<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

In silico analysis of R835Q mutation in human ventricular AP model (ten Tusscher model).

<p>Simulated human ventricular model of (<b>A</b>) WT and (<b>B</b>) R835Q action potentials (APs) at cycle lengths (CL) of 1000 and 350 ms. The R835Q mutation has a greater impact on M-cell AP morphology than on that of epicardial or endocardial cells. At CL 1000 ms, M-cell APD₉₀ is prolonged from 412 ms in WT to 428 ms in R835Q mutants, while epi/endo APD₉₀ values are nominally prolonged from 308/306 ms to 316/314 ms, respectively. At CL 350 ms, epi/endo APD₉₀ values increase from 232/236 ms in WT to 240/242 ms in mutant; M-cell APD₉₀ is stable at 278 ms in WT, but displays APD alternans in R835Q-mutant APs with APD₉₀ values of 330/236 ms.</p

Representative I_KCNH2 recordings (protocol in inset A) obtained from CHO cells transiently transfected with KCNH2-WT and KCNH2-R835Q cDNA (A–B).

<p>Currents recorded from cells with Kv11.1-R835Q (n = 13 cells) or Kv11.1-WT with R835Q (n = 9) were similar in size to the tail currents from cells transfected with Kv11.1-WT (n = 13, C). D, mean±SEM deactivating tail current density recorded upon repolarization to −50 mV. Ordinate and abscissa scale bars represent 10 pA/pF and 1 sec, respectively, for all current examples. WT: wild type; n.s.: not significant; TP: test potential.</p

Dynamic restitution and failure to capture at fast rates in R835Q-mutant AP model.

<p>(<b>A</b>) Dynamic restitution curves for WT and R835Q show that APD₉₀ is moderately increased in R835Q as compared to WT. APD prolongation and restitution steepening are most pronounced in ventricular M-cells. (<b>B</b>) Stimulation at 350 ms induces APD alternans in mutant M-cell but not WT APs. As stimulation CL decreases from 350 to 340 ms, APD₉₀ is stable in WT M-cells; however, in R835Q-mutant M-cells subsequent stimulation encroaches on the refractory period of the previous long AP, failing to capture.</p

Pedigree and ECGs of the index patient and his deceased brother.

<p><b>A,</b> arrows indicate genotype-positive family members; filled black square represents the index patient. Grey symbols represent ungenotyped family members. <b>B,</b> DNA sequence chromatograms indicating homozygous KCNH2 mutation in the index patient. The mutation is located on the C-terminus of the KCNH2 gene, causing a guanine to alanine change (c.G2504A, p.R835Q) at a conserved location. <b>C,</b> ECG of the index patient and his brother at initial presentation. Paper speed and leads of ECGs are indicated. The index patient (IV 5) had a QTc of ∼506 ms with notched T waves. <b>D,</b> illustrates patient characteristics. Yrs.: years; SCD: sudden cardiac death.</p

Family trees for the three Cases.

<p>The arrow indicates the proband; “+” indicates the presence of the mutation; na = DNA not available. For Case III no relatives were recruited at the time of this report.</p

Immunoblot detection of CDC73.

<p>Total protein cell lysates from cell transfected with WT and mutants (R77P, delVV, delENIP) vectors were detected with Anti-Flag antibody showing that the mutant proteins are poorly expressed with respect to the WT: the same assay performed in presence of the proteasome inhibitor, MG132 (25 μM, final concentration), led to the partial recovery of the mutant proteins.</p

qRT-PCR expression of Flag parafibromin WT and mutant mRNAs.

<p>The assay, performed in presence and absence of cycloheximide (CHX 500 μM, final concentration) shows that the inhibition of the degradation process led to the total recovery of the mutant mRNAs.</p

MTT test.

<p>Proliferation assay at different time points (24, 48 and 72 h). The WT vector limited the cell growth, while all the three mutant vectors caused cell overgrowth in presence of the endogenous parafibromin, suggesting a dominant interfering effect.</p

CHX Chase assay.

<p>The western blot performed in presence of MG132, after 48 h from transfection showed that the mutated proteins were degraded (a) up to the 80% with respect to the WT, with short half-lives around the 2,5 h (b).</p