Mazzanti, M, Warton, K, Tonini, R, Lorenzi, G, Fairlie, WD, Matthews, J, Valenzuela, S, Qiu, M, Wu, W, Pankhurst, S, Bauskin, AR, Campbell, TJ, Curmi, PM & Breit, SN 2002, 'Ncc27 (clic1) Interacts With Artifical Bylayer In A Ph Dependent Manner To Form Chloride Ion Channels', Biophysical Journal, vol. 82, no. 1, pp. 1-1.
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Singarayar, S, Singleton, C, Tie, H, Wyse, K, Bursill, J, Bauskin, A, Wu, W, Valenzuela, S, Breit, S & Campbell, T 2002, 'Effects of Components of Ischemia on the Kv4.3 Current Stably Expressed in Chinese Hamster Ovary Cells', Journal of Molecular and Cellular Cardiology, vol. 34, no. 2, pp. 197-207.
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We investigated the effects of three components of ischemia: external acidosis (pH=6.0), extracellular hyperkalemia ([K+]=20 mmol/l), and resting membrane depolarization to -60 mV, on Kv4.3 current stably expressed in Chinese Hamster Ovary cells. We used single electrode whole cell patch clamp techniques to study changes in the current elicited. External acidosis caused a positive shift in the steady state activation curve from -13.4±2.1 mV to -3.3±1.5 mV (n=8, P=0.004) and the steady state inactivation curve from -56.5±0.4 mV to -46.7±0.5 mV (n=14, P<0.0001). Acidosis also caused an acceleration of recovery from inactivation with the t1/2 decreasing from 306 ms (95% CI 287327 ms) to 194 ms (95% CI 182207 ms), (n=14, P<0.05). Hyperkalemia did not affect any of these parameters. Combined acidosis and hyperkalemia produced effects similar to those seen with acidosis. Changing the holding potential from -90 mV to -60 mV with test potentials of +5 and +85 mV decreased the peak currents by 34.1% and 32.4% respectively (n=14). However, in the presence of external acidosis the decrease in peak currents induced by changing the holding potential was less marked. In acidotic bath the peak current at -60 mV was reduced by only 13.6% at a test potential of +5 mV and 12.3% at a test potential of +85 mV (n=14). Taken together our data suggest that the membrane depolarization and changes in pH which occur under ischemic conditions would be accompanied by relative preservation of Kv4.3 currents and provide a molecular basis for the observation of preserved epicardial Ito and epicardial action potential duration (APD) shortening in ischemia.
Wang, D, Jin, D, Wu, Z, Zou, W, Xu, D, Zheng, Z & Liu, X 2002, '[Therapeutic effects of human interleukin 10 gene transfer on severe acute pancreatitis in rats, an experimental study].', Zhonghua Yi Xue Za Zhi, vol. 82, no. 12, pp. 844-847.
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OBJECTIVE: To study the therapeutic effects of human interleukin 10 (IL-10) gene transfer on severe acute pancreatitis (SAP) in rats. METHODS: Twenty healthy SD rats were injected intraperitoneally with SA liposome, SA liposome/pcDNA3 or SA liposome/pcDNA3-IL-10. Another twenty SD rats were randomly divided into five groups: rats in one group underwent laparotomy only (normal control), and SAP was induced in the other 4 groups induced by homogeneous injection of sodium taurocholate beneath the pancreatic capsule. Among the 4 SAP groups, one group did not receive any drugs, and liposomes, pcDNA3 or pcDNA3-IL-10 complexed with cationic liposomes were administered to the other groups. Drugs were administered by a single intraperitoneal injection thirty minutes after SAP had been induced. The levels of IL-10 in pancreas, liver and lungs were determined by ELISA kits. The level of serum amylase, histology, and tissue tumor necrosis factor (TNF) were assessed and mortality rate was observed in different groups for one week. RESULTS: The levels of IL-10 in the pancreas, liver and lung 24 hours after IL-10 gene transfer, increased significantly (all > 350 pg/g), and then gradually decreased, however, the levels of IL-10 were still significantly higher that those in the control groups (P < 0.05) 96 hours later and decreased to normal in one week. The levels of IL-10 of transfer control group were not significantly different from those of the normal control group. The levels of IL-10 expression in pancreas, liver and lungs were increased significantly in the gene therapy group, compared with the SAP group. The serum amylase level was (4 300 +/- 700) U/L in normal control group, increased to (20 300 +/- 1 100) U/L 24 hour after SAP induction without a difference between the therapy control group and SAP group, and decreased to (6 800 +/- 700) U/L after IL-10 gene therapy (P < 0.05). The histological score of pancreas was 4.1 +/- 0.2 24 hours after the induction of...
Warton, K, Tonini, R, Fairlie, WD, Matthews, JM, Valenzuela, SM, Qiu, MR, Wu, WM, Pankhurst, S, Bauskin, AR, Harrop, SJ, Campbell, TJ, Curmi, PMG, Breit, SN & Mazzanti, M 2002, 'Recombinant CLIC1 (NCC27) Assembles in Lipid Bilayers via a pH-dependent Two-state Process to Form Chloride Ion Channels with Identical Characteristics to Those Observed in Chinese Hamster Ovary Cells Expressing CLIC1', Journal of Biological Chemistry, vol. 277, no. 29, pp. 26003-26011.
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CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.
