海马脑片盲法膜片钳全细胞记录技术

本文较为详细地介绍了海马脑片盲法膜片钳全细胞记录技术,对其关键步骤和需要注意的问题进行了重点说明,同时对CA1区锥体神经元突触活动的特点,电压门控性Ca^2 通道以及谷氨酸（glutamate,Glu)γ－氨基丁酸（GABA）受体通道电流性质等进行了观察和分析,实验结果为采用海马脑片盲法膜片钳全细胞记录技术研究海马神经元离子通道动力学性质和中枢神经系统药物对突触活动的影响提供了可靠的依据。     

Determination of the induced ELF electric field distribution in a two layer in vitro system simulating biological cells in nutrient solution

In-vitro studies of biological effects of electromagnetic fields are often conducted with cultured cells either in suspension or grown in a monolayer. In the former case, the exposed medium can be assumed to be homogeneous; however, eventually the cells settle to the bottom of the container forming a two layer system with different dielectric and conductive properties. In the present work the effect of this separation on the electric field distribution is calculated and experimentally measured at selected positions for a commonly used exposure configuration. The settled cell suspension is modeled by a well-defined two layer system placed in a rectangular container with the base of the container parallel to the direction of the magnetic field. Theoretical calculations based on numerical techniques are done for various two layer systems with different conductivities in each layer. The agreement between the theoretical calculations and the experimental measurements is within ± 1.5 mV/m, or 10% of the maximum induced field when the conductivity of the lower layer is ten times that of the upper layer. This result is well within experimental error. When the thickness of one of the layers is small compared to the thickness of the other layer, it is found that the electric field distribution is essentially that of the homogeneous case. The latter situation corresponds to a typical cell exposure condition. © 1993 Wiley-Liss, Inc.     

Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

The T-cell receptor (TCR) mutation assay for in vivo somatic mutations is a sensitive indicator of exposure to ionizing radiation. However, this assay cannot be immediately applied after radiation exposure because expression of a mutant phenotype may require as long as several months. In the present study, we eliminate this time lag by stimulating lymphocytes with a mitogen that can accelerate the turnover of TCR protein expression in T-cells. When lymphocytes obtained from healthy donors were irradiated with various doses of X-rays and cultured with human interleukin-2 after phytohemagglutinin (PHA) pulse stimulation, the mutant frequency (MF) of CD4⁺ T-cells increased dose dependently during the first 7 days, then decreased rapidly due to the growth disadvantage of mutant cells. This suggests that PHA stimulation can shorten the expression time of a mutant phenotype to within a week after radiation exposure. The relationship between radiation dose and TCR MF on the seventh day was best fitted by a linear-quadratic dose–response model. We applied this improved TCR mutation assay to gynecological cancer patients who received 5 days of localized radiotherapy, totaling about 10 Gy. The in vivo TCR MF in the patients did not change within a week after radiotherapy, whereas the in vitro TCR MF of PHA-stimulated lymphocytes from the same patients significantly increased 7 days after initiating culture. The estimated mean radiation dose to the peripheral blood lymphocytes of the cancer patients was about 0.9 Gy, based on the in vitro linear-quadratic dose–response curve. This estimated dose was close to that described in a previous report on unstable-type chromosome aberrations from cervical cancer patients after receiving the same course of radiotherapy. On the basis of these findings, we propose that the improved TCR mutation assay is a useful biological dosimeter for recent radiation exposure.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

Tuning,coupling and matching of a resonant cavity in microwave exposure system for biological objects

A new microwave exposure system for biological experiments with well-defined exposure conditions and improved control of the exposure parameters consisting of variable frequency power source, coaxial to waveguide transition, matching network and single-mode resonant cavity with movable shorting plunger was fabricated and characterized. The introduction of a biological sample into a resonant cavity has a large impact on its field configuration and port impedance. As such, the properties, geometry and position of the biological sample become a part of the electrical properties of the microwave circuit. With that change, the electrical properties of the resonant cavity, such as impedance, quality factor and resonant frequency, also change. In this study, an appropriate coupling system with effective power transfer and an algorithm to tuning and coupling of resonant cavity in resonance before and after the introduction of biological sample have been proposed. This procedure will lead to a known dose distribution within the biological sample and allow a better comparison with other studies. Coupling of the electromagnetic energy into a resonant cavity was experimentally investigated. Graphical representation of cavity impedance in case of undercoupled, critically coupled and overcoupled cavity has been presented. Critical coupling of an empty resonant cavity has been accomplished at voltage standing wave ratio (VSWR) 1.01, at resonance frequencies 900 and 947.5 MHz. Critical coupling with the introduction of a biological sample has been accomplished at VSWR ≤ 1.07 for frequency bandwidth 1 MHz and VSWR ≤ 1.5 for frequency bandwidth up to 5 MHz with central frequency 947.5 MHz.     

Use of the modified Robbins device to study the in vitro biofilm removal efficacy of NitrAdine, a novel disinfecting formula for the maintenance of oral medical devices

Aims:  To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine^TM, a disinfectant for the maintenance of oral medical devices.
Methods and Results:  Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine^TM and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine^TM revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus ) and allowed the determination of the optimal conditions for its use.
Conclusion:  The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine^TM exhibits high activity against biofilms formed by the micro-organisms tested.
Significance and Impact of the Study:  Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.