Novel Protein Interactions of G Protein-Coupled Receptor Kinase 5 (GRK5) Searched with Yeast Two-Hybrid System

BACKGROUND AND OBJECTIVES: G protein-coupled receptors were considered to be the only natural substrates of G protein-coupled receptor kinases (GRKs). However, it was recently demonstrated that GRKs can also bind to other signal molecules. The purpose of this study was to investigate new molecules that might interact with the GRK5 using a yeast two-hybrid system to screen the cDNA library. MATERIALS AND MEDTHODS: For the yeast two-hybrid system, the "bait" was constructed to generate a LexA-GRK5 fusion protein in the EGY48 yeast strain. Rat library cDNA was inserted into the "prey". The first step in the library screening was performed by a galactose dependent leucine orthotrophism. For the second step screening, a beta-galactosidase dependent discoloration of colonies was used. Sequencing and searching of the gene bank was undertaken to characterize the clones. RESULTS: We screened a total of 1.3X10 6 clones from the cDNA library. On the first screening, 162 clones were identified by leucine orhotrophism. Another 54 clones were identified on the second screening by beta-galactosidase activation. Seven clones were selected by PCR and restriction patterns. Sequencing of seven molecules revealed that four of the clones were emerin fragments, with 2 of the remaining 3 clones being: an ID2 protein and a mitochondrial cytochrome c oxidase subunit II, with the last one remaining an unknown molecule. For the emerin fragments, their interactions with the GRK5 were confirmed by immunoprecipitation. CONCLUSION: We describe the novel protein-protein interactions of the GRK5, specifically, with three molecules. At first, these proteins may modulate the activation of the GRK5 through this specific protein-protein interaction desensitizing the beta-adrenergic receptors. Conversely, the localization of these molecules inside the cell indicates a potential new physiological role for the GRK5.

Clinical Characteristics of Paradoxical Response to Chemotherapy in Pulmonary Tuberculosis / 결핵

BACKGROUND: The paradoxical response refers to an enlargement of old lesions or unexpected new ones during apparently adequate antituberculous therapy. This response has been reported in cases of intracranial tuberculoma, tuberculous lymphadenopathy, tuberculous pleurisy and pulmonary tuberculosis. However, there are few reports on its frequency and clinical characteristics. METHOD: This study enrolled 205 patients who were treated with first line antituberculous agents for more than 6 months. We retrospectively studied 155 patients with pulmonary tuberculosis and 57 patients with pleural tuberculosis (7 patients had both) from July 1998 to March 2000. The patients were divided into the paradoxical response group and the non-paradoxical group. The clinical characteristics of the paradoxical group were investigated. Statistical analysis was done with an independent sample T-test and Chi-squared test. RESULT: 29 of the 205 patients(14.1%) had paradoxical response. Among the 29 patients, there were 19 pulmonary tuberculosis, 8 tuberculous pleurisy(2 patients had both). Paradoxical response appeared 32 days (mean 35 days in pulmonary tuberculosis, mean 25 days in tuberculous pleurisy) after the beginning of chemotherapy. The duration to regress less than half of initial chest lesion was 114 days in pulmonary tuberculosis and 124 days in tuberculous pleurisy, respectively. Most common clinical manifestation of paradoxical response patients was coughing in both pulmonary tuberculosis and tuberculous pleurisy. Male sex, high blood WBC count and high level of pleural fluid LDH were related with paradoxical response. CONCLUSION: These findings suggest that presponse usually appears 1 month and disappears within 4 months after the beginning of anti-tuberculous chemotherapy. Paradoxical response was relatively correlated with male sex, high blood WBC count and high level of pleural fluid LDH.

Influence of the Angiotensin II AT1 Receptor Antagonist on Reperfusion Injury in Rat Myocardial Ischemia Model

BACKGROUND AND OBJECTIVES: The protective effect of angiotensin converting enzyme (ACE) inhibitor against ischemia/reperfusion injury has been demonstrated in animal models, however the effect of AT1 receptor antagonist is contradictory. The present study was designed to investigate the myocardial protective effects of the AT1 receptor antagonist irbesartan during myocardial ischemia/reperfusion in vivo. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to a 45-minute left coronary artery ligation followed by a 2-hour re-perfusion. An inert vehicle (group I:n=14) or irbesartan (50 mg/kg/day:group II, n=12) was administered for 3 days before coronary occlusion. The ratio of the myocardial infarct area to the ischemic area at risk was assessed through triphenyltetrazolium chloride staining. Apoptosis was evaluated by analyzing DNA fragmentation and TdT-mediated dUDP nick end labeling staining. Western blot analysis was performed for MAP Kinases (ERK1/2 and p38) and Bcl-2 and Bax. RESULTS: The ratio of the infarct area to the ischemic area at risk of group II was smaller than that of group I (42.6+/-2.7% vs. 64.1+/-4.6%;p<0.005). Agarose gel electrophoresis revealed discrete DNA laddering in the ischemic zone of group I, however DNA ladder formation was attenuated in group II. The expressions of ERK1 MAPK and Bcl-2 were increased in the ischemic area of group II compared to that of group I. CONCLUSION: AT1 receptor antagonist was effective in reducing myocardial reperfusion injury in vivo. This effect can at least be partially attributed to the attenuation of cardiomyocyte apoptosis, and this anti-apoptotic effect appears to be related to the increased expression of Bcl-2 and alterations in MAP kinase signaling.