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J R Lepock

Publications and source records attributed to J R Lepock.

90 records · Page 5Linked to original sources
PubMed1980

Water permeability of mammalian cells as a function of temperature in the presence of dimethylsulfoxide: correlation with the state of the membrane lipids.

The water permeability of V-79 Chinese hamster lung fibroblasts was determined by measuring the rate of cell shrinkage in hypertonic medium using a cell sizer. The water permeability appears to follow Arrhenius kinetics as a function of temperature with a sharp discontinuity at 21 degrees C. An activation energy of 7.0 +/- 1.6 kcal/mole was found below 21 degrees C and 22.8 +/- 3.1 kcal/mole above 21 degrees C. The correlation time of rotation of the spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide was measured as a function of temperature in the cellular membranes, and shows a break at 20 degrees C. A discontinuity was also found in the membrane to water partitioning of the spin label 2,2-dimethyl-5-pentyl-5-butyloxazolidine-N-oxide near 20 degrees C. These breaks may correspond to a membrane lipid phase transition. Dimethylsulfoxide, in the concentration range of 0.2--0.5 M, decreases the water permeability by a factor of two.

Animals↗
PubMed1980

Thermotolerance as a possible cause of the critical temperature at 43 degrees in mammalian cells.

Most mammalian cell lines appear to have a critical temperature near 43 degrees in their response to hyperthermia. This critical temperature is usually defined as the temperature at which a break occurs in an Arrhenius plot of the rate of cell killing. Below this temperature, thermotolerance (the appearance of a more heat-resistant subpopulation during the survival curve) is also observed. This critical temperature may indicate a real change in the cellular response to heat or may be due to thermotolerance. For example, an apparent (measured) increase in the Do relative to the actual Do of the normal or sensitive population can be due to the following: (a) the presence of a normal (or sensitive) subpopulation and a thermotolerant subpopulation: (b) the induction with time of thermotolerance at a hyperthermic temperature in a defined subpopulation; or (c) the induction of thermotolerance with time in the surviving population. Thus, the critical temperature at 43 degrees may be an artifact due to this increase in the measured Do instead of being due to a change in the mode of inactivation of cells at that temperature. If this is true, the 43 degrees critical temperature should be considered as the maximum temperature at which thermotolerance can be induced and not as an infection point indicating two modes of cell killing.

Cell Survival↗
PubMed1979

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells.

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37 degrees C to 22 degrees C with an activation energy of 11.8 +/- 0.2 kcal/mol in this region. There is a sharp decrease in agglutination rate below 22 degrees C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 (2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.

Agglutination↗
PubMed1979

Effect of salt solutions on the radiosensitivity of mammalian cells as a function of the state of adhesion and the water structure.

The radiation isodose survival curve of attached Chinese hamster (V79) cells, subjected to a wide concentration range of salt or sucrose solutions, is characterized by two maxima separated by a minimum. Cells are radioprotected at the maxima (high and low hypertonic salt concentrations) while they are radiosensitized at the minimum (intermediate hypertonic salt concentrations). Both cations and anions can alter the cellular radiosensitivity above and beyond the (osmotic) effect observed for cells treated with sucrose solutions. However, the basic curve shape, except in the case of sulphate salts, remains the same. When these experiments are repeated with single cells in suspension, the isodose survival curve is quite different in that high salt concentrations (greater than 0.9 M) do not protect cells in suspension unlike the case with attached cells. The curve shape is also altered in that the second maximum is absent with many salt solutions. If multicellular spheroids are used for these experiments, the data resemble those for single cell suspensions rather than for attached cells. The radiation survival data for cells in suspension in salt solutions correlate with water proton spin-lattice relaxation time (T1) and, in hypo- and iso-tonic solutions, with cell volume.

Animals↗
PubMed1978

The effects of cotyledon senescence on the composition and physical properties of membrane lipid.

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.

Fabaceae↗
PubMedDate not supplied

Thermal destabilization of transmembrane proteins by local anaesthetics.

