Cold shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures

Abstract Brief exposure to low (0^oC) or high (40^oC) temperature elicits a protective response that prevents injury when the flesh fly, Sarcophaga crassipalpis Macquart, is subjected to more severe cold (-10^oC) or heat (45^oC). Both the low and high temperature responses were found in all developmental stages of the fly, but were most pronounced in the pupal and pharate adult stages. The protective responses generated by brief exposure to 0 or 40^oC appear similar in that both result in a rapid acquisition of cold or heat tolerance and a loss of protection after the flies are returned to 25^oC. The protection generated by chilling is obvious within 10 min of exposure to 0^oC while a 30 min exposure to 40^oC is required to induce the high temperature protection. High temperature protects against cold shock injury within a narrow range (around 36^oC) but we have no evidence that low temperature can protect against heat injury. We previously demonstrated that the rapid increase in cold tolerance correlates with concomitant increases in glycerol concentration, but in this study we found no significant elevation in glycerol in heat-shocked flies. Thus the physiological and biochemical bases for the rapid responses to cold and heat appear to be different.     

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

Distance distributions in native and random-coil troponin I from frequency-domain measurements of fluorescence energy transfer

We used time-dependent fluorescence energy transfer to determine the distribution of donor-to-acceptor distances in native and denatured troponin I(TnI). The single tryptophan residue (Trp 158) of TnI served as the donor (D), and the acceptor (A) was a labeled cysteine residue (Cys 133). The time-dependent intensity decays of the donor were measured by the frequency-domain method from 10 to 320 MHz. The frequency response of the donor emission, in the absence and presence of acceptor, was used to recover the distribution of D to A distances, using an algorithm that accounts for the intrinsic multiexponential decay of the donor. In the native state the D–A distribution is characterized by an average distance of 23 Å and a half-width of 12 Å. Denaturation results in a modest increase in the average distance to 27 Å, and a dramatic increase in half-width to 47 Å. We believe the ability to recover distance distributions will have numerous applications in the characterization of biological macromolecules.     

The aggregation state of mellitin in lipid bilayers. An energy transfer study

We used fluorescence non-radiative energy transfer to measure the self-association of melittin in solution and when bound to lipid bilayers. Energy transfer occurred from the tryptophan residue of unlabeled melittin to an N-methyl anthraniloyl residue covalently bound to a basic lysine residue on melittin. The extent of energy transfer from tryptophan to the label was found to increase severalfold upon the salt-induced tetramