Application of fluorescence resonance energy transfer to examine EnvZ/OmpR interactions.
ABSTRACT The EnvZ/OmpR two-component regulatory system is best known for regulating the porin genes ompF and ompC in response to changes in the osmolarity of the growth medium. In response to an unknown signal, EnvZ is autophosphorylated by ATP on a histidine residue. The phosphoryl group is subsequently transferred to a conserved aspartate residue on OmpR. Phosphorylation of OmpR increases its affinity for the regulatory regions of the porin genes, altering their expression. Phosphorylation also alters the interaction with EnvZ and OmpR. In order to study the interactions of EnvZ and OmpR, we employed a full-length EnvZ construct fused to the green fluorescent protein (GFP) that was overexpressed and targeted to the inner membrane. Spheroplasts were prepared and lysed in microtiter plates containing purified, fluorescent-labeled OmpR protein. Fluorescence resonance energy transfer (FRET) from the GFP donor to fluorescein- or rhodamine-conjugated OmpR acceptor occurred, indicating that the two proteins interact. We then used FRET to further characterize the effect of phosphorylation on the interaction parameters. Results indicate that the full-length EnvZ behaves similarly to the isolated cytoplasmic domain EnvZc alone. Furthermore, the phospho-OmpR protein has a reduced affinity for the EnvZ kinase. This chapter describes general considerations regarding such experiments and provides detailed protocols for quantitatively measuring them.
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ABSTRACT: This study was undertaken to compare long-term results of various types of surgical repairs for either congenitally corrected transposition of the great arteries or double outlet right ventricle with discordant atrioventricular connections, and to analyze the risk factors that affect early and late mortality and reintervention. Between January 1972 and September 2005, a total of 189 patients (median age 8.3 years, range 2 months to 47 years old) with congenitally corrected transposition of the great arteries or double outlet right ventricle with discordant atrioventricular connections underwent definitive repairs. The definitive repairs comprised a conventional repair (atrial septal defect, or ventricular septal defect closure with or without pulmonary stenosis release, or isolated tricuspid valve surgery) in 36 patients (group I), conventional Rastelli in 31 patients (group II), double-switch operation (atrial switch plus arterial switch) in 15 patients (group III), atrial switch plus intraventricular rerouting (with or without extracardiac conduits) in 69 patients (group IV), and a Fontan-type repair in 38 patients (group V). The mean follow-up period was 10.1 years. Hospitalization and late mortality and reoperation were indicated as events. Risk factors for these events were analyzed by logistic regression for hospital death and a Cox proportional hazards model for late events. The Kaplan-Meier survival including hospital and late mortality was 62.4% at 32 years in group I, 78.5% at 27 years in group II, 74.5% at 15 years in group III, 80% at 16 years in group IV, and 79.3% at 22 years in group V. The reoperation-free ratio was 64.2% in group I, 76.6% in group II, 84.4% in group III, 89.6% in group IV, and 91.3% in group V. Risk analyses showed that the risk for hospital death was preoperative in patients with more than moderate tricuspid regurgitation and a cardiopulmonary bypass time of more than 240 minutes. A risk for late mortality was the presence of tricuspid regurgitation. Risks for reoperation were preoperative cardiomegaly, preoperative tricuspid regurgitation of more than grade II, ventricular septal defect enlargement, and body weight less than 10 kg. Risks for pacemaker implantation, as indicated by multivariate analysis, were ventricular septal defect enlargement during operation and age less than 3 years. There were no statistical differences between long-term survival rates of patients who underwent conventional surgical repair versus those of patients who underwent anatomic surgical repair. Results of conventional repair were satisfactory except in patients with significant tricuspid regurgitation. Results of anatomic repair were also satisfactory even for patients with significant tricuspid regurgitation, and therefore, anatomic repair should be the procedure of choice for those patients.The Journal of thoracic and cardiovascular surgery 06/2007; 133(5):1318-28, 1328.e1-4. · 3.41 Impact Factor
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ABSTRACT: In congenital heart defects where the morphologic right ventricle (RV) supports the systemic circulation, RV failure is common yet develops gradually. We hypothesized that patients who are asymptomatic may have unrecognized RV dysfunction. Conventional and Doppler tissue/strain echocardiography were performed on consecutive patients with asymptomatic systemic RV caused by congenitally corrected transposition of the great arteries (ccTGA) and on age-matched control subjects. RV index of myocardial performance was measured using conventional echocardiography. Longitudinal tissue velocities, strain rate, and strain of the basal RV free wall were measured using Doppler tissue/strain echocardiography and compared with nonsystemic RV of the control subjects. Mean age was 39.5 +/- 14.6 (n = 13) and 36 +/- 24 (n = 10) years for ccTGA and control groups, respectively. Mean RV index of myocardial performance was higher in patients with ccTGA than in control subjects (0.66 +/- 0.25 vs 0.28 +/- 0.12, P < .001). Mean RV tissue displacement (10.8 +/- 4.5 vs 20.3 +/- 3.9 mm, P < .0001), peak systolic strain rate (-1.16 +/- 0.3 vs -2.23 +/- 0.9 s(-1), P = .005), and peak systolic strain (-17.3 +/- 8.0 vs -30.6 +/- 11.0%, P = .008) were significantly lower in patients with ccTGA compared with control subjects, respectively. Patients with asymptomatic ccTGA have quantifiable RV dysfunction by echocardiography.Journal of the American Society of Echocardiography: official publication of the American Society of Echocardiography 08/2006; 19(8):1033-7. · 2.98 Impact Factor
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ABSTRACT: This study was undertaken to determine the outcomes of patients with congenitally corrected transposition of the great arteries after restoration of the morphologically left ventricle to the systemic circulation. Between November 1991 and June 2001, a total of 54 patients (median age 3.2 years, range 7 weeks-40 years) with either congenitally corrected transposition of the great arteries (n = 51) or atrioventricular discordance with double-outlet right ventricle (n = 3) underwent anatomic repair. This comprised a Senning procedure in all cases plus arterial switch (double-switch group) in 29 cases (53.7%), plus a Rastelli procedure (Rastelli-Senning group) in 22 cases (40.7%), and plus intraventricular rerouting (Senning-tunnel group) in 3 cases (5.6%). Left ventricular training by PA banding was performed before the double-switch operation in 9 of 29 cases (31%). Follow-up is complete (median 4.4 years). Early mortality was 5.6% (n = 3), with 2 late deaths. Kaplan-Meier survivals (+/- SEM) were 94.4% +/- 3.1% at 1 year and 89.7% +/- 4.4% at 9 years. Survivals at 7 years were 84.9% +/- 7.1% in the double-switch group and 95.5% +/- 4.4% in the Rastelli-Senning group (P =.32). Of the 49 survivors, 46 (94%) were in New York Heart Association functional class I. Six have acquired new left ventricular dilatation or impaired systolic ventricular function. Four patients in the double-switch group had moderate aortic valve regurgitation develop, and 2 of them required valve replacement. Overall freedoms from reoperation at 1 and 9 years were 94.2% +/- 3.3% and 77.5% +/- 9.0%, with no significant difference between the groups (P =.60). Anatomic repair of congenitally corrected transposition of the great arteries can be carried out with low early mortality. Excellent functional status can be achieved, with good midterm survival. Continued surveillance is necessary for patents with valved conduits and to determine the longer-term function of the aortic valve and the morphologically left ventricle in the systemic circulation.Journal of Thoracic and Cardiovascular Surgery 07/2003; 125(6):1229-41. · 3.53 Impact Factor