<資料>新西蘭,加奈陀,印度の中央銀行設立計畫

<p><b>Panels A–C,</b> representative images obtained from confocal microscopy of transiently transfected HEK cells. R835Q mutant channels do not appear differently distributed in comparison to WT KCNH2. <b>D</b>, Immunoblots using anti-erg1 (2, 5 µg/mL) of crude membrane extracts from heterologous expression in HEK cells, indicating equal protein expression level. Illustrated below are endoplasmic reticulum and plasma membrane fraction with respective markers of equal protein loading (calnexin for endoplasmic reticulum, spectrin for plasma membranes). Exemplary Western blots of preparations at physiological temperature (37°C) and 40°C (to simulate febrile illness of the index patient’s brother) are shown. No differences were observed in Kv11.1-WT or Kv11.1-R835Q plasma membrane representation of the two proteins under the two conditions. ER: endoplasmic reticulum fraction; PM: plasma-membrane fraction; WT: wild type; NT: non-transfected cells.</p

Comparison of in vivo and ex vivo micro-CT.

<p>Lumen area profile after partial ligation of the LCCA. (A, B) Lumen profile of the LCCA and RCCA was evaluated by in vivo (AuroVist Nanoparticles) and ex vivo (MicroFil) micro-CT at postoperative day 14 (n = 5). Lumen area was evaluated from the aortic arch (1) to the bifurcation (9). Error bars indicate the mean ± SEM. (C) Correlation between mean lumen area of LCCA and RCCA of each mouse examined by in vivo and ex vivo micro-CT. Linear regression parameters and correlation coefficients were calculated. (D) Volume rendering of the LCCA and RCCA examined by in vivo and post mortem micro-CT with corresponding visualization of the virtual elastic sphere path (grey). BA, brachiocephalic artery; LCCA, left common carotid artery; RCCA right common carotid artery; RSA, right subclavian artery branch; STA, superior thyroid artery.</p

Lumen profile evaluation by in vivo micro-CT and histology

<p>(A) Volume rendering of a micro-CT angiography of a ApoE^-/- mouse infused with AuroVist nanoparticles at postoperative day 14 after partial LCCA (left common carotid artery) ligation. A virtual elastic sphere (yellow) is fitted through the segmented LCCA lumen (left panel) from the aortic arch to the bifurcation. The LCCA was segmented into nine equidistant parts (right panel). (B) Side dependent vascular lumen profile of the LCCA from the aortic arch (1) till the bifurcation (9) Shown are the averages of all mice (n = 10) in orange and mouse M1, corresponding to (A), in blue. (C) H/E stainings of LCCA at postoperative day 14 after partial ligation of the LCCA. Lumen, tunica intima (red dashed line), tunica media, tunica externa, inner layer of tunica media (yellow dashed line) and plaque are clearly seen. (D) H/E stainings of LCCA (M1) at postoperative day 14 near the aortic arch (1), the bifurcation (9) and each 500 μm in between. (E) Histological evaluation of LCCA lumen (upper panel) and plaque area (lower panel) from the aortic arch (1) to the bifurcation (9) (n = 10). Squares show the mean ± SEM of all mice. Dashed blue line shows one individual mouse (M1). Scale bars in (C) and (D) represent 100 μm.</p

Experimental design.

<p>Partial ligation of the LCCA in ApoE^-/- mice at day 0. Timeline of high fat diet (HFD) treatment and overview showing analysis methods. Comparison of in vivo micro-CT and histology was performed by in vivo micro-CT scan and subsequent histological examination (d7, n = 4, d14, n = 10, d28, n = 9). Comparison of in vivo micro-CT and ex vivo micro-CT was performed by in vivo micro-CT scan and subsequent perfusion with the post mortem contrast agent MicroFil (n = 5); H/E, hematoxylin/eosin.</p

Comparison of in vivo micro-CT and histology vascular lumen profile.

