Localization and quantification of net fluxes of H+ along maize roots by combined use of pH-indicator dye videodensitometry and H+-selective microelectrodes

Two methods for measuring proton fluxes along intact maize roots grown with NH ₄ ⁺ or NO ₃ ⁻ at pH 6.5 were compared. Videodensitometric measurement of changes in a pH-indicator dye by video camera was used to map pH around roots and determine the amounts of protons released by various root regions. This method was compared with potentiometric determination of the concentration of H⁺ in the unstirred layer at the root surface using ion-selective microelectrodes. With NH ₄ ⁺ the roots released large amounts of H⁺ in preferential regions where the rate of flux can reach 1.4 or even 2.5 nmol m⁻¹ s⁻¹. Videodensitometry indicated a first region of root acidification in the subapical zone, but this was more difficult to localize with microelectrodes. With NO₃ ⁻ both methods showed that the roots released small amounts of H⁺ and that the apical region took up H⁺ in the first 10 mm then sometimes released H⁺ over the following 10 mm of root. The H⁺ flux profiles obtained by both methods were in good agreement in terms of both order of magnitude of the fluxes and spatial differences along the root. These results suggest that videodensitometry, which is easier to use than potentiometry, can be used to screen different plant species or cultivars under various experimental conditions. The microelectrode technique is indispensable, however, for studying the underlying mechanisms of net H⁺ fluxes. This revised version was published online in June 2006 with corrections to the Cover Date.     

反义寡核苷酸pH敏前体脂质体的制备及性质分析

为了提高反义寡核苷酸的稳定性和生物利用度及避免在溶酶体内降解,采用旋转蒸发-薄膜水化、超声-挤压、冻干三步法完成pH敏前体脂质体的制备,研究了体外释药规律;以反义寡核苷酸为实验对象,测定了包封率.制得的pH敏前体脂质体复水后形成的pH敏脂质体形态圆整,粒径12.3～389.9 nm范围内,平均粒径为22.7 nm;三批pH敏脂质体的平均包封率为68.3%;体外释药方程为Q=1.8382－2.5186×10^－2T (r=0.9913);结果说明制备的pH敏前体脂质体水合形成的pH敏脂质体粒度适宜,可用于反义寡核苷酸的包封.     

Membrane potential of rat hepatoma cells in culture: Influence of factors affecting amino acid transport

The effect has been studied of various media, hormones and of amino acids on the membrane potential of rat hepatoma cells in culture measured by microelectrode impalement. Cells in Eagle's minimal essential medium plus 5% serum had a value which varied daily from about 5–8 mV, inside negative. The membrane potential of rat hepatocytes was measured to be 8.7 ± 0.2mV, inside negative. The membrane potential of the hepatoma cells was decreased by insulin and increased by glucagon. Membrane potential was unaffected by change of medium to Hanks' or Earle's balanced salt solutions or deprivation of serum. It was, however, reduced in cells in phosphate-buffered saline and by reduction of pH. The former effect was shown to be due to the higher [Na⁺] of phosphat-buffered saline as opposed to the other media. Addition of alanine, glycine, serine, proline and methylaminoisobutyrate all reduced membrane potential by 2–3 mV. Smaller decreases were seen with methionine, leucine and phenylalanine, but none with glutamine, threonine, BCH (2-aminonorborane-2-carboxylic acid) and D-alanine. The results are compared with the effects of similar conditions on aminoisobutyrate uptake. Whilst there was a correlation under some conditions there was not under others. It is concluded that for the hepatoma cells factors additional to the membrane potential must exert some influence on the capacity for amino acid transport.     

Microfabricated nerve–electrode interfaces in neural prosthetics and neural engineering

Neural interfaces and implants are finding more clinical applications and there are rapid technological advances for more efficient and safe design, fabrication and materials to establish high-fidelity neural interfaces. In this review paper, we highlight new developments of the microfabricated electrodes and substrates with regard to the design, materials, fabrication and their clinical applications. There is a noticeable trend towards integration of microfluidic modules on a single neural platform. In addition to the microelectrodes for neural recording and stimulation, microfluidic channels are integrated into a nerve–electrode interface to explore the rich neurochemistry present at the neural interface and exploit it for enhanced electrochemical stimulation and recording of the central and peripheral nervous system.     