Local anaesthetics, in addition to anaesthesia, induce the synthesis of heat shock proteins (HSPs), sensitize cells to hyperthermia, and increase the aggregation of nuclear proteins during heat shock. Anaesthetics are membrane active agents, and anaesthesia appears to be due to altered ion channel activity; however, the direct effect of heat shock is protein denaturation. These observations suggest that local anaesthetics may sensitize cells to hyperthermia by interacting with and destabilizing membrane proteins such that protein denaturation is increased. It is shown, using differential scanning calorimetry (DSC), that the local anaesthetics procaine, lidocaine, tetracaine and dibucaine destabilize the transmembrane domains of the Ca2+ -ATPase of sarcoplasmic reticulum and the band III anion transporter of red blood cells. The transmembrane domain of the Ca2+ -ATPase has a transition temperature (Tm) of denaturation of 61 degrees C which is decreased, for example, to 53 degrees C by 15 mM lidocaine. The degree of destabilization (deltaTm) by each anaesthetic is proportional to the lipid to water partition coefficient, and the increased sensitization by anaesthetics with larger partition coefficients and at higher pH suggests that the uncharged forms of the anaesthetics are responsible for destabilization. A Hill analysis of deltaTm for the Ca2+ -ATPase as a function of the concentration of anaesthetic in water gives dissociation constants (Kd) on the order of 10(-4) M, if binding occurs directly from the aqueous phase. This demonstrates moderate affinity binding. However, dissociation constants of 1-3 M are obtained, if binding occurs through the lipid phase, which demonstrates low affinity binding. Thus, the interaction of local anaesthetics with the Ca2+ -ATPase may be moderately specific or non-specific depending on the mechanism of interaction. The observation that local anaesthetics also destabilize the transmembrane domain of the band III protein of erythrocytes suggests that destabilization of transmembrane proteins is a general property of anaesthetics, which is at least in part a mechanism of sensitization to hyperthermia.

Anesthesia, Local↗
PubMedDate not supplied

Hyperthermia-induced inhibition of respiration and mitochondrial protein denaturation in CHL cells.

Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40 degrees C when plotted in the Arrhenius fashion but is strongly inhibited above 40 degrees C. The protein of mitochondria isolated from V79 cells undergoes structural transitions at 28 and 40 degrees C. This is supported by changes in the fluorescence excitation spectrum of conjugated pyrene maleimide and, to a lesser extent, intrinsic protein fluorophores. Electron spin resonance labelling studies with a derivative of tempo maleimide imply that extensive protein unfolding coincides with the 40 degrees C transition. The structural transition at 40 degrees C correlates well with inhibition of O2 consumption, is irreversible and is probably due to protein denaturation, while the change at 28 degrees C is reversible and has no effect on O2 consumption. Previous studies indicate the presence of a broad lipid transition extending from approximately 8 to 30 degrees C in mitochondrial membranes with all lipids being in the liquid-crystalline state above 30 degrees C. Thus, the onset of the lipid transition may induce the observed protein conformational change at 28 degrees C, but inhibition of respiration above 40 degrees C can be explained by protein denaturation alone. The region from 28 to 40 degrees C of stable protein conformation corresponds to the temperature range of V79 cell growth.

Animals↗
PubMedDate not supplied

Hyperthermia (heat shock)-induced protein denaturation in liver, muscle and lens tissue as determined by differential scanning calorimetry.

Protein denaturation has been shown to occur in cells during heat shock and is closely correlated with the cellular responses to hyperthermia; however, little is known about protein denaturation in tissue. This study describes an analysis of endothermic transitions in the hyperthermic region using differential scanning calorimetry (DSC) in liver, white muscle, and lens tissue from Wistar rat, New Zealand white rabbit, and Rainbow trout. Complex DSC profiles consisting of several transitions were obtained for each tissue. Evidence is given that these transitions are due primarily to protein denaturation. Onset temperatures of denaturation (Tl) for rat liver, muscle, and lens are about 38, 39 and 48 degrees C, respectively. Thus, significant protein denaturation occurs in liver and muscle during mild hyperthermia (40-45 degrees C) with lens considerably more stable. The values of Tl for the same tissue from the different animals correlates well with body temperature (rabbit 39.4, rat 38.2, and trout grown at 11 degrees C); Tl increased in the same order as the body temperature for each tissue. Thus, there is correlation between the onset temperature for protein denaturation in these tissues and body temperature.

Animals↗