Donald C. Chang

Nuclear Magnetic Resonance Transverse Relaxation Times of Water Protons in Skeletal Muscle

The observation of the spin-echo decay in a long time domain has revealed that there exist at least three different fractions of non- (or slowly) exchanging water in the rat gastrocnemius muscle. These fractions of water are characterized with different nuclear magnetic resonance (NMR) relaxation times and are identified with the different parts of tissue water. The water associated with the macromolecules was found to be approximately 8% of the total tissue water and not to exchange rapidly with the rest of the intracellular water. The transverse relaxation time (T(2)) of the myoplasm is 45 ms which is roughly a 40-fold reduction from that of a dilute electrolyte solution. This fraction of water accounts for 82% of the tissue water. The reduced relaxation time is shown neither to be caused by fast exchange between the hydration and myoplasmic water nor by the diffusion of water across the local magnetic field gradients which arise from the heterogeneity in the sample. About 10% of the tissue water was resolved to be associated with the extracellular space, the relaxation time of which is approximately four times that of the myoplasm. Mathematical treatments of the proposed mechanisms which may be responsible for the reduction of tissue water relaxation times are given in this paper. The results of our study are consistent with the notion that the structure and/or motions of all or part of the cellular water are affected by the macromolecular interface and this causes a change in the NMR relaxation rates.

Cell poration and cell fusion using an oscillating electric field

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.

High efficiency gene transfection by electroporation using a radio-frequency electric field

In order to develop a safe and effective way to introduce exogenous genes into cells, we have experimented with a new method of electroporation which uses a radio-frequency (RF) electric field to permeabilize the cell membrane. This RF method has several advantages over the conventional electroporation method which uses a direct current (DC) field. We have shown that the RF electroporation method can be used to introduce marker genes into a wide variety of cell lines, including COS-M6, CV-1, CHO, 3T3 and hepatocytes, and is able to increase substantially the efficiency of gene transfection. (For example, the amount of DNA required for transfecting two million COS-M6 cells can be as low as 0.1 microgram). The transfection efficiency is shown to be affected by a number of factors, including cell type, field strength, pulse protocol and medium buffer. Because of its wide range of applications, high transfection efficiency and lack of harmful side-effect, the RF electroporation method would be particularly useful for introducing genes into human cells for gene therapy.

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene beta-gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the beta-gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiently using the in situ method. Furthermore, we have measured the time required to express the beta-gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing beta-gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.

Cell Fusion and Cell Poration by Pulsed Radio-Frequency Electric Fields

Cell fusion plays a very important role in modern biotechnology. For example, one key procedure in genetic engineering is the introduction of exogenous genetic material into a host cell. Such insertion of genes is accomplished by either permeabilizing the cell membrane to allow entry of genetic material, or fusing the host cell with a cell containing the desired genetic material. Furthermore, cell fusion is important in the production of monoclonal antibodies, which requires fusion of antibody-producing cells with continuously dividing cancer cells such as myeloma cells (Galfre et al., 1977; Lo et al., 1984). Also, cell fusion can be used as a microinjection technique to deliver drugs which normally cannot enter a cell. One can simply fuse the cell with liposomes or red blood cell ghosts that have been preloaded with specific drugs (Schlegel and Lieber, 1987).

Study of anisotropy in nuclear magnetic resonance relaxation times of water protons in skeletal muscle.

The anisotropy of the spin-lattice relaxation time (T1) and the spin-spin relaxation times (T2) of water protons in skeletal muscle tissue have been studied by the spin-echo technique. Both T1 and T2 have been measured for the water protons of the tibialis anterior muscle of mature male rats for theta = 0, 55, and 90 degrees, where theta is the orientation of the muscle fiber with respect to the static field. The anisotropy in T1 and T2 has been measured at temperatures of 28, -5 and -10 degrees C. No significant anisotropy was observed in the T1 of the tissue water, while an average anisotropy of approximately 5% was observed in T2 at room temperature. The average anisotropy of T2 at -5 and -10 degrees C was found to be approximately 2 and 1.3%, respectively.

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0 We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of water in cysts hydrated with D(2)O have been measured for hydrations between 1.5 and 0.1 g of D(2)O per gram of dry solids. When the relaxation rates (I/T(1), I/T(2)) of (2)H and (17)O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D(2)O (or H(2) (17)O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The (2)H and (17)O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H(2)O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T(1) as a function of hydration in the vicinity of 0.6 g H(2)O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

A comparative study of the effects of tetrodotoxin and the removal of external Na+ on the resting potential: Evidence of separate pathways for the resting and excitable Na currents in squid axon

Summary1.To investigate whether the Na permeability of the resting membrane is determined predominantly by the excitable Na channel, we examined the effects of tetrodotoxin (TTX) and the complete removal of external Na+ on the resting potential.2.In the intact squid axon bathed in K-free artificial seawater, both TTX and the removal of Na+ produced small hyperpolarizations. The effect of Na removal, however, was larger than that of TTX.3.In the perfused squid axon, the hyperpolarization produced by the removal of external Na+ was greatly enhanced when the internal K concentration ([K+]i) was reduced. The effect of TTX, on the other hand, was not sensitive to the [K+]i or to the membrane potential. For [K+]i = 50 mM and [K+]o = 0, the average hyperpolarization produced by TTX was 1.2 mV, while the hyperpolarization produced by Na removal was approximately 21 mV.4.The difference between these two effects suggests that the majority of the resting Na current passes through pathways other than the excitable Na channel.

Ultrastructure of the squid axon membrane as revealed by freeze-fracture electron microscopy.

Summary1.The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy.2.Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed.3.Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation.4.We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm.5.The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.6.On the basis of existing physiological and biochemical information about the estimated size and density of the so-called sodium-channel proteins, we suspect that some of the IMP at the P-face of the axolemma, especially those with diameters between 9 and 11 nm, may be associated with the intramembrane component of the sodium channels.