Biomedical subjects

J R Lepock

Publications and source records attributed to J R Lepock.

At least 55 records · Page 3Linked to original sources

PubMed1992

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance.

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.

Adaptation, Physiological↗

PubMed1991

Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum.

The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.

Amino Acids↗

PubMed1991

Cycloheximide increases the thermostability of proteins in Chinese hamster ovary cells.

Protein denaturation resulting from temperatures between 42.0 degrees C and 50 degrees C has been observed and implicated as the lethal lesion for hyperthermic cell killing. A logical corollary is that protection against hyperthermic killing requires stabilization of cellular proteins against thermal denaturation. To test this, Chinese hamster ovary cells were treated with the heat protector cycloheximide and then subjected to differential scanning calorimetry to measure protein denaturation. Cycloheximide stabilized proteins that denatured between 42 degrees C and 52 degrees C in control cells by increasing their transition (denaturation) temperature by an average of 1.3 degrees C. In addition, cycloheximide reduced the cytotoxicity of actinomycin D and adriamycin, suggesting that protein stabilization protects cells against stresses other than hyperthermia.

Animals↗

PubMed1991

Factors influencing survival of mammalian cells exposed to hypothermia. V. Effects of hepes, free radicals, and H2O2 under light and dark conditions.

Cytotoxicity resulting from the interaction of fluorescent light from a flow hood with Hepes-buffered cell culture medium at room temperature was demonstrated. Toxicity was prevented by keeping both cells (V79 Chinese hamster) and medium shielded from direct fluorescent light ("dark conditions") or by supplementing the medium with 10 micrograms/ml catalase; this suggests that extracellular hydrogen peroxide is a major cause of the lethal effect under "lighted conditions." No sensitization resulted from the exposure of cells in a sodium bicarbonate (SBC)-buffered medium to fluorescent light, nor in a catalase supplemented SBC-buffered medium. The Hepes/light reaction during routine cell manipulations presensitized cells to hypothermia damage in the dark with the presensitization being more severe for 5 than for 10 degrees C hypothermic exposure. Presensitization was prevented by performing the complete experiment under dark conditions or by supplementing the medium with 10 micrograms/ml catalase. However, catalase did not improve the hypothermic survival when experiments were performed under dark conditions. Hence, 10 micrograms/ml catalase does not protect cells from hypothermic (5 and 10 degrees C) damage per se, but rather from Hepes/light sublethal damage which interacts with hypothermic sublethal damage to result in lethal lesions. Additionally, under dark conditions, superoxide dismutase (SOD), allopurinol, catalase plus SOD, DMSO, or mannitol did not improve survival when present during hypothermic storage, suggesting that extracellular superoxide anion, hydrogen peroxide, or hydroxyl radicals are not the cause of cell killing under conditions of pure hypothermia uncomplicated by prehypothermic ischemia or hypoxia.

Animals↗

PubMed1990

Contribution of conformational stability and reversibility of unfolding to the increased thermostability of human and bovine superoxide dismutase mutated at free cysteines.

The conformational stability and reversibility of unfolding of the human dimeric enzyme Cu Zn superoxide dismutase (HSOD) and the three mutant enzymes constructed by replacement of Cys6 by Ala and Cys111 by Ser, singly and in combination, were determined by differential scanning calorimetry. The differential scanning calorimetry profile of wild-type HSOD consists of two components, which probably represent the unfolding of the oxidized and reduced forms of the enzyme, with denaturation temperatures (Tm) of 74.9 and 83.6 degrees C, approximately 7 degrees lower than those for bovine superoxide dismutase (BSOD). The conformational stabilities of the two components of the mutant HSOD's differ only slightly from those of the wild type (delta delta Gs of -0.2 to +0.8 kcal/mol of dimer), while replacement of the BSOD Cys6 by Ala is somewhat destabilizing (delta delta G of -0.7 to -1.3 kcal/mol of dimer). These small alterations in conformational stability do not correlate with the large increases in resistance to thermal inactivation following substitution of free Cys in both HSOD and BSOD (McRee, D.E., Redford, S.M., Getzoff, E.D., Lepock, J.R., Hallewell, R.A., and Tainer, J.A. (1990) J. Biol. Chem. 265, 14234-14241 and Hallewell, R.A., Imlay, K.C., Laria, I., Gallegos, C., Fong, N., Irvine, B., Getzoff, E.D., Tainer, J.A., Cubelli, D.E., Bielski, B.H.J., Olson, P., Mallenbach, G.T., and Cousens, L.S. (1991) Proteins Struct. Funct. Genet., submitted for publication). The reversibility of unfolding was determined by scanning part way through the profile, cooling, rescanning, and calculating the amount of protein irreversibly unfolded by the first scan. The order of reversibility at a constant level of unfolding is the same as the order of resistance to inactivation for both the HSOD and BSOD wild-type and mutant enzymes. Thus, the greater resistance to thermal inactivation of the superoxide dismutase enzymes with free Cys replaced by Ala or Ser is dominated by a greater resistance to irreversible unfolding and relatively unaffected by changes in conformational stability.

