Marking Individual Nerve Cells Through Electrophoresis of Ferrocyanide from a Microelectrode

Microelectrodes filled with an aqueous mixture containing 0.5 M potassium ferrocyanide and 2.5 M KCl were used to electrophoretically mark single neurones in the snail brain. After a physiological experiment, 3-4 μa at 20 v were allowed to flow for 10-15 min and carry the ferrocyanide into the cell. Cells from 40 μ to 130 μ have been marked. There is no diffusion of the Prussian blue (formed by soaking 10-15 min in 1.1 M FeCl₃) outside the cell. The marked cell can be studied both in the whole brain and in sections. In many cases a length of axon is stained also, and it can be traced through successive sections of the brain.     

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.     

A Reinterpretation of Davenport's Game Theory Analysis: Brief Communication

Davenport's classic application to anthropology of game theory is reinterpreted so as to separate the mathematics of his model from the criticized game theory interpretation of that mathematics. The reinterpretation implies that the fishing pattern of a certain Jamaican fishing village may be the one that produces the steadiest average net income for the village as a whole.     

Localization of Branching Enzyme in Potato Tuber Cells with the Use of Immunoelectron Microscopy

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.     

A Solid State Chloride-selective Microelectrode for use with Plant Tissues1

A solid state Cl^–selective microelectrode is describedand its performance in test solutions compared with that ofa microelectrode embodying a Cl^–selective liquid ion-exchanger.The solid state microelectrode was found to be more selectiveto Cl^– against an interfering background of common inorganicion species than the ion-exchanger microelectrode. It performedsatisfactorily in the presence of NO^–₃ and thus is suitablefor use with plant tissues.     

Cellular Damage to the Callus Cells of Potato Subjected to Freezing

Callus cells of potato (Solanum tuberosum L.) cv. Désirée were exposed to various subzero temperatures and examined for the freezing damage. In the cells subjected to –3 °C, plasma membranes appeared to be intact, while tonoplast seemed to be damaged and organelles to be swollen. After freezing at –6 °C, the damage became severe and plasma membranes were ruptured. After exposure to –10 °C, the damage was so severe that the cell organelles could not be recognised and cytoplasm became fragmented.     

神经球结合异种细胞生长的实验研究

目的 研究神经球在形成过程中是否能结合异种细胞,并与之形成杂合细胞球.方法 分别用绿色荧光蛋白(EGFP)标记的神经球来源细胞、大鼠神经胶质瘤细胞C6、HER293细胞,同正在形成中的神经球共培养,检测异种细胞是否能与神经球形成杂合细胞球.结果 神经球在形成过程中,能与异种细胞形成杂合细胞球,杂合细胞不改变原神经球细胞的特性,且杂合细胞球的形成能促进杂合细胞的增殖. 结论 神经球能与异种细胞形成杂合细胞球,并能为细胞生长分裂提供特殊的微环境,这为神经球微环境的研究提供实验依据.     

蛇毒对肿瘤细胞的体外抑制实验

目的 探讨蝮蛇毒及蝮蛇与眼镜蛇混合毒对肿瘤细胞的抗癌活性,方法 应用蝮蛇毒及蝮蛇与眼镜蛇混合毒人源肿瘤细胞进行体外细胞毒试验。结果 蝮蛇毒及蝮蛇与眼镜蛇混合毒对传代细胞株（Ｎｏｖｉｋｏｆｆ及Ｈｅｐ－２）的抑制作用随蛇毒剂理的增加而增强,剂量为５μｇ／ｍｌ时,蝮蛇毒对传代细胞Ｎｏｖｉｋｏｆｆ及Ｈｅｐ－２的抑制率分别为５０．３％和４７．５％,蝮蛇与眼镜蛇混合毒对Ｎｏｖｋｏｆｆ及Ｈｅｐ－２的抑制率分别为     

Potato Lectin: A Cell-Wall Glycoprotein

The activity and the amount of potato lectin were measured inpotato tuber slices (Solanum tuberosum cv. Huinkul) aeratedfor 48 h. Lectin was found in a soluble form, liberated to themedium and associated with insoluble structures. Polyacrylamidegel electrophoresis in denaturating conditions and immunologicaltechniques indicated that the lectins associated to cell wall,soluble or liberated to the medium, were identical. The cell-wallfraction was found to contain 65% of total lectin in the tuber.The possible role of potato lectin in tubers was discussed. (Received June 5, 1985; Accepted September 3, 1985)     

