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Sonoporation has been exploited as a novel strategy for intracellular drug and gene delivery. In sonoporation, ultrasound application generates transient pores or openings in the cell membrane that allow entry of extracellular agents normally not permeable to the cell membrane. In order to improve the sonoporation outcome, we seek to obtain improved understanding of the sonoporation mechanism and investigate the factors affecting sonoporation process. We established a voltage clamp technique for real time measurement of sonoporation at single cell level using Xenopus oocytes as a model system. As both cell survival and intracellular delivery efficiency of drug or genes depend on the sonoporation dynamic process, and Calcium plays important roles in cellular processes, we focus on studying of the effect of extracellular Calcium concentration on the formation, extension, and resealing of membrane pores in sonoporation. We obtained experimental results demonstrating that the cell membrane reseals in the order of seconds in the presence of physiological level of extracellular [Ca]. We measured the resealing as function of extracellular [Ca] (0–1.8mM) and observed that the resealing rate decreases as extracellular [Ca] decreases from normal physiological level. No resealing was demonstrated when 1mM EGTA was added in the extracellular medium to chelate the [Ca] extracellularly. Our experimental findings suggest that extracellular Calcium plays an important role in controlling membrane resealing in sonoporation and thus the sonoporation outcome such as cell survival and delivery efficiency. © 2006 American Institute of Physics [ABSTRACT FROM AUTHOR]

As sea levels rise, salinity will increase into freshwater areas near the coast. Increases in salinity could adversely affect amphibian embryos. Sea salt is produced through the evaporation of ocean water. The chemical composition of the sea salt compromises mostly of sodium chloride and 0.2 to 10% of other salts. It is used as a seasoning in foods, cooking, cosmetics and for preserving foods. This objective of this research was to determine potential developmental effects of sea salts to frog embryos. The effects of the concentration of the sea salt were evaluated using the Frog Embryo Teratogenesis Assay- Xenopus (FETAX). The early embryonic stages of South African clawed frog, Xenopus laevis, were exposed to the concentrations of the sea salt with FETAX solution for 4 days from small cell blastula to a free-living tadpole. The sea salt was mixed with 20 mL of FETAX solution to make different concentrations of sea salt solution. Five different test solutions were used in sets of plastic petri dishes containing 20 embryos. Two control groups of 4 plastic petri dishes were used, each of 8 mL FETAX solution without sea salt solution. Dead embryos were removed after every 24 hours and solution were renewed. The mortality, malformation and length of tadpole were recorded for all petri dishes at the end of 4 days. The same experiment was repeated three times, and the results were compared individually and combined. The 96-h LC50 ranged from 3.8 to 5.8 ppt. The 96-h EC (malformation)50 ranged from 4.5 to 6.2 PPT. The results show that higher the concentration of the sea salt, the more likely to increase the mortality of the tadpole rather than cause malformation or reduction of embryo growth. These results also indicate that salinities increase to above 3.8 ppt would likely critically impact amphibian reproduction. [ABSTRACT FROM AUTHOR]

P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular loop, transmembrane segments and intracellular termini to the action of partial agonists (higher potency and efficacy of BzATP and AP5A at P2X1 compared to P2X2 receptors) and antagonists (NF449 ∼ 1,500 fold more potent at P2X1 compared to P2X2). P2X receptors and chimeras were expressed in Xenopus oocytes and currents recorded with TEVC. Replacing the extracellular loop of the P2X1 receptor with that from P2X2 (P2X1-2EXT) produced a receptor that had low sensitivity to NF449 equivalent to that at the WT P2X2 receptor. Sensitivity similar to WT P2X1 receptors was recorded in the reciprocal P2X2-1EXT chimera for NF449. These data show that antagonist sensitivity was conferred by the extracellular loop, and indicates that these chimeras do not result in major conformational changes. In contrast the effectiveness of partial agonists was increased above P2X1 levels for both the loop transfers suggesting interactions with the rest of the receptors played an important role in regulating agonist sensitivity and efficacy. Swapping TM1 increased the efficacy of AP5A and BzATP at P2X1-2TM1 and P2X2-1TM1 chimeras. Replacement of the TM2 region had reciprocal effects on partial agonist efficacy; P2X1- 2TM2 showed partial agonist sensitivity similar to that at P2X2 and P2X2-1TM2 showed partial agonist sensitivity intermediate between P2X1 and P2X2. These results suggest a complex interaction between the TMs and the rest of the receptor in regulating partial agonist efficacy. Exchanging the intracellular N- terminus had no effect on agonist potency, but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras. This demonstrates that potency and efficacy can be independently regulated. Further subdivision of the N- terminus showed amino acid residues 1- 16 predominate in the control of efficacy. The C-terminal chimera P2X2-1C had no effect of partial agonist sensitivity, whereas the reciprocal chimera (P2X1-2C) had partial agonist efficacy intermediate between P2X1 and P2X2. Chimeras and point mutations also identified residues in the C-terminus that regulated recovery from channel desensitization. In summary this study shows that interactions between the extracellular, transmembrane and intracellular portions of the P2X receptor regulate channel properties and suggest that transitions to channel opening, the behaviour of the open channel and recovery from the desensitized state can be controlled independently. [ABSTRACT FROM AUTHOR]

Blood cells are turned over rapidly and we carry a relatively modest number of stem cells to regenerate them. One of the key properties to understand is how these cells stably maintain the undifferentiated state. To study this, we have taken the approach of determining how these cells are programmed. We have lineage traced the cells in Xenopus and zebrafish embryos to determine their history and the niches that they occupy. We have then searched these niches for the signals that programme the cells. We have also begun to identify the nuclear responses to these signals that lead to the establishment of a stable networks of transcription factors that specify cell fate. We have taken a similar approach to the myocardium, and more recently have begun to compare normal development with regeneration. We are attempting to identify the cells, signals and transcriptional networks involved in this process, an understanding of which could inform regenerative strategies in the human heart. [ABSTRACT FROM AUTHOR]

TRPM8 channels are a polymodal receptors activated by voltage, low temperatures and agonists. The structural data suggests that the distal C-terminus (1064-1104 amino acid residues) form a coiled-coil motif. Our aim was to investigate the importance of this structure in the thermo-sensitivity of TRPM8. TRPM8 wild type and deletion mutants of the coiled-coil region of this channel were cloned and expressed in Xenopus oocytes. Macroscopic currents were recorded by patch-clamp technique in cell-attached configuration at different temperatures (10°C-40°C). An analysis of the conductance-voltage curves determined from tail currents showed that values of the voltage at which the open probability is 0,5 (V1/2 ) saturate at high and low temperatures. These results and limiting slope analysis of the open probability-voltage curves demonstrated that TRPM8 channel gating is best explained using an allosteric gating model for both voltage and temperature dependence. On the other hand, gradual deletions of the coiled-coil region (from 1064 to 1104 residues) promoted a gradual decrease in the temperature sensitivity of TRPM8 channel, without affecting the voltage dependence. The decrease in the enthalpy promoted by gradual deletions of the distal C-terminus suggests that coiled-coil region is a structural determinant of the thermo sensitivity in TRPM8 channels. Our results also indicate that the voltage and temperature sensors are localized in different structural domains. [ABSTRACT FROM AUTHOR]

Hydroxyzine is a potent first-generation histamine H1 receptor antagonist that can increase action potential duration and induce QT prolongation in several animal models. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of leading causes of acquired long QT syndrome, we investigated the acute effects of hydroxyzine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of hydroxyzine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Hydroxyzine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 of hydroxyzine-dependent hERG block in Xenopus oocytes increased progressively relative to the degree of depolarization. Hydroxyzine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutation Y652A attenuated the hERG current block. The IC50 value of hydroxyzine-dependent hERG block increased from 5.9 µM to 35.1 µM by the mutation from tyrosine to alanine at 652 amino acid of the channel. These results suggest that the H1 antihistamine, hydroxyzine is a blocker of the hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects. [ABSTRACT FROM AUTHOR]