Chie Yasuoka

Antiapoptotic Activity of Akt Is Down-regulated by Ca2+ in Myocardiac H9c2 Cells EVIDENCE OF Ca2+-DEPENDENT REGULATION OF PROTEIN PHOSPHATASE 2Ac

Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2+]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. Akt was down-regulated when [Ca2+]i was elevated by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, but was up-regulated when it was suppressed by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), a cell permeable Ca2+ chelator. The inactivation of Akt was well correlated with the susceptibility to oxidant-induced apoptosis in H9c2 cells. To investigate the mechanism of the Ca2+-dependent regulation of Akt via the regulation of PP2A, we examined the transcriptional regulation of PP2Acα in H9c2 cells with Ca2+ modulators. Transcription of the PP2Acα gene was increased by thapsigargin but decreased by BAPTA-AM. The promoter activity was examined and the cAMP response element (CRE) was found responsible for the Ca2+-dependent regulation of PP2Acα. Furthermore, phosphorylation of CRE-binding protein increased with thapsigargin but decreased with BAPTA-AM. A long term change of [Ca2+]i regulates PP2Acα gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis.

Tyrosine phosphorylation of clathrin heavy chain under oxidative stress

In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.

Nε-(Carboxymethyl)lysine induces γ-glutamylcysteine synthetase in RAW264.7 cells

Advanced glycation end products (AGEs) play an important role in the development of angiopathy in diabetes mellitus and atherosclerosis. Here, we show that adducts of N(epsilon)-(carboxymethyl)lysine (CML), a major AGE, and bovine serum albumin (CML-BSA) stimulated gamma-glutamylcysteine synthetase (gamma-GCS), which is a key enzyme of glutathione (GSH) synthesis, in RAW264.7 mouse macrophage-like cells. CML-BSA stimulated the expression of gamma-GCS heavy subunit (h) time- and dose-dependently and concomitantly increased GSH levels. CML-BSA also stimulated DNA-binding activity of activator protein-1 (AP-1) within 3h, but the stimulatory effect decreased in 5h, and nuclear factor-kappaB (NF-kappaB) with a peak activity at 1h and the stimulatory effect diminished in 3h. Studies of luciferase activity of the gamma-GCSh promoter showed that deletion and mutagenesis of the AP-1-site abolished CML-BSA-induced up-regulation, while that of NF-kappaB-site did not affect CML-BSA-induced activity. CML-BSA also stimulated the activity of protein kinase C, Ras/Raf-1, and MEK/ERK1/2. Inhibition of ERK1/2 abolished CML-BSA-stimulated AP-1 DNA-binding activity and gamma-GCSh mRNA expression. Our results suggest that induction of gamma-GCS by CML adducts seems to increase the defense potential of cells against oxidative stress produced during glycation processes.

Sustained Cytomegalovirus-Specific CD4+ T Cell Response Associated with Prevention of Recurrence of Cytomegalovirus Retinitis without Secondary Prophylaxis after Highly Active Antiretroviral Therapy in Patients with AIDS

It has been demonstrated that the cytomegalovirus (CMV)-specific CD4(+) T cell response could be restored after ganciclovir and highly active antiretroviral therapy (HAART) in AIDS patients. In this study, we first confirmed the above observation cross-sectionally. We then performed a prospective longitudinal study over a period of 48 weeks. The second study included nine patients. All patients had received HAART. Five patients had a history of retinitis that was, however, under control after discontinuation of anti-CMV therapy more than 1 year before this study (group A). The other four had active CMV retinitis at the start of this study and anti-CMV therapy was required to control retinitis (group B). Median periods between commencement of HAART and the start of this study in group A and in group B were 27 and 4.5 months, respectively. Within both groups, the number of CD4(+) T cells that produced tumor necrosis factor alpha in response to CMV antigen did not vary throughout the observation period (Friedman test; p > 0.05). However, the median number of responsive CD4(+) T cells in group A patients was significantly higher than in group B (p < 0.05). Our results demonstrate that the number of CMV-responsive CD4(+) T cells increased when HIV was well controlled with HAART and was then maintained, and suggest that these cells may play an important role in the control of retinitis in patients with AIDS.

A Case of Radiofrequency Catheter Ablation of Ventricular Tachycardia Associated with an Old Myocardial Infarction Guided by a Noncontact Mapping System

We describe here a 72‐year‐old man with ventricular tachycardia (VT) associated with an old myocardial infarction, in whom noncontact mapping guided radiofrequency catheter ablation (RFCA) successfully eliminated the VT. Right ventricular pacing induced 3 VTs with different QRS morphologies and axes, 2 of which were hemodynamically unstable. A dynamic virtual activation map constructed during the VTs superimposed on a virtual voltage map constructed during sinus rhythm demonstrated that all VTs shared a single large myocardial scar in the inferolateral portion of the left ventricle which served as a slow conduction zone. All VTs were eliminated by RFCA at the exit or within the critical slow conduction zone. The patient has been free from any VT recurrences during a follow‐up period of 22 months.

Nonsustained VT in a Patient with Genetically Proven Andersen-Tawil Syndrome (LQT 7)

Andersen-Tawil syndrome, LQT 7, is a genetic disorder associated with mild QT prolongation and ventricular tachyarrhythmia (especially bidirectional VT) which is well known to be notoriously difficult to suppress by antiarrhythmic drugs. We here report a 41 year-old female patient with LQT 7, who carried a missense mutation of R 67 W in KCNJ2. She admitted to our hospital because of syncope. She had none of periodic paralysis, dysmorphic features and family history. Frequent PVCs and nonsustained VT (NSVT) have been pointed out since 13 years old although she was asymptomatic until her admission to our hospital. Twelve-lead ECG exhibited marked QT (QU) prolongation of 720 ms associated with frequent PVCs, which were easily escalated into NSVT by exercise and usually presented as a bidirectional form. A 24-hour Holter recording revealed more than 35,000 beats of PVCs/NSVTs. Combination of oral flecainide (200 mg, bid) and bisoprolol (1.25 mg, qd) remarkably reduced daily number of PVCs/NSVTs to less than 1,000 beats, and rendered exercise-induced bidirectional NSVT before the medication to infrequent PVCs after the medication. No syncope recurred during 6 month follow-up. In conclusion, combination of flecainide and bisoprolol suppressed bidirectional VT in an LQT 7 patient.