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Biomedical subjects

J Kruuv

Publications and source records attributed to J Kruuv.

At least 37 records · Page 2Linked to original sources
PubMed1986

Effects of pre- and post-thaw cell-to-cell contact and trypsin on survival of freeze-thaw damaged mammalian cells.

When multicellular spheroids, which simulate small bits of tissue, are exposed to a freeze-thaw (FT) cycle, the survival of the individual cells in the spheroid is higher if the cells of the spheroid are trypsinized and plated as single cells immediately after thawing than if the spheroid is allowed to remain intact for 4 hr and then trypsinized for plating. The results imply either that cell-to-cell contact inhibits repair of potentially lethal damage (PLD) or that accumulation of additional lethal or sublethal damage during the post-thaw period for cells in contact is taking place. Pre- and post-FT trypsinization of single cells indicate that trypsin does not enhance repair of PLD caused by a FT cycle.

Animals↗
PubMed1986

Effects of butylated hydroxytoluene on membrane lipid fluidity and freeze-thaw survival in mammalian cells.

Butylated hydroxytoluene (BHT) increases the fluidity of membrane lipids in the hydrocarbon but not the polar regions, as measured by electron spin resonance spin label probes. BHT also sensitizes nucleated mammalian cells to freeze-thaw damage as measured by colony formation survival assays. Furthermore, the membranes of BHT-exposed cells are more susceptible to physical stress, as reflected by the BHT-induced sensitization to hypotonic stress. Since others have shown that BHT induces hexagonal phase lipids in lipid bilayers, this phenomenon may also influence the above survival results.

Animals↗
PubMed1985

Structural analyses of various Cu2+, Zn2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy.

The thermal denaturation profile of the Cu2+, Zn2+ metalloenzyme, bovine superoxide dismutase, consists of two primary components, the major component denatures irreversibly at Tm = 104 degrees C with a total enthalpy (delta Hcal) of 7.30 cal/g. Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers Tm to 96 degrees C and delta Hcal to 6.96 cal/g. The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 degrees C with an enthalpy only one-half that of the holo-form. The reduced thermal stability, which indicates a greater ability to change conformation, may explain the previously observed much greater membrane binding of the apo-enzyme. Reconstitution with Zn2+, Cu2+, or Zn2+ and Cu2+ raises Tm to 80, 89, or 102 degrees C, respectively, with corresponding increases in the enthalpy. Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation. The effect of Cu2+ alone is greater than that of Zn2+, although both are needed for full stability. Raman spectroscopy indicates little difference in secondary structure between the apo- and holo-forms, implying that the increased stability due to metal binding is not caused by an extreme structural reorganization. The value of Tm of canine and yeast superoxide dismutase is also lowered by reduction of Cu(II). The reduced form of the yeast enzyme denatures irreversibly, as do all forms of the bovine and canine enzymes, but the oxidized form is unique in that it denatures reversibly. Thus, the copper ion must be oxidized for renaturation and appears to act as a nucleation site.

Animals↗
PubMed1985

Factors influencing survival of mammalian cells exposed to hypothermia. II. Effects of various hypertonic media.

Survival of Chinese hamster lung (V79) cells, exposed as a function of time to hypothermia in tissue culture, in isosmotic and various hypertonic media was measured using a colony assay. The mechanism of hypothermic cell killing is different above and below 7 degrees C in this cell line. Addition of NaCl or mannitol to increase the tonicity to 400 mOsm greatly decreased the survival at 10 degrees C while addition of KCl had no significant effect. When these experiments were repeated at 5 degrees C, addition of either NaCl, KCl, or mannitol was detrimental to long-term cell survival. Furthermore, addition of mannitol to the medium did not improve survival when cells were stored at 7 degrees C. Addition of KCl at 5 or 10 degrees C or NaCl at 5 degrees C only affected the cells' ability to accumulate sublethal damage, while addition of mannitol at 5 or 10 degrees C affected both of the above and the cold sensitivity of the cells. Addition of NaCl at 10 degrees C only affected the latter. These experiments suggest that prevention of cell swelling by these conditions, while possibly necessary during clinical hypothermic organ storage, is detrimental to single cell survival at these temperatures.

Animals↗
PubMed1983

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers.

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.

Animals↗
PubMed1983

Rotational diffusion of TEMPONE in the cytoplasm of Chinese hamster lung cells.

The correlation time for rotational diffusion (tau R) of 2,2,6,6-tetramethyl-4-piperidone-N-oxide (TEMPONE) in Chinese hamster lung (V79) cells has been measured. For these cells in an isosmotic solution at 20 degrees C, tau R = 4.18 X 10(-11) s, approximately 3.6 times greater than tau R = 1.17 X 10(-11) s in water. The relationship between tau R and viscosity was investigated in a number of glycerol-water (0-50%) and sucrose-water (20-40%) solutions and a constant Stokes-Einstein volume of 44 A3 was found for TEMPONE in solutions of less than 20% glycerol and sucrose. This gives an average shear viscosity (for rotation of a small molecule) of 0.038 poise for the cytoplasm. When nonsecular terms were used in the calculation of tau R, the activation energies for rotation of TEMPONE in the above solutions correlated well with the activation energies for shear viscosity. The viscosity increases as the cell is shrunk in hypertonic solutions. It also increases with decreasing temperature with an activation energy of 3.7 kcal/mol, about the same as the activation energy for the viscosity of pure water. The rotational correlation times were carefully calculated considering inhomogeneous line broadening, non-Lorentzian line shapes, the need for accurate tensor values and nonsecular terms.