Animals↗

PubMed1990

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Bacillus↗

PubMed1990

Changes in crystallographic structure and thermostability of a Cu,Zn superoxide dismutase mutant resulting from the removal of a buried cysteine.

In principle, protein thermostability depends on efficient interior packing of apolar residues and on avoidance of irreversible denaturation in the unfolded state. To study these effects, the single free cysteine in the highly stable enzyme bovine Cu,Zn superoxide dismutase was mutated to alanine (Cys6----Ala), and the recombinant protein was expressed in yeast, purified, characterized for reversible and irreversible denaturation, crystallized isomorphously to the wild-type enzyme, and used to determine the atomic structure. Removal of the chemically reactive thiol significantly decreased the rate of irreversible denaturation (as monitored by thermal inactivation at 70 degrees C), but the observed energetic cost (delta delta G of 0.7-1.3 kcal/mol as determined by differential scanning calorimetry) was much less than predicted from either the change in hydrophobicity or packing due to removal of the interior sulfur atom. X-ray diffraction data were collected to 2.1-A resolution using an area detector, and the atomic model for the mutant enzyme was determined by fitting to electron density difference maps, followed by reciprocal space refinement both with stereochemical restraints using PROLSQ and with molecular dynamics using X-PLOR. The refined 2.1-A resolution crystallographic structure suggests that small concerted and compensating shifts (less than 0.5 A) of the surrounding side chains and of the adjacent N- and C-terminal beta-strands significantly reduced the energetic cost of the interior mutation by improving packing and stereochemistry in the mutant enzyme. Taken together, these results differentiate between the effects of reversible and irreversible processes as they impact the design of thermostable proteins and suggest that relatively subtle concerted shifts can significantly reduce the energetic cost of evolutionary variation in internal residues of proteins with Greek key beta-barrel folds.

Alanine↗

PubMed1990

Thermal denaturation of the Ca2(+)-ATPase of sarcoplasmic reticulum reveals two thermodynamically independent domains.

Inactivation of Ca2+ uptake and ATPase activity of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was measured and compared to the thermal denaturation of the enzyme as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. Two fluorophores were monitored: intrinsic tryptophan (localized in the transmembrane region) and fluorescein isothiocyanate (FITC)-labeled Lys-515 (located in the nucleotide binding domain). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Activation energies (EA) and frequency factors (A) for inactivation were obtained for the enzyme in 1 mM EGTA and 1 mM Ca2+. These were transformed to a transition temperature for inactivation, Tm (defined as the temperature of half-inactivation when temperature is scanned upward at 1 degree C/min). All denaturation profiles were fit with an irreversible model to obtain EA and Tm for each transition, and the values of these parameters for denaturation were compared to the values for inactivation. In EGTA, denaturation obeys a single-step model (Tm = 49 degrees C), but a two-step model is required to fit the DSC provile of the enzyme in 1 mM Ca2+. The specific locations of tryptophan and the fluorescein label were used to demonstrate that denaturation in Ca2+ occurs through two distinct thermodynamic domains. Domain I (Tm = 50 degrees C) consists of the nucleotide binding region and most likely the phosphorylation and transduction regions [MacLennan, D. H., Brandl, C. J., Korczak, B., & Green, N. M. (1985) Nature 316, 696-700].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry.

Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC.

Animals↗

PubMed1990

Factors influencing survival of mammalian cells exposed to hypothermia. IV. Effects of iron chelation.

