Peck-Sun Lin

Surface Modification of T-Lymphocytes Observed during Rosetting

Scanning electron microscopy has been used to characterize alterations of lymphocyte surface topology that occur on contact with erythrocytes during the rosetting reaction. Molt-4 cells, a line of leukemic human lymphocytes, defined as T-cells through their ability to form rosettes with sheep erythrocytes, were used for this. Unreacted Molt-4 cells exhibit surfaces that are virtually smooth and carry few microvilli. In contrast, Molt-4 cells in rosettes display a time-dependent modification of surface topology, characterized by the emergence of numerous, long microvilli, particularly in areas of red cell contact.

Hyperthermic treatment (43 °C) rapidly impedes attachment of fibroblasts to culture substrates

Hyperthermic (43 degrees C) exposure of Chinese hamster fibroblasts for more than 30 min, either in suspension or as a monolayer, significantly inhibits their ability to attach to the culture substrate. This finding as well as the effect of hyperthermia on membrane proteins and on surface topology are rapidly expressed and differ from the inhibition of colony formation which is a later effect of hyperthermia on reproductive ability.

Effect of heat on the microtubule disassembly and its relationship to body temperatures

Summary Microtubule proteins extracted from brain tissues of mouse, rat, calf and chicken have been used to study the heat induced disassembly process. The disassembly temperatures were 39.5°C, 41.5°C, 41.6°C, and 45°C respectively, to each species. These are within the range of fever temperatures of these species. Temperatures lethal to these animals cause denaturation of the microtubule proteins of the corresponding species. Purified microtubule protein is less sensitive to heat, but will reestablish its temperature sensitivity by addition of the supernatant obtained during the purification process. This study suggests that microtubule protein may have an important role in determining cellular function in the elevated temperature environment.

Stability of heating temperature on cytotoxicity

Chinese hamster V79 cells were used to test the cytotoxic effect of heat which alternates between high and low temperatures during the treatment period. This type of temperature fluctuation is often encountered in clinical hyperthermia. Two sets of heating protocols were used in the experiments: Temperature alternates (a) between 42 and 43 degrees and (b) between 42 and 44 degrees in cycle with equal total thermal dose, that is heating temperature X heating time. The effectiveness of these heating protocols expressed in average percentage of cell survival depend on the initial temperature attained and the length of the time of this temperature alternation. If the period of temperature change is short such as every 5 or 10 minutes, the cytotoxicity shows very little difference whether the initial treatment temperature is low or high. However, when the period is longer than 20 minutes, the difference in cell survival between the initial temperature at 42 degrees and at higher temperatures is substantial. Cells obtained from logarithmic or plateau phases of growth yield the same result. The difference is likely resulted from a combination of thermotolerance and step-down heating mechanisms. In addition, the effects of heating temperature fluctuation on cytotoxicity is not altered by a 500 cGy of gamma-ray radiation applied either immediately before or after the heat treatment.

Perfusion of cell spheroids for study by NMR spectroscopy

Abstract Many anchorage-dependent types of cells can be cultured in suspension as small clumps of defined size known as spheroids. These spheroids are particularly suited for study with NMR spectroscopy because, unlike suspensions of single cells, they can be perfused without being carried along with the perfusate, thus eliminating the need for filters or gels. We have successfully maintained human breast cancer cells (MCF-7) and Chinese hamster cells (V 79) in a physiological steady state as spheroids for more than 80 hours in a 10 mm NMR tube using a simple and inexpensive perfusion setup which requires no modification of NMR instrumentation. Although we were primarily interested in obtaining 31P NMR spectra, cell mass was sufficiently high that natural abundance 13C NMR spectra were also readily obtained.

Peak temperature influences on heating rate effect in hyperthermic cytotoxicity

Exponentially growing Chinese hamster V79 cells were heated from 37 degrees C up to 42 degrees C or 43 degrees C at four different heating rates: immediate exposure, 1/2 hour, 1 hour, and 2 hours. The D0s of the heat survival curves for both 42 degrees C and 43 degrees C were determined for the different heating rates. The results show that the decrease in toxicity to cells at 43 degrees C resulting from a slower heating rate appears to be less pronounced than that at 42 degrees C. However, the thermotolerance ratio is found to depend on the specific heating time (the reciprocal of the heating rate), in a linear fashion and it does not seem to depend on the peak temperature. We propose that the difference in the heat exchange on cell membranes at different rates of heating may contribute to the causes for the difference in cell sensitivity to hyperthermia.

Diminution of L cell microvilli following exposure to 25-hydroxycholesterol

The effect of sterol depletion on the topology of mouse L cells was studied by scanning electron microscopy. Treatment of cells with 25-hydroxycholesterol inhibited the synthesis of cellular sterol and diminished the number of microvilli on the cell surface. Simultaneous addition of mevalonic acid or cholesterol to cells during treatment with the inhibitor prevented the loss of microvilli. These results demonstrate that cholesterol is important in maintaining the ultrastructure of the surface membrane of nucleated mammalian cells.

Altered surface topology and membrane functions of rat thymocytes eluted from nylon woll columns

(1) Following incubation of thymocytes with nylon wool at 37 degrees C, the eluted cells showed an increase in the number of microvilli per cell and a concominant elongation of the microvilli (0.22 mum versus 1.15 mum. (2) Cyclic adenosine monophosphate (cylic AMP) levels were lowered by 30-50% in nylon wool-treated thymocytes. (3) Nylon wool-treated cells showed an impaired Na+-dependent amino acid transport system (2-aminoisobutyrate) whereas the Na+-independent amino acid transport system (1-aminocyclopentane-1-carboxylate) was unaffected.

Action of Halothane on Human Erythrocytes Mechanisms of Cell Lysis and Production of Sealed Ghosts

1. Suspension of human erythrocytes in halothane-saturated physiological media induces major cell deformation. 2. Release of halothane from erythrocyte suspensions after equilibrium with the anaesthetic produces complete hemolysis. 3. The membrane fragments isolated after halothane release are in the form of biconcave ghosts, impermeable to macromolecules (lactoperoxidase) and small molecules (ATP). 4. The protein composition of the membranes differs from that of ghosts produced by hypotonic lysis in the lack of components previously shown to be adsorbed at low ionic strength. 5. A hypothesis is presented explaining the action of halothane in terms of both its action on membranes and its capacity to modify water structure. 6. Halothane-induced hemolysis constitutes a simple method for the large-scale production of hemoglobin-depleted, sealed erythrocyte ghosts under physiological ionic conditions.

Effect of the superoxide anion (O2post−) on Na+-dependent amino acid transport

Superoxide anion (O2-) generated either by the autoxidation of dihydroxyfumaric acid (DHF) or enzymatically by the xanthine-xanthine oxidase system inhibited the uptake of 2-aminoisobutyric acid (AIB) in thymocytes. The transport of this non-metabolizable amino acid in thymocytes is mediated by a Na+-dependent mechanism. Inhibition of this transport system by O2- was similar to that observed when radiosensitive lymphocytes are subjected to ionizing radiation. As in irradiated thymocytes, O2- generation affected primarily the maximal rate of uptake of the amino acid (i.e. Vmax). No change was observed in the apparent affinity of the amino acid for its carrier (i.e. Km) or the efflux rate of the amino acid. The data suggests that the superoxide anion may be one of the major species responsible for the observed radiation damage to radiosensitive lymphoid cells.