Animals↗
PubMed1983

Thermotropic lipid and protein transitions in chinese hamster lung cell membranes: relationship to hyperthermic cell killing.

Exposure of mammalian cells to hyperthermic temperatures (ca. 41-45 degrees C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40-41.5 degrees C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7-8 and 23-36 degrees C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40-41.5 degrees C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 degrees C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 degrees C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

Animals↗
PubMed1983

Characterization and radiation response of a heat-resistant variant of V79 cells.

A thermoresistant variant of the established cell line V79-S171-W1 was isolated after treatment with nitrosoguanidine and repeated heat treatments at 42.6 to 43 degrees C, and showed an enhanced ability to survive at 42.6, 43.5, and 44.5 degrees C. The rates of inactivation of the normal and heat-resistant lines differed by approximately a factor of 2 over this temperature range. This level of thermoresistance was stable for the first 80 doublings, but was lost by 120 doublings. This may have been due to a reversion to the normal V79 line since there was no continuous selection pressure and the thermoresistant variant, which was designated at HR7, had a longer average doubling time. Transient thermotolerance was induced in both the V79 and HR7 cells by a 10-min exposure to 44.5 degrees C. After 3 hr incubation at 37 degrees C, both cell lines had an identical sensitivity to further exposure to 44.5 degrees C. Thus the long-term thermoresistance of the HR7 cells may be due to a permanent induction of a low level of thermotolerance. The (ionizing) radiation survival curves and the ability to repair sublethal radiation damage were identical for the thermoresistant variant and the parent cell line.

Animals↗
PubMed1981

Perturbation of lipid membranes by organic pollutants.

The ability of a range of organic pollutants--hexachlorobenzene, mirex(1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachlorooctahydro-1,3,4-metheno-1H cyclobuta(cd) pentalene), 1,3,5-trichlorobenzene, 2,4,6-trichlorophenol, p-nitrophenol, p-chlorophenol, DDT, and pentachlorophenol--to perturb liposomes of dipalmitoyl phosphatidylcholine (DPPC) has been measured by differential scanning calorimetry. The degree of perturbation was measured by the increase in breadth of the main DPPC phase transition in both heating and cooling scans. DDT and the phenol derivatives were effective perturbers of phospholipid, broadening the transition by as much as 12-fold. Hexachlorobenzene and mirex did not perturb at all when mixed with DPPC at concentrations as high as 20 mol%, although 1,3,5-trichlorobenzene caused slight broadening of the main transition at this concentration. Perturbation is facilitated by the presence of a hydroxyl group on the benzene ring and hindered by increasing degrees of chloride substitution. An apparent correlation exists between the extent of phospholipid perturbation measured by differential scanning calorimetry and LD50 values for these compounds taken from the literature. This suggests the possibility of formulating an "index of perturbation" which could be used to screen certain classes of organic compounds for potential biological toxicity on a routine basis.

Animals↗
PubMed1981

Effect of membrane lipid perturbers on the temperature dependence of repair of sublethal and potentially lethal radiation damage.

The repair of sublethal radiation damage (SLD) in Chinese hamster (V79) cells was investigated as a function of temperature in the presence and absence of the membrane lipid perturbers, butylated hydroxytoluene (BHT) or adamantanone, which decrease the viscosity of the membrane lipids. Addition of 0.01 mM BHT to the cells significantly increased the amount of repair of SLD over controls from 0 to 25 degrees C but not from 30 to 37 degrees C. The amount of repair of SLD decreased as a function of temperature to about 20 degrees C and remained relatively constant thereafter till about 2.5 degrees C where it began to decrease again. The change in the amount of repair at approximately 20 degrees C coincides with a membrane lipid phase transition, as seen by spin labelling, in these cells. The repair of potentially lethal damage (PLD) was not increased by BHT at the two temperatures investigated (5 and 20 degrees C). In fact, additional PLD was expressed when cells were incubated post-irradiation at these temperatures. The results imply that at least a component of the SLD repair system is membrane associated and that the SLD and PLD repair systems are independent.

Adamantane↗
PubMed1981

Nuclear magnetic resonance spin-lattice times of normal and transformed cultured mammalian cells and of normal and neoplastic animal tissues.

Proton spin-lattice relaxation times (T1) for normal tissues and cultured cells, and for neoplastic tissues and transformed culture cells, were measured. Increases in T1 of neoplastic tissue and normal tissues of mice with neoplasms were observed. No difference was found between T1 of normal 3T3 cells and of an SV40 viral transformed cell line. These data imply that changes in T1 of neoplastic tissue may not be due to intrinsic properties of cancerous cells but rather to physiological interaction of the neoplasm with its physiological environment. The measurement of several tissues of rat showed a good correlation between T1 and tissue water content, and addition of water by glass capillary increased T1 of samples. For samples in sealed NMR tubes, however, changes in T1 were incurred by boiling, freezing, or storage at various temperatures. These data imply that changes in T1 may be influenced by cellular water content as well as macromolecular conformation.

Adenocarcinoma↗
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↗