Survival of V-79 Chinese hamster cells was assessed by colony growth assay after hypothermic exposure in the presence of iron chelators. At 5 degrees C, maximum protection from hypothermic damage was achieved with a 50 microM concentration of the intracellular ferric iron chelator Desferal. A 3-hr prehypothermic incubation with 50 microM Desferal followed by replacement with chelator-free medium at 5 degrees C also provided some protection. This was not observed when the extracellular chelator DETA-PAC (50 microM) was used prior to cold storage. Treating 5 degrees C-stored cells with Desferal just prior to rewarming was ineffective, but treating cells with Desferal during hypothermia exposure after a significant period of unprotected cold exposure ultimately increased the surviving fraction. Submaximal protection during hypothermia was achieved to various degrees with extracellular chelators at 5 degrees C, including 50 microM DETAPAC and 110 microM EDTA. EGTA (110 microM) had little effect. The sensitization of cells at 5 degrees C with 200 microM FeCl3 could be reduced or eliminated with Desferal in accordance with a 1:1 binding ratio. At 10 degrees C, 50 microM Desferal, 50 microM DETAPAC, and 110 microM EDTA were as or less effective in protecting cells than at 5 degrees C. An Arrhenius plot of cell inactivation rates shows a break at 7-8 degrees C, corresponding to maximum survival for control cells and cells in 50 microM Desferal; however, the amount of protection offered by the chelator increases with decreasing temperature below about 19 degrees C, and sensitization increases above that point. It has not previously been shown that iron chelators protect against cellular hypothermia damage which is uncomplicated by previous or simultaneous ischemia. This may be relevant to the low-temperature storage of transplant organs, in which iron of intracellular origin and in the perfusate may be active and damaging.

Animals↗

PubMed1990

Mechanism of freeze-thaw damage to liver alcohol dehydrogenase and protection by cryoprotectants and amino acids.

Multiple freeze-thaw (FT) cycles, with complete melting between cycles, resulted in an exponential decline in liver alcohol dehydrogenase (LADH) enzyme activity. The reduction in activity of LADH as a result of FT damage was proportional to the decrease in the intensity of the tryptophan fluorescence of the enzyme. Treatment with urea resulted in a similar relationship between tryptophan fluorescence intensity and inactivation. Evidence from fluorescence and activity studies from the same sample, as well as gel electrophoresis, indicates that damage to LADH from a FT cycle, resulting in inactivation, is likely an unfolding of the enzyme rather than separation of subunits or aggregation of enzymes at the enzyme concentrations and cooling rates used. A nonexponential decline in enzyme activity, as a function of the number of FT cycles, can be achieved if complete melting between cycles is not allowed or if the samples are stored at +4 degrees C for 24 hr following the last FT cycle, prior to assay. In the latter case, a partial recovery in enzyme activity is seen. "Seeding," while lowering the enzyme activity, is desirable to achieve consistent results without the artifacts that are introduced if not used. Amino acids were tested for their effectiveness as cryoprotectants. From the results of this study, the mean fractional area loss of amino acid residues upon incorporation in globular proteins (f) is inversely proportional to the FT protection by these free amino acids. Thus, amino acid residues which tend to be found at the surface of proteins (e.g., glutamate) improve the FT survival of LADH, when added as the free amino acid, while those amino acids which are found in the interior of proteins (e.g., valine, leucine) sensitize LADH to FT damage. The pattern of protection ("fingerprint") of LADH by various amino acids is different from that of living cells. Furthermore, unlike the case with cells, glutamine and DMSO do not act independently when protecting LADH.

Alcohol Dehydrogenase↗

PubMed1989

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins.

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗

PubMed1989

Protein degradation in CHL V79 cells during and after exposure to 43 degrees C.

Cellular protein degradation during and following hyperthermia should be altered due to increased enzymatic activity at elevated temperatures, inhibition of protein synthesis, and denaturation of proteins. We have previously demonstrated by differential scanning calorimetry that approximately 1-2% of total CHL V79 cellular protein denatures during a 10- to 15-min exposure to 43 degrees C (J. E. Lepock et al., J. Cell. Physiol. 137, 14-24 (1988)). Proteolysis was measured during and after exposure to 43 degrees C. The decay curves of the degradation of [3H]Leu-labeled proteins are fit well by a double exponential; however, each component is the sum of the decay curves of a large number of proteins, probably with a distribution of rates of degradation. At 37 degrees C a fast-decaying component (T1/2 congruent to 1.3 h), representing short-term proteins, and a slow-decaying component (T1/2 congruent to 50 h), representing long-term proteins, are observed. At 43 degrees C the rate of degradation of the fast-decaying component is stimulated three- to fivefold (to T1/2 = 0.27-0.45 h). After return to 37 degrees C, the rate of degradation of the slow-decaying component is depressed twofold (to T1/2 = 109-141 h). The period of depression is dose dependent (i.e., time at 43 degrees C) and recovers at approximately the same time as resumption of protein synthesis and growth. Overall stimulation of degradation lasts for approximately 15 min at 43 degrees C and, coupled with an inhibition of synthesis, leads to the loss of at least a small percentage of total cellular protein. It is likely that the initial stimulated degradation is in part due to increased substrate in the form of denatured protein, further supporting the denaturation of proteins during hyperthermia.

