水性残基のクラスター (Phe153，Ile160，Ile163，Phe164)に結合後のアデニン環が疎水的相互作用により侵入することが見いださ されている。そこでこのシュミレーションをもとに上記残基の部位特異的変異解析を行ったところ、 Phe164を除く上記の残基が透過機能に必須であることが明らかとなった。以上の結果は、このルー プM2内ループ(Arg151～Asp167)領域がbhAACの主たる基質結合部位であることを強く示唆した。 以上の研究成果より、AACの基質輸送には、マトリックス側の巨大な親水性ループが深く関与 していることが明らかとなった。また、これらはそれぞれが異なる構造と機能的役割を有しており、 これらループの協調的な構造転移がAACの基質輸送の本体であることが強く示唆された。このこ とは、一般的な輸送担体による物質輸送にも応用できると考えられる

Selective expression of KCNS3 potassium channel α-subunit in parvalbumin-containing GABA neurons in the human prefrontal cortex

The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV) or somatostatin (SST). Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K+ channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K+ channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with 35S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia. © 2012 Georgiev et al

Calcifying aponeurotic fibroma of the elbow : a case report

Calcifying aponeurotic fibroma is a rare soft tissue tumor that typically occurs in the distal extremities of children and adolescents. Because a calcifying aponeurotic fibroma is an ill-defined mass and has a tendency to infiltrate the surrounding tissue, local recurrence rates are 50%. We report a case of calcifying aponeurotic fibroma occurring in the elbow region, an uncommon site. The patient is followed up for 6 years without local recurrence of the tumor

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate “revertants” viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria

H1821+643: The most X-ray and infrared luminous AGN in the Swift/BAT survey in the process of rapid stellar and supermassive black hole mass assembly

H1821+643 is the most X-ray luminous non-beamed AGN of $L_\mathrm{14-150 keV}= 5.2\times 10^{45}$ erg s$^{-1}$ in the Swift/BAT ultra-hard X-ray survey and it is also a hyper-luminous infrared (IR) galaxy $L_\mathrm{IR} = 10^{13.2} L_\odot$ residing in the center of a massive galaxy cluster, which is a unique environment achieving the rapid mass assembly of black holes (BH) and host galaxies in the local universe. We decompose the X-ray to IR spectral energy distribution (SED) into the AGN and starburst component using the SED fitting tool CIGALE-2022.0 and show that H1821+643 consumes a large amount of cold gas ($\dot{M}_\mathrm{con}$) with star-formation rate of $\log ( \mathrm{SFR}/M_{\odot}~\mathrm{yr}^{-1}) = 3.01 \pm 0.04$ and BH accretion rate of $\log (\dot{M}_\mathrm{BH}/M_{\odot}~\mathrm{yr}^{-1}) = 1.20 \pm 0.05$. This high $\dot{M}_\mathrm{con}$ is larger than the cooling rate ($\dot{M}_\mathrm{cool}$) of the intra-cluster medium (ICM), $\dot{M}_\mathrm{con}/\dot{M}_\mathrm{cool} \gtrsim 1$, which is one to two order magnitude higher than the typical value of other systems, indicating that H1821 provides the unique and extreme environment of rapid gas consumption. We also show that H1821+643 has an efficient cooling path achieving from $10^7$ K to $10^2$ K thanks to [OIII] 63 $\mu \mathrm{m}$, which is a main coolant in low temperature range ($10^4$ K to $10^2$ K) with a cooling rate of $\dot{M}_{\mathrm{cool}}=3.2\times 10^5\ M_{\odot}\mathrm{~yr^{-1}}$, and the star-forming region extends over 40 kpc scale.Comment: 20 pages, 8 figures, accepted for publication in Ap

Current status of clinical background of patients with atrial fibrillation in a community-based survey: The Fushimi AF Registry

AbstractBackgroundAtrial fibrillation (AF) increases the risks of stroke and death, and the prevalence of AF is increasing significantly. Until recently, warfarin was the only oral anticoagulant for stroke prevention, but novel anticoagulants are now under development.Methods and resultsThe Fushimi AF Registry is a community-based survey of AF patients. We aimed to enroll all of the AF patients in Fushimi-ku, which is located at the southern end of the city of Kyoto. Fushimi-ku is densely populated with a total population of 283,000, and is assumed to represent a typical urban community in Japan. On the basis of the general prevalence of AF in the Japanese (0.6%), we estimated the total number of AF patients as 1700. A total of 76 institutions, a large proportion of which were private clinics, participated in the study. At present, we have enrolled 3183 patients from March 2011 to June 2012 (approximately 1.12% of total population). The mean age was 74.2±11.0 years, and 59.3% of subjects were male. The mean body weight was 58.5±13.2kg, and the proportions with a body weight of less than 50kg and 60kg were 25.7% and 55.0%, respectively. The type of AF was paroxysmal in 46.0%, persistent in 7.3%, and permanent in 46.7%. Major co-existing diseases were hypertension (60.6%), heart failure (27.9%), diabetes (23.2%), stroke (19.4%), coronary artery disease (15.0%), myocardial infarction (6.4%), dyslipidemia (42.4%), and chronic kidney disease (26.4%). The mean CHADS2 score was 2.09±1.35: 0 in 11.8% of patients, 1 in 27.1%, and 2 in 29.1%. Warfarin was prescribed in only 48.5% of patients, whereas anti-platelet drugs, mainly aspirin, were prescribed for more than 30% of the patients.ConclusionsThe Fushimi AF Registry provides a unique snapshot of current AF management in an urban community in Japan