Effect of dehydration and rehydration of the pH-sensitive liposomes containing chimeric gag-V3 virus like particle on their long-term stability

One of the practical limitations with the use of liposomes for delivery of the pharmaceutical substances such as antigens is that liposomes are relatively unstable in storage. In order to extend the stability of liposome in storage without affecting their functional activity, solution-type liposomes were dehydrated to form a structurally intact dry liposomes. Comparative immunological evaluation was carried out for both dry and solution-type liposomes containing gag-V3 chimera, consequently it was found that dry liposomes elicited both humoral and cellular response as efficiently as solution-type liposomes did against the same gag-V3 antigen. Especially, long-term stability of the liposomes was remarkably enhanced by the dehydration made to liposomes without a significant change in its ability to elicit immune responsein vivo. These results indicate that dry pH-sensitive liposome may become an effective delivery and adjuvant system for general vaccine development.     

High efficiency,Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes^1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies^9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies¹³. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells^14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies^{19, 24-26}. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection²⁷. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures^17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.     

豚鼠不同部位微动脉平滑肌细胞电生理学特性的比较

本研究应用电生理技术在豚鼠离体小脑前下动脉(anterior inferior cerebellar artery,AICA)、肠系膜动脉(mesenteric artery,MA)和耳蜗螺旋动脉(spiral modiolar artery,SMA)分支(直径小于100μm)上比较微动脉平滑肌细胞电生理学特性的异同。结果显示:(1)应用细胞内微电极记录技术测得AICA、MA和SMA细胞静息膜电位分别为(-68±1.8)(n=65)、(-71±2.4)(n=80)和(-66±2.9)mV(n=58),各微动脉间无统计学差异。(2)一段血管微动脉标本全细胞膜片钳记录的平滑肌细胞膜电容和膜电导都远大于单个细胞标本,且微动脉间存在统计学差异,大小顺序为MAAICASMA。应用缝隙连接阻断剂2-APB(100μmol/L)后记录一段微动脉平滑肌细胞膜电容和膜电导与单个细胞十分接近。(3)AICA、MA和SMA单个平滑肌细胞膜电流I/V关系呈明显的外向整流特性,都对1mmol/L4-AP和10mmol/LTEA敏感。当指令电压为+40mV时,AICA、MA和SMA血管平滑肌细胞电流密度分别为(26±2.0)、(24±1.7)和(18±1.3)pA/pF,SMA和AICA、MA间存在统计学差异。上述结果提示,豚鼠不同部位微动脉平滑肌细胞在缝隙连接耦联力和电流密度等电生理特性存在差异。     

Instrumental analysis of microbiologically influenced corrosion

Appropriate application of techniques for detection andmonitoring of microbiologically influenced corrosion isessential for understanding the mechanistic nature of theinteractions and for obtaining control methods. This paperreviews techniques and methods applied tomicrobiologically influenced corrosion in recent years.The techniques presented in this paper includeelectrochemical noise measurement, concentric electrodes,scanning vibrating electrode mapping, electrochemicalimpedance spectroscopy, atomic force microscopy,confocal laser microscopy, Fourier transform infraredspectroscopy, x-ray photoelectron spectroscopy, Augerelectron spectroscopy, extended x-ray absorption finestructure and utilization of piezoelectric materials. Thesetechniques are reviewed regarding the heterogeneouscharacteristics of microbial consortia and their possibleinfluences on metal substrata. We hope this review willmotivate application and combination of new techniquesfor practical detection and on-line monitoring of theimpact of biofilms on engineering alloys.     

豚鼠冠状动脉微动脉细胞静息膜电位的分布特性及机制

目的:观察冠状动脉微动脉细胞静息膜电位(RP)的分布特性及形成机制.方法:离体豚鼠冠状动脉微动脉(直径小于100 μm)上,应用细胞内微电极技术记录细胞RP.结果:①成功记录到112个细胞,细胞平均RP为(-65±4.2)mV,应用高斯函数拟合后细胞RP呈双峰状分布,两个峰值分别为-43和-74 mV,分别称为高和低R...