Thermostability study of SCF^1–141 and SCF^1–165 by molecular modeling simulation.

<p>(A) Potential energy of SCF^1–141 and SCF^1–165. The root mean square deviation (RMSD) of SCF^1–141 and SCF^1–165 at (B) 25°C; (C) 70°C; and (D) 90°C. (E) Snapshots of the evolving SCF^1–141 structure at different time points at the indicated temperatures.</p

Temperature-dependent changes in circular dichroism spectrum from 190 to 250 nm of (A) rhSCF^1–141 and (B) rhSCF^1–165.

<p>Conditions: 300 µg/mL in 100 mM phosphate buffer (pH 7.0); 1, 25°C as control; 2, 70°C for 10 min; 3, 90°C for 15 min; 4, 90°C for 30 min; and 5, 90°C for 60 min.</p

The expression and purification of rhSCF^1–141 and rhSCF^1–165.

<p>(A) PCR analysis of pPICZαC-rhSCF^1–141 (1011 bp) and pPICZαC/SCF^1–165 (1083 bp) using <i>5′AOX I</i> and <i>3′AOX I</i> primers (arrow). (B) Coomassie blue-stained SDS-PAGE of rhSCF^1–141 and rhSCF^1–165overexpressed from <i>Pichia</i> culture supernatants. Lane M: molecular weight markers. Numbers above the lanes represent hours after methanol induction. Arrows indicate the overexpressed rhSCF^1–141 or rhSCF^1–165 (C) Silver-stained 15% SDS–PAGE of purified rhSCF^1–141 and rhSCF^1–165 produced in <i>P. pastoris</i>. Lane M, molecular weight markers. Lanes 1 and 2, purified rhSCF^1–141 (200 and 100 ng, respectively); Lanes 3 and 4, purified rhSCF^1–165 (20 and 50 ng, respectively). (D) Western blot analysis of rhSCF^1–141-6His and rhSCF^1–165-6His from culture supernatants. The recombinant proteins were 6xHis tagged and detected with anti-His antibodies.</p

The effect of temperature and time on the viability of TF-1 cells treated with rhSCF¹⁴¹ or rhSCF¹⁶⁵.

<p>(A) The effect of temperature on the viability of TF-1 cells treated with rhSCF^1–141 or rhSCF^1–165. TF-1 cells were incubated with 100 ng/mL rhSCF^1–141(solid circle) or rhSCF^1–165 (hollow triangle) in PBS (pH 7.4) for 10 minutes and the cell viability assayed. (B) Thermostability of rhSCF^1–141 and rhSCF^1–165 over time as indicated by cell viability. TF-1 cells were incubated with 100 ng/mL rhSCF^1–141(solid circle) or rhSCF^1–165 (hollow triangle) in PBS (pH 7.4) at 90°C for the times indicated. Results are expressed as mean ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001</p

Effects of rhSCF^1–141 and rhSCF^1–165 on downstream signaling targets MAPK and Akt.

<p>Cells were incubated with rhSCF^1–141 or rhSCF^1–165 in PBS (pH 7.4) at the concentrations indicated. A and B are Western blots using the indicated antibodies. Similar results were obtained in 3 independent experiments. C is the quantitation results of the Western blots shown in A and B.</p

SDS-PAGE analysis of limited proteolytic digestion of rhSCF^1–141 and rhSCF^1–165.

<p>Purified (A) rhSCF^1–141 or (B) rhSCF^1–165 (15 µg each in 0.1 M-Tris/HCl buffer, pH 8.2) was incubated with trypsin at 25°C or 90°C for 10 minutes (lanes 2 and 3), or preheated at 90°C 10 minutes (lanes 4 and 5), 90 minutes (lanes 6 and 7), or 120 minutes (lanes 8 and 9) and then treated with trypsin for 10 minutes at 25°C or 90°C as indicated above. Lanes 1 and 10 are rhSCF and trypsin only.</p

Uremic Retention Solute Indoxyl Sulfate Level Is Associated with Prolonged QTc Interval in Early CKD Patients

<div><p>Total mortality and sudden cardiac death is highly prevalent in patients with chronic kidney disease (CKD). In CKD patients, the protein-bound uremic retention solute indoxyl sulfate (IS) is independently associated with cardiovascular disease. However, the underlying mechanisms of this association have yet to be elucidated. The relationship between IS and cardiac electrocardiographic parameters was investigated in a prospective observational study among early CKD patients. IS arrhythmogenic effect was evaluated by in vitro cardiomyocyte electrophysiological study and mathematical computer simulation. In a cohort of 100 early CKD patients, patients with corrected QT (QTc) prolongation had higher IS levels. Furthermore, serum IS level was independently associated with prolonged QTc interval. In vitro, the delay rectifier potassium current (IK) was found to be significantly decreased after the treatment of IS in a dose-dependent manner. The modulation of IS to the IK was through the regulation of the major potassium ion channel protein Kv 2.1 phosphorylation. In a computer simulation, the decrease of IK by IS could prolong the action potential duration (APD) and induce early afterdepolarization, which is known to be a trigger mechanism of lethal ventricular arrhythmias. In conclusion, serum IS level is independently associated with the prolonged QTc interval in early CKD patients. IS down-regulated <i>I_K</i> channel protein phosphorylation and the <i>I_K</i> current activity that in turn increased the cardiomyocyte APD and QTc interval in vitro and in the computer ORd model. These findings suggest that IS may play a role in the development of arrhythmogenesis in CKD patients.</p></div

Ventricular cardiomyocyte action potential (AP) and pseudo-ECG constructed by the O'Hara-Rudy dynamic human ventricular model.

<p>The suppression of inward rectifier potassium current (<i>I</i>_{<b><i>K</i></b>}) mimics the effect of indoxyl sulfate toxicity to ventricular cardiomyocyte AP. The AP duration was gradually increased and the QT interval was also prolonged with the increment of <i>I</i>_{<b><i>K</i></b>} suppression. The early afterdepolarization was noted in the higher suppression of <i>I</i>_{<b><i>K</i></b>} especially in the mid-myocardial cardiomyocyte (arrow). The ventricular arrhythmias like ECG was also noted when the <i>I</i>_{<b><i>K</i></b>} was severely suppressed (empty arrow).</p

Multiple logistic regression analysis with presence of prolonged QTc interval as the dependent variable.

<p>BMI, body mass index; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.</p><p>Multiple logistic regression analysis with presence of prolonged QTc interval as the dependent variable.</p

Clinical and biochemical variables associated in univariate analysis with log indoxyl sulfate.

<p>HDL: high-density lipoprotein, LDL: low-density lipoprotein.</p><p>Clinical and biochemical variables associated in univariate analysis with log indoxyl sulfate.</p