Further Evidence for the Uptake of Exogenous DNA into Potato Leaf Cells

The fate of a DNA-fluorescent dye complex rubbed on potato leaveswas followed by fluorescence microscopy. DNA was taken up bythe leaf cells through the scars of broken hairs and throughstomata. The significance of this observation is discussed inrelation to the DNA-mediated resistance of potatoes to Phytophthorainfestans. ³Present address: Plantech Research Institute, 1000 Kamoshida-cho,Midori-ku, Yokohama 227, Japan. (Received August 9, 1983; Accepted March 8, 1984)     

Microelectrode Measurements on Red Beet Vacuole : Biological Effect of Na OR NO(3) Ions, Diffusing from the Microelectrode

Glass microelectrodes filled with 3 molar KCl are widely used to measure intracellular potentials and it is usual to try to minimize their electrolyte loss. In these experiments we have used the ionic leak of our microelectrodes, filled with various salt solutions, to introduce a given ion into the red beet vacuole. This allowed us to show that NO₃⁻ ions reduce the magnitude of the current spectral density while they do not change the resistance of the tonoplast. This is true when NO₃⁻ is either added to the external medium or used as the microelectrode filling solution. This can be interpreted by a NO₃⁻ effect on the vacuolar side of the tonoplast, resulting in an inhibition of the ion transporting ATPase. Replacing K⁺ by Na⁺ ions in the medium has no effect on tonoplast resistance (R_s). On the contrary, when ions leaking from the microelectrode are H⁺, Li⁺ or K⁺, R_s is close to 4 kilohm square centimeter, whereas R_s is of the order of 30KΩ square centimeter when Na⁺ are the leaking ions. We also found a possible correlation between the presence of a Lorentzian in the current spectral density (cut-off frequency = 2 hertz) and a Cl⁻ efflux from the vacuole. This could be explained by the existence of Cl⁻ channels on the tonoplast.     

Design and Implementation of a Microelectrode Assembly for Use on Noncontact In Situ Electroporation of Adherent Cells

In situ electroporation of adherent cells provides significant advantages with respect to electroporation systems for suspension cells, such as causing minimal stress to cultured cells and simplifying and saving several steps within the process. In this study, a new electrode assembly design is shown and applied to in situ electroporate adherent cell lines growing in standard multiwell plates. We designed an interdigitated array of electrodes patterned on copper with printed circuit board technology and covered with nickel/gold. Small interelectrode distances were used to achieve effective electroporation with low voltages. Epoxy-based microseparators were constructed to avoid direct contact with the cells and to create more uniform electric fields. The device was successful in the electropermeabilization of two different adherent cell lines, C2C12 and HEK 293, as assessed by the intracellular delivery of the fluorescent dextran FD20S. Additionally, as a collateral effect, we observed cell electrofusion in HEK 293 cells, thus making this device also useful for performing cell fusion. In summary, we show the effectiveness of this minimally invasive device for electroporation of adherent cells cultured in standard multiwell plates. The cheap technologies used in the fabrication process of the electrode assembly indicate potential use as a low-cost, disposable device.     

Immunocytochemical Localization of ADPglucose Pyrophosphorylase in Developing Potato Tuber Cells

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.     

Experiments with genitalia: a commentary

There has been a recent burst of studies of the function of genitalia, many of which share several important shortcomings. Given that further studies on this topic are likely (there are probably millions of species showing rapid genital divergence), I discuss the studies critically to promote clear formulation of hypotheses and interpretation of results in the future. I also emphasize some possibly important but neglected variables, including female stimulation, phylogenetic contexts, and the behavior of male genitalia, and outline simple techniques that could improve future studies.     

毒胡萝卜素诱导A549细胞凋亡的实验研究

目的:探讨内质网应激诱导剂毒胡萝卜素(thapsigargin TG)对体外培养人肺泡II型细胞来源的A549细胞生长及凋亡的影响。方法:体外培养A549细胞,经不同浓度TG(1μM、5μM、10μM)干预24小时,MTT检测细胞存活率、Hochest/PI染色观察细胞凋亡形态学、Wersten-blot检测活化caspase-3水平。结果:与对照组比较,经TG作用24小时后,活化caspase-3表达显著增加,细胞存活率下降,凋亡率增加,均呈浓度依赖性,P0.005。结论:毒胡萝卜素能抑制A549细胞生长,诱导细胞凋亡。