Animals↗

PubMed1988

Amino acid pools in CHL V79 cells during induction of thermotolerance: reduction in free intracellular glutamine.

The amino acid pools in Chinese hamster lung V79 cells were measured as a function of time during hyperthermic exposure at 40.5 degrees and 45.0 degrees C. Sixteen of the 20 protein amino acids were present in sufficient quantity to measure accurately. The total amino acid pool and all individual amino acids, except glutamine, remained relatively constant for at least 90 min at 40.5 degrees C and for 30 min at 45 degrees C. The glutamine pool decreased rapidly to 20% of its control value within 30 min at 40.5 degrees C with a T1/2 = 15 min. At 45 degrees C, the decrease was 36%. Thermotolerance developed at 40.5 degrees C with a T1/2 = 30 min; thus, glutamine depletion preceeds the development of thermotolerance. The depletion of glutamine is probably due to increased metabolism and oxidation of glutamine through the TCA cycle at hyperthermic temperatures. Glutamine, as is true for other amino acids, was shown to protect proteins from thermal inactivation and V79 cells from hyperthermic killing when added in excess (4-10 mM) to the medium during heat stress. However, the stability of the total amino acid pool during the development of thermotolerance indicates that resistance to heat does not result from the accumulation of amino acids which then protect against thermal damage. The effects of the large decrease in the glutamine pool are unknown, although glutamine depletion may act as a signal for part of the heat shock response.

Amino Acids↗

PubMed1988

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response.

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.

Alcohols↗

PubMed1988

Protective effect of L-glutamine against freeze-thaw damage in mammalian cells.

L-Glutamine at 18 mM protects mammalian cells against freeze-thaw (FT) damage by a factor of about 6, depending on FT conditions, in balanced salt solutions. While not nearly as effective a cryoprotectant as dimethyl sulfoxide (DMSO) or propylene glycol (PG), the mechanism of protection by glutamine appears to be independent from that of DMSO or PG; thus, 18 mM glutamine is effective at reducing FT damage in combination with these agents. These combinations allow lower concentrations of the more toxic agents DMSO and PG to be used in FT medium. There is no pre-FT or post-FT effect of glutamine when cells are exposed to a FT cycle in balanced salt solutions. Hence, protection is due to its presence during the FT-cycle. The presence of 2 mM L-glutamine in Eagle's basal medium is sufficient to account for cryoprotection by this medium.

Animals↗

PubMed1987

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography.

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.

Amino Acids↗

PubMed1987

Protection of the membrane calcium adenosine triphosphatase by cholesterol from thermal inactivation.

There is correlative evidence that one mechanism of cellular thermoresistance is an increased level of membrane cholesterol. The hypothesis that cholesterol protects membrane proteins from thermal inactivation was tested using Ca-ATPase as a model. The intracellular Ca2+- and Mg2+-dependent ATPase from muscle sarcoplasmic reticulum was reconstituted into lipid mixtures containing different amounts of cholesterol [cholesterol/phospholipid molar ratio (C/PL) = 0.1 or 0.3]. The rate of thermal inactivation of calcium uptake activity of the reconstituted vesicles with C/PL = 0.3 was found to be significantly lower than those with C/PL = 0.1 in the temperature range 43-47 degrees C where hyperthermic cell killing occurs. At 43 degrees C, this is equivalent to a 3 degrees C temperature shift. ATP hydrolysis of Ca-ATPase was found to be substantially heat resistant in reconstituted vesicles with C/PL = 0.1 or 0.3. Glycerol (10%) protects while ethanol (2.5%) and the local anesthetics dibucaine, tetracaine, and procaine sensitize the thermal inactivation of calcium uptake. To investigate the molecular mechanisms of thermal inactivation and cholesterol protection, the responses of the physical state of the lipid and protein conformation to hyperthermic sensitizers and protector were monitored using fluorescent and spin label probes and circular dichroism, respectively. The calcium uptake inactivation appears to be due to a direct thermotropic conformational change (denaturation) of the protein. Cholesterol raises the temperature of inactivation, as does glycerol, while ethanol and the local anesthetics lower it.

Adenosine Triphosphate↗