Differential modulation of late sodium current by protein kinase A in R1623Q mutant of LQT3

AimsIn the type 3 long QT syndrome (LQT3), shortening of the QT interval by overdrive pacing is used to prevent life-threatening arrhythmias. However, it is unclear whether accelerated heart rate induced by β-adrenergic agents produces similar effects on the late sodium current (I_Na) to those by overdrive pacing therapy. We analyzed the β-adrenergic-like effects of protein kinase A and fluoride on I_Na in R1623Q mutant channels.Main methodscDNA encoding either wild-type (WT) or R1623Q mutant of hNa_v1.5 was stably transfected into HEK293 cells. I_Na was recorded using a whole-cell patch-clamp technique at 23 °C.Key findingsIn R1623Q channels, 2 mM pCPT-AMP and 120 mM fluoride significantly delayed macroscopic current decay and increased relative amplitude of the late I_Na in a time-dependent manner. Modulations of peak I_Na gating kinetics (activation, inactivation, recovery from inactivation) by fluoride were similar in WT and R1623Q channels. The effects of fluoride were almost completely abolished by concomitant dialysis with a protein kinase inhibitor. We also compared the effect of pacing with that of β-adrenergic stimulation by analyzing the frequency-dependence of the late I_Na. Fluoride augmented frequency-dependent reduction of the late I_Na, which was due to preferential delay of recovery of late I_Na. However, the increase in late I_Na by fluoride at steady-state was more potent than the frequency-dependent reduction of late I_Na.SignificanceDifferent basic mechanisms participate in the QT interval shortening by pacing and β-adrenergic stimulation in the LQT3.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Chondrosarcoma of the urinary bladder and establishment of a human chondrosarcoma cell line (OCUU-6)

Chondrosarcoma is a very rare tumor of the urinary bladder, with only 4 cases reported to date. In this study, we report on a case of a 73-year-old male who presented bladder mass and right hydroureteronephrosis. Radical cystectomy, right nephrectomy and left ureterocutaneoustomy were performed, and histological study disclosed chondrosarcoma of the urinary bladder. As reported in other cases, the tumor was highly aggressive with a short clinical course, and the patient died of carcinomatous pleuritis at one month after surgery. Subsequently, we successfully established a human chondrosarcoma cell line (OCUU-6) from the pleural effusion of the patient.     

Gicerin,a cell adhesion molecule,promotes the metastasis of lymphoma cells of the chicken

Gicerin is an immunoglobulin superfamily cell adhesion molecule purified from chicken gizzards. This molecule displays an adhesive interaction with a laminin-like protein as well as with gicerin itself. Gicerin appears in embryonic tissues and plays a role in chick development through its cell adhesive properties. An increase in gicerin expression is found in some sporadic tumors of the chicken. To elucidate the possible role of gicerin in tumor progression in chickens, we introduced gicerin cDNA into an endogenous gicerin negative lymphoma MDCC-MSB1 cell line, and subsequently analyzed them for changes in their metastatic potentials. After intravenous implantation of the gicerin transfectants into chickens, the metastatic potential to the lung, liver and kidney was enhanced compared with parental MDCC-MSB1 cells. Self-aggregation activity was increased in gicerin transfectants. In addition, adhesive and migratory activities of the gicerin transfectants to the gicerin ligands were enhanced in vitro. These findings indicate that gicerin can contribute to the malignancy and metastatic properties of lymphoma.This work was supported in part by a Grant-in-Aid for Scientific Research (No. 13760210), and a grant for Scientific Research on Priority Areas "Cancer" (No. 12215133), from the Ministry of Education, Science, Sports and Culture, Japan, grants from the Uehara Memorial Foundation and Senri Life Science and a Grant-in-Aid for Advanced Scientific Research from Osaka Prefecture University     

Glycoprotein Ib distribution on the surface of platelets in resting and activation states: An electron microscope study

Summary Using a monoclonal antibody (TM60) against glycoprotein (GP) Ib, we determined immunocytochemically how GPIb is distributed on the platelet surface. When glutaraldehyde-fixed platelets were incubated with TM60, a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm. The number of ferritin particles on the surface of one side were 2070–4150 (2940 ± 790; mean ±s.d.,n = 10) under the scanning electron microscope. The distribution of ferritin particles was somewhat disarranged on the surface of unfixed platelets incubated with TM60 compared to that in the fixed platelets. Cluster-like structures of ferritin particles were observed in several places. When platelets were activated with ristocetin or thrombin, the distribution of ferritin particles was disturbed and cluster formation was observed in several places on the surface. These findings suggest that GPIb is uniformly distributed on the surface of platelets in the resting state, and that cluster formation occurs during activation of platelets.     

Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca²⁺ imaging of cells loaded with the Ca²⁺ indicator Fluo-4 enabled us to examine intracellular Ca²⁺ handling properties, and we found a significantly higher incidence of irregular Ca²⁺ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca²⁺ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na^+/Ca²⁺exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca²⁺ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (I_NCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of I_NCX.     

Supersusceptibility to hydrophobic antimicrobial agents and cell surface hydrophobicity in Branhamella catarrhalis

To clarify the cause of the supersusceptibility of Branhamella catarrhalis to macrolide antibiotics, which are well-known to be inactive to most Gram-negative bacteria, we determined its cell surface hydrophobicity by the partition experiment between water and hydrocarbons. Its cell surface was found to be markedly more hydrophobic than that of Escherichia coli or Pseudomonas aeruginosa cells. This suggested that the outer membrane of B. catarrhalis plays no role as a diffusion barrier towards hydrophobic agents.