<p>LCCA and RCCA lumen profiles evaluated by in vivo micro-CT and histology at postoperative days 7 (n = 4), 14 (n = 10) and 28 (n = 9) after LCCA ligation. (A) In vivo micro-CT evaluation of LCCA (left panel) and RCCA (middle panel) lumen area starting from the aortic arch (1) to the bifurcation (9). Average lumen areas evaluated by micro-CT (right panel). (B) Histological evaluation of LCCA (left panel) and RCCA (middle panel) lumen area profiles. Averages of carotid lumen areas evaluated by histology (right panel). (C) Comparison between mean lumen areas of LCCA and RCCA of each mouse evaluated by in vivo micro-CT and histology. Linear regression and correlation coefficient were calculated (C, left panel). Correlation of lumen areas of LCCA and RCCA of each mouse for each time point (C, middle panel). Bland-Altman plot depicting the degree of agreement between micro-CT and histology (C, right panel). Bars indicate the mean ± SEM. **, p<0.01; ***, p<0.001; LCCA left common carotid artery; RCCA right common carotid artery.</p

NS1619 attenuates PDGF-induced PASMC proliferation.

<p>(A) BK channel opening agent NS1619 (100 µmol/l) reduces PDGF-induced PASMC proliferation (n = 21 each column; * p<0.05). (B,C) In cultured PASMCs, phosphorylation state of protein kinases ERK1, ERK2 and AKT increased significantly 4 minutes after PDGF-BB stimulation (n = 3–6, *:p<0.05). Afterwards, phosphorylation states of ERK1 and ERK2 significantly increased within the period between 4 and 8 minutes after PDGF stimulation (n = 3–6; *:p<0.05) whereas AKT phosphorylation state rose by trend. BK channel opening by NS1619 (100 µmol/l) treatment does not affect phosphorylation of any kinase analysed.</p

Blood gas analyses.

<p>12 µM and 100 µM NS1619 improved oxygenation (PaO₂) considerably 120 minutes after inhalation and 100 µM and ∑NS1619 data exhibits a significant increase already 30 minutes after NS1619 inhalation. Carbon dioxide levels (PaCO₂) were attenuated 120 minutes after NS1619 inhalation. 12 µM and ∑NS1619 data demonstrate an elevated pH 120 minutes after inhalation. Base excess (BE) and bicarbonate concentration were not altered significantly by NS1619 although HCO₃⁻ was attenuated in NS1619 treated animals at least by trend. Data obtained after inhalation were subtracted from individual basis values and individual value differences (Δ) were calculated for each animal and time point. (Solvent: n = 8; 12 µM: n = 6, 100 µM: n = 6, ∑NS1619: n = 12; *: p<0.05).</p

Left ventricular work.

<p>Left ventricular work was estimated as the product of heart rate (HR) and systolic arterial pressure (bp sys). Relative cardiac output was estimated as the product of heart rate and relative stroke volume (integral of the arterial curve). dP/dt _max of the arterial pressure curve did not show a NS1619-dependent variation. Data obtained after inhalation were subtracted from individual basis values and individual value differences (Δ) were calculated for each animal and time point. (Solvent: n = 10; 12 µM: n = 7, 100 µM: n = 7; ∑NS1619: n = 14;*: p<0.05).</p

Basic values.

<p>The basic values for each of the parameter recorded did not differ significantly between the three groups (Solvent, 12 µM NS1619, 100 µM NS1619).</p

Systemic hemodynamic parameters.

<p>Heart rate, systolic (bp sys), mean (bp mean), diastolic (bp dia) arterial blood pressure were determined before (basis) as well as 30 and 120 minutes after inhalation of two different NS1619 concentrations, respectively solvent. Data obtained after inhalation were subtracted from individual basis values and individual value differences (Δ) were calculated for each animal and time point. None of the parameters investigated were affected by NS1619 inhalation in a significant manner. According to the character of the study as “a proof of principle”, pooled NS1619 (∑NS1619) data was compared with the control group (Solvent = Solv.). No NS1619-dependent effect on systemic hemodynamic parameters was discovered.</p