Risk factors for sternal wound infection

Background Although the utility of flaps for the treatment of sternal wound infections following median sternotomy has been reported for 30 years, there have been few reports on the risk factors for complications after reconstruction. The objective of this investigation was to identify factors related to complications after the reconstruction of sternal wound infections. Methods A retrospective analysis of 74 patients with reconstructive surgery after sternal wound infection over a 5-year period was performed. Clinical data including age, sex, body mass index (BMI), comorbidities, bacterial culture, previous cardiac surgery, wound depth, mortality rate, type of reconstructive procedure, and complication rate were collected. Results The patients' BMI ranged from 15.2 to 33.6 kg/m2 (mean, 23.1±3.74 kg/m2). Wound closure complications after reconstructive surgery were observed in 36.5% of the cases. The mortality rate was 2.7%. Diabetes mellitus significantly affected the rate of wound closure complications (P=0.041). A significant difference in the number of complications was seen between Staphylococcus aureus (S. aureus) and coagulase-negative Staphylococci (P=0.011). There was a correlation between harvesting of the internal thoracic artery and postoperative complications (P=0.048). The complication rates of the pectoralis major flap, rectus abdominis flap, omentum flap, a combination of pectoralis major flap and rectus abdominis flap, and direct closure were 23.3%, 33.3%, 100%, 37.5%, and 35.7%, respectively. Conclusions Diabetes mellitus, S. aureus, harvesting of the internal thoracic artery, and omentum flap were significant factors for complications after reconstruction. The omentum flap volume may be related to the complications associated with the omentum flap transfer in the present study

Magnetotelluric and temperature monitoring after the 2011 sub-Plinian eruptions of Shinmoe-dake volcano

Three sub-Plinian eruptions took place on 26–27 January 2011 at Shinmoe-dake volcano in the Kirishima volcanic group, Japan. During this event, GPS and tiltmeters detected syn-eruptive ground subsidence approximately 7 km to the WNW of the volcano. Starting in March 2011, we conducted broad-band magnetotelluric (MT) measurements at a site located 5 km NNW of the volcano, beneath which the Shinmoe-dake magma plumbing system may exist. In addition, temperature monitoring of fumaroles and hot-springs near the MT site was initiated in July 2011. Our MT data record changes in apparent resistivity of approximately ±5%, along with a ±1◦ phase change in the off-diagonal component of the impedance tensor (Zxy and Zyx ). Using 1-D inversion, we infer that these slight changes in resistivity took place at relatively shallow depths of only a few hundred meters, at the transition between a near-surface resistive layer and an underlying conductive layer. Resistivity changes observed since March 2012 are correlated with the observed temperature increases around the MT monitoring site. These observations suggest the existence beneath the MT site of pathways which enable volatile escape

Influence of Thermodynamic Effect on Blade Load in a Cavitating Inducer

Distribution of the blade load is one of the design parameters for a cavitating inducer. For experimental investigation of the thermodynamic effect on the blade load, we conducted experiments in both cold water and liquid nitrogen. The thermodynamic effect on cavitation notably appears in this cryogenic fluid although it can be disregarded in cold water. In these experiments, the pressure rise along the blade tip was measured. In water, the pressure increased almost linearly from the leading edge to the trailing edge at higher cavitation number. After that, with a decrease of cavitation number, pressure rise occurred only near the trailing edge. On the other hand, in liquid nitrogen, the pressure distribution was similar to that in water at a higher cavitation number, even if the cavitation number as a cavitation parameter decreased. Because the cavitation growth is suppressed by the thermodynamic effect, the distribution of the blade load does not change even at lower cavitation number. By contrast, the pressure distribution in liquid nitrogen has the same tendency as that in water if the cavity length at the blade tip is taken as a cavitation indication. From these results, it was found that the shift of the blade load to the trailing edge depended on the increase of cavity length, and that the distribution of blade load was indicated only by the cavity length independent of the thermodynamic effect