Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations

OCTN1 (SLC22A4) transports cationic compounds such as tetraethylammonium in a pH-sensitive and sodium-independent manner in cultured cells, and is expressed in wide variety of tissues, including kidney, muscle, placenta, heart, and others. This study focused on the clarification of its subcellular distribution in kidney and on its driving force to throw light on the pharmacological and physiological roles of OCTN1. Uptake of [14C]tetraethylammonium by membrane vesicles prepared from HEK293 cells stably transfected with human OCTN1 cDNA was osmolarity-sensitive, and the Km of tetraethylammonium was 1.28 mM at intravesicular and extravesicular pH values of 6.0 and 7.4, respectively. Tetraethylammonium uptake was pH-dependent, and overshoot uptake was observed in the presence of an outwardly directed proton gradient. A protonophore and membrane potential affected the overshoot uptake. Furthermore, preloading tetraethylammonium in the vesicles significantly increased the rate of uptake of [14C]tetraethylammonium. In mouse kidney, OCTN1 was expressed predominantly at the apical membrane of cortical proximal tubular epithelial cells. It was concluded that OCTN1 is involved in renal excretion of organic cations across the apical membrane in a pH-dependent, membrane potential-sensitive manner and is affected significantly by the organic cations on the trans side, showing counter transport activity.     

Screening of the Interaction Between Xenobiotic Transporters and PDZ Proteins

PURPOSE: Xenobiotic transporters have been proposed to be involved in membrane penetration of various therapeutic agents. As little information is available on molecular mechanism of their functional regulation, we have attempted to clarify the protein-protein interactions of such transporters as a first step to identify their regulators. METHODS: Yeast two-hybrid screening was performed to examine the interaction between carboxylic terminus of various xenobiotic transporters and PDZ (PSD95, D1g and ZO1) domain-containing proteins. The interaction and functional regulation were also evaluated in pull-down, immunoprecipitation and transport studies. RESULTS: Specific interaction with PDZ proteins was identified for several xenobiotic transporters including PEPT1, PEPT2, OCT3, OCTN1, OCTN2, OAT4, OATP-A, OATP-D, and OATP-F. The potent interaction was observed between PEPT2 and PDZK1, and deletion of the last four amino acids of the PEPT2 C-terminus almost completely abrogated such interaction. Recombinant PEPT2 C-terminus fusion protein can bind to purified His6-tagged PDZK1, confirming the involvement of two of four PDZ domains within PDZK1 in the interaction. Alanine-scanning mutation in PEPT2 revealed the presence of a consensus sequence (-T-X-L) that is responsible for the PDZK1 interaction. Transfection of PDZK1 increased the uptake of glycylsarcosine by PEPT2, whereas such stimulation was not observed for PEPT2 with the last four amino acids deleted. CONCLUSIONS: These results first identified the interaction between PDZ proteins and the cytosolic tail of various xenobiotic transporters. PDZK1 directly interacts with PEPT2, exerting functional regulation of its transporting activity. The current findings imply the localization of PEPT2 within a protein network constructed from PDZK1 and other transporter proteins.     

Ion channel gene expression in human lung,skin, and cord blood-derived mast cells

Immunoglobulin E (IgE)-dependent activation of human mast cells (HMC) is characterized by an influx of extracellular calcium (Ca(2+)), which is essential for subsequent release of preformed (granule-derived) mediators and newly generated autacoids and cytokines. In addition, flow of ions such as K(+) and Cl(-) is likely to play an important role in mast cell activation, proliferation, and chemotaxis through their effect on membrane potential and thus Ca(2+) influx. It is therefore important to identify these critical molecular effectors of HMC function. In this study, we have used high-density oligonucleotide probe arrays to characterize for the first time the profile of ion channel gene expression in human lung, skin, and cord blood-derived mast cells. These cells express mRNA for inwardly rectifying and Ca(2+)-activated K(+) channels, voltage-dependent Na(+) and Ca(2+) channels, purinergic P2X channels, transient receptor potential channels, and voltage-dependent and intracellular Cl(-) channels. IgE-dependent activation had little effect on ion channel expression, but distinct differences for some channels were observed between the different mast cell phenotypes, which may contribute to the mechanism of functional mast cell heterogeneity.     

Use of the Leeds-Keio prosthetic ligament for repair of patellar tendon rupture after total knee arthroplasty

We describe a technique for repair of the patellar tendon rupture after total knee arthroplasty (TKA), using the Leeds-Keio (L-K) prosthetic ligament. Two patients were followed for a mean of 38 months. Knee extension power and improved function were obtained in both cases. Use of the L-K ligament is a treatment option for repair of patellar tendon rupture following TKA.     

Detection of Japanese yam mosaic virus by RT-LAMP

Summary. A rapid and simple procedure is described to detect the genomic RNA molecule of Japanese yam mosaic potyvirus (JYMV). This method, named RT-LAMP, allows direct detection of RNA from infected plants without careful RNA extraction, rapid thermal cycling and gel electrophoresis. RT-LAMP was successfully applied to leaves, propagules and roots of Japanese yam infected with JYMV. One of the characteristics of the RT-LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophospate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of the amplification of JYMV genomic RNA without gel electrophoresis.Received January 20, 2003; accepted April 16, 2003 Published online July 2, 2003     

Prognostic value of nonlinear heart rate dynamics in hemodialysis patients with coronary artery disease

BACKGROUND: Although altered nonlinear heart rate dynamics predicts death in patients with coronary artery disease (CAD), its prognostic value in chronic hemodialysis patients with CAD is unknown. METHODS: We analyzed 24-hour electrocardiogram for nonlinear heart rate dynamics and heart rate variability in a retrospective cohort of 81 chronic hemodialysis patients with CAD. RESULTS: During a follow-up period of 31 +/- 20 months, 19 cardiac and 8 noncardiac deaths were observed. Cox hazards model, including diabetes, left ventricular ejection fraction, and the number of diseased coronary arteries, revealed that abnormal alpha2 (defined as both increase and decrease in alpha2 because of its J curve relationship with cardiac mortality), decreased approximate entropy and decreased heart rate variability (triangular index and ultra-low frequency power) were significant and independent predictors of cardiac death. No significant and independent predictive power for noncardiac death was observed in either the heart rate dynamics or the heart rate variability measures. The predictive power of alpha2 and approximate entropy was independent of that of triangular index and ultra-low frequency power. Combinations of two categories of measures improved the predictive accuracy; overall accuracy of approximate entropy + ultra-low frequency power for cardiac death was 87%. CONCLUSION: Altered nonlinear heart rate dynamics are independent predictors of cardiac death in chronic hemodialysis patients with CAD and their combinations with decreased heart rate variability provide clinically useful markers for risk stratification.     

Involvement of multiple transport systems in the disposition of an active metabolite of a prodrug-type new quinolone antibiotic, prulifloxacin

Prulifloxacin is a prodrug-type new quinolone. The purpose of this study is to clarify the mechanism of biliary excretion and brain distribution of its active metabolite, UFX. UFX was efficiently excreted into the bile in rats, with its concentration in the bile being 30-60 times higher than that in plasma. The in vivo disposition study revealed that multidrug resistance-associated protein 2 (MRP2) was involved in the biliary excretion of glucuronide metabolite, but not of the unchanged UFX. A transport study using a P-glycoprotein (P-gp) overexpressing cell line, LLC-GA5-COL150, showed that UFX was a substrate of P-gp. Nevertheless, the biliary clearance (CLbile) of UFX in P-gp-gene-deficient mice was not different from that in the normal mice, although the concentration in the liver was slightly higher than that in the normal mice. These observations suggest that multiple transport systems are involved in the biliary excretion of UFX, with minor contribution of P-gp. The distribution of UFX in the rat brain was quite low, and its tissue to plasma concentration ratio (Kp) in the brain was much less than the unity and was increased by cyclosporin A. The Kp in the brain of mdr1a/1b(-/-) mice was higher than that in the normal mice, suggesting that efflux by P-gp played a major role in the limited brain distribution of UFX.