Kenneth W. Minton

Temperature-induced homeoviscous adaptation of Chinese hamster ovary cells.

Exponential and plateau phase Chinese hamster ovary cells were maintained for 3 days at 32, 37, 39 or 41 degrees C. The effect of growth temperature on the fluidity and composition of the cellular membranes, and on the ability of the cells to resist a subsequent heat treatment at 43 degrees C, was measured. Cells grown at temperatures above 37 degrees C displayed increased resistance or tolerance to a 43 degree C heat treatment, whereas cells grown at 32 degrees C were sensitized to heat. Extensive cell division was not required for expression of heat tolerance. Membrane fluidity, as determined by the degree of rotational mobility of the fluorescent probe diphenylhexatriene, decreased with increasing growth temperatures, but the relationship did not hold in exponential phase cells grown at 32 degrees C. The cholesterol : phospholipid molar ratio correlated with the fluorescence polarization values, suggesting that the cells are able to adjust membrane fluidity by varying the concentration of cholesterol. The results are compatible with the concept of homeoviscous adaptation: that organisms strive to maintain an optimal level of membrane fluidity and when grown at a different temperature will alter the lipid composition in order to maintain this level. Up until now, cholesterol has not been implicated in this process.

Survival and concanavalin-A-induced capping in CHO fibroblasts after exposure to hyperthermia, ethanol, and X irradiation.

Two parameters, concanavalin-A-induced capping and clonogenicity, were examined in HA-1 fibroblasts after exposure to heat, ethanol, x irradiation, and heat plus deuterium oxide. Capping and clonogenicity were shown to decrease over time in samples exposed to 43, 44, and 45/sup 0/C, while both parameters remained unaffected at 37, 41, and 42/sup 0/C. Ethanol exposure showed a survival decrease at a concentration of 8% but no significant effect on survival at 4, 6, and 7%. Capping, however, decreased over time at ethanol concentrations of 6, 7, and 8%. X irradiation had no effect on capping over a dose range of 0 to 1400 rad, while survival was markedly decreased. At 37/sup 0/C, exposure to 20, 40, and 80% deuterium oxide showed no effect on either survival or capping. However, at 43/sup 0/C, the same concentrations of deuterium oxide markedly increased both capping and clonogenicity over that of cells exposed to 43/sup 0/C without deuterium oxide. Potential mechanisms accounting for the heat- and ethanol-induced impairment of capping ability are inhibition of cellular energy metabolism and/or alteration of plasma membrane composition.

Lack of correlation between membrane lipid composition and thermotolerance in Chinese hamster ovary cells

Abstract Membrane composition and fluidity, and survival of Chinese hamster ovary fibroblasts have been examined following various thermal exposures. It has been found that enhanced thermal resistance following brief exposure to 43°C is not accompanied by detectable membrane lipid alterations. This is in contrast to membrane alterations that occur following adaptation to elevated temperatures compatible with growth (39°C and 41°C).

DNA Deoxyribophosphodiesterase and an Activity That Cleaves DNA Containing Thymine Glycol Adducts in Deinococcus radiodurans

Deinococcus radiodurans is the most radioresistant bacterium discovered to date. Recently it has been demonstrated that this organism contains the DNA repair enzyme uracil-DNA glycosylase and an apurinic/apyrimidinic (AP) endonuclease that may function as part of a DNA base excision repair pathway. We demonstrate here that a DNA deoxyribophosphodiesterase activity that acts on incised AP sites in DNA to remove deoxyribose-phosphate groups is found in lysates prepared from D. radiodurans cells. The partially purified activity was found to be smaller in size than the E. coli dRpase activity, with an estimated molecular weight of 25-30 kDa. In addition, an activity that recognizes and cleaves DNA containing thymine glycols was also detected, with a molecular weight of approximately 30 kDa. This enzyme may be analogous to the thymine glycol glycosylase/AP lyase endonuclease III of E. coli.

Conformational studies of aqueous melittin: collisional quenching of tryptophan-19 fluorescence in melittin

Abstract Conformational changes in melittin during self-association were investigated by monitoring the quenching of fluorescence of the single tryptophan residue. Acrylamide and the monovalent ions, Cs + and I − , quench melittin by a diffusion-controlled, collisional mechanism, as judged by the viscosity dependence of the process. The quenching constants ( K Q ) for monomeric melittin suggest the tryptophan residue is fully solvent exposed. Formation of a soluble melittin tetramer decreases the accessibility of tryptophan to quenching, most dramatically for Cs + . The relationship of K Q and pH indicates that monomeric melittin has ionization constants of 10 −6.8 and 10 −8.7 M which affect quenching. The acidic and basic forms of melittin have conformations which hinder collisional quenching. The melittin tetramer also has a pH-dependent change in Trp-19 quenching, with two protonic ionizations of 10 −7.4 M which lead to a conformation with lower Trp exposure upon deprotonation. The p K values obtained for collisional quenching are in excellent agreement with values obtained by chemical reactivity (Quay, S.C. and Tronson, L.P. (1983) Biochemistry 22, 700), and suggest that Lys-21 and -23 are responsible for the pH-dependent conformational changes which affect Trp-19 accessibility. The presence of phosphate or pyrophosphate ion leads to melittin conformations with lowered solvent exposure of Trp-19. The lowered accessibility of Trp-19 upon tetramerization supports the hypothesis that tetramer formation involves association of the nonpolar face of the amphipathic helix of melittin by hydrophobic interactions.

Enzymes involved in thymine dimer excision in bacteriophage T4-infected Escherichia coli.

A study was made of the kinetics of excision of thymine-containing pyrimidine dimers from prelabeled phage T4 during infection of Escherichia coli . In wild-type hosts excision of dimers was observed immediately after infection, but ceased at some maximal level five to seven minutes later. In host cells defective in DNA polymerase I (either polymerizing activity, 5′→3′ exonuclease activity, or both) there was a significant reduction in both the rate and the final extent of excision during the first five to seven minutes after infection. Mutation in the rec B gene was found to have no effect on the kinetics of excision. The kinetics of dimer excision in cells infected with a T4 mutant defective in DNA synthesis were no different from that observed with wild-type phage. Thus the cessation of dimer excision at about five to seven minutes after infection is not obviously related to the onset of phage DNA replication.

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).