Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing.

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.

[Effects of repetitive myocardial ischemia on collateral circulation, ST deviation and epicardial wall motion].

The effects of repetitive myocardial ischemia on collateral circulation, ST deviation and epicardial wall motion were examined in 12 patients undergoing percutaneous transluminal coronary angioplasty (PTCA) for single left anterior descending artery disease. Rentrop collateral filling grade was assessed using contrast injection to the contralateral artery during the first and final episodes of coronary occlusion. ST deviation was measured by intracoronary electrocardiography. Epicardial wall motion (%pre PTCA) was measured by guide wire motion analysis according to the centerline method. Collateral filling grade was assessed 30 sec after balloon inflation. The ST segment and the epicardial wall motion were measured 60 sec after balloon inflation and deflation, respectively. There was no change in the collateral filling grade between the first and final episode of coronary occlusion. Patients with collateral filling grade I (R-I group, n = 4), II (R-II group, n = 3) and III (R-III group, n = 5) during coronary occlusion showed mean ST segment shifts of 13.2, 9.4, and 0.9 mm, respectively, and mean epicardial wall motion of 41.4%, 67.2%, and 78.5%, respectively. The collateral filling grade correlated with ST deviation and epicardial wall motion, and there was a significant correlation between epicardial wall motion and ST deviation (r = -0.67). Comparison of the R-I group or severe ischemia (n = 4) and the R-III group or slight ischemia (n = 4) during coronary occlusion for the fourth time showed the effect of preconditioning was obtained in R-I group. More R-III group patients than R-I group had hyperkinetic epicardial wall motion during coronary reperfusion. Stunned myocardium was demonstrated in both R-I group and R-III group patients. Epicardial wall motion was poorer in the R-I group than R-III group. We concluded the following: There is no change in the grade of collaterals during repetitive coronary occlusion, and there is a relationship between the grade of collateral and degree of myocardial protection; there is good correlation between ST segment and epicardial wall motion; ischemic preconditioning is obtained during repetitive severe myocardial ischemia; recovery from brief episodes of slight myocardial ischemia is associated with hyperkinesia of epicardial wall motion; the reduction of stunned myocardium is related to the degree of premyocardial ischemia; preconditioning is sufficient to cause myocardial stunning, but myocardial stunning is insufficient to cause preconditioning.

Percutaneous dye image cardioscopy for detection of endocardial lesions.

Endocardial lesions are caused not only by inflammatory processes but also by myocardial ischemia, resulting in endocardial thrombosis and cerebral embolism. We deviced a method for direct visualization of endocardial damages by a novel dye image cardioscopy with Evans blue and examined its feasibility in patients with heart disease. The dye was injected into the left ventricle before and after endomyocardial biopsy. Endocardial surface was stained in dark blue in 63% of patients with angina pectoris before biopsy. After biopsy, the biopsied portions were stained in blue in all. The results indicate that endocardium is damaged even in apparently intact LV in patients with ischemic heart disease and that endomyocardial biopsy causes severe endocardial damages.