Electroporation of cells

By measuring uptake of the membrane impermeable dye. phenosafranine, it can be shown that the plasma membrane of intact cells within cell aggregates can be reversibly permeabilized by electroporation. However, the plant cell wall is a barrier to DNA uptake by intact cells, although under certain circumstances expression of DNA, electroporated into intact cells, can be demonstrated. The level of expression is about 20–50 times lower than that obtained by electroporation of protoplasts, and depends on cell wall properties and pretreatments of cell aggregates. In contrast, efficient transformation of whole cells of bacteria and yeasts can be achieved by electroporation. Factors which influence DNA transfer into whole plant cells and the possibility of stable transformation are discussed.     

The nature of parsimony and instrumentalism in systematics

This paper traces the historical and philosophical roots of the principle of parsimony, and its incorporation in empiricist philosophy of science. It is argued that phylogeny reconstruction looks back to the empiricist tradition in its application of parsimony. The radical coherentism that motivates the application of parsimony in direct optimization of sequence alignment results in an instrumentalist approach to phylogeny reconstruction. A similarly instrumentalist perspective motivated the 'barcoding' initiative in taxonomy and bioconservation. This is contrasted with a realist conception of systematics.     

苦参碱对哇巴因诱导钠电流改变的调节作用

目的:探讨苦参碱拮抗哇巴因诱导的心律失常的作用机制。方法:应用全细胞膜片钳技术记录哇巴因对单个豚鼠心室肌细胞的Na+电流和动作电位时程作用后,观察苦参碱对哇巴因诱导Na+电流和动作电位时程改变的恢复作用。结果:1 5μmol·L-1哇巴因延长APD50从给药前476±40.7 ms增加到744±62.9 ms(n=6,P0.05),APD90从给药前499±84.9 ms增加到775±87.7 ms(n=6,P0.01),100μmol·L-1苦参碱恢复APD50至603±79.0 ms(n=6,P0.05),APD90至630±81.6 ms(n=6,P0.05);2 5μmol·L-1哇巴因可增加钠电流的峰值,在-20 m V电压条件下,5μmol·L-1哇巴因增加INa,由正常-40.9±2.32 p A/p F增加到-55.2±2.26 p A/p F(n=8,P0.05),100μmol·L-1苦参碱减少INa至-34.6±2.14 p A/p F(n=8,P0.05);5μmol·L-1哇巴因右移钠通道的激活曲线,并左移钠通道的失活曲线从而改变通道动力学特性;100μmol·L-1苦参碱可抑制哇巴因诱导的INa的增加,并恢复Na+通道动力学特性接近正常。结论:苦参碱拮抗哇巴因诱导的心律失常机制与其抑制哇巴因诱发细胞水平Na+电流的增加,缩短哇巴因诱发APD的延长有关。     

Translational aspects of cardiac cell therapy

Cell therapy has been intensely studied for over a decade as a potential treatment for ischaemic heart disease. While initial trials using skeletal myoblasts, bone marrow cells and peripheral blood stem cells showed promise in improving cardiac function, benefits were found to be short‐lived likely related to limited survival and engraftment of the delivered cells. The discovery of putative cardiac ‘progenitor’ cells as well as the creation of induced pluripotent stem cells has led to the delivery of cells potentially capable of electromechanical integration into existing tissue. An alternative strategy involving either direct reprogramming of endogenous cardiac fibroblasts or stimulation of resident cardiomyocytes to regenerate new myocytes can potentially overcome the limitations of exogenous cell delivery. Complimentary approaches utilizing combination cell therapy and bioengineering techniques may be necessary to provide the proper milieu for clinically significant regeneration. Clinical trials employing bone marrow cells, mesenchymal stem cells and cardiac progenitor cells have demonstrated safety of catheter based cell delivery, with suggestion of limited improvement in ventricular function and reduction in infarct size. Ongoing trials are investigating potential benefits to outcome such as morbidity and mortality. These and future trials will clarify the optimal cell types and delivery conditions for therapeutic effect.     

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.     

5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches

Background

5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5?HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K_ATP (sarcK_ATP) channel activity.

Methods

Single sarcK_ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK_ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A.

Results

5-HD inhibited (IC₅₀ ∼ 30 μM) K_ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK_ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK_ATP channel activation by either metabolic inhibition or rilmakalim.

General significance

SarcK_ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK_ATP and mitoK_ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK_ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.     

Direct shoot regeneration from intact leaves of Arnebia euchroma (Royle) Johnston using thidiazuron

The present study highlights the importance of preculture time and concentration of TDZ (thidiazuron) for direct regeneration from in vitro leaves (attached to shoots) in Arnebia euchroma. Shoot buds proliferated to form multiple shoots on MS medium (Murashige and Skoog medium) with 5.0 μM Kn. Different additives viz. ascorbic acid, PVP (polyvinylpyrrolidone), PVPP (polyvinylpolypyrrolidone) or activated charcoal (50, 100 and 250 mg/l each) were used to check the phenolic exudations. Direct shoot regeneration was obtained when shoots were initially precultured for 40 days on medium with a higher concentration of TDZ (20.0 μM) and then transferred to a lower concentration (5.0 μM TDZ). The identity of shoot buds was confirmed by histological studies. Regenerated shoots were cultured for 30 days on medium containing Kn (5.0 μM) for proliferation and then transferred to IBA (0.25 μM)‐containing medium for rooting. Rooted plantlets were transferred to greenhouse with 45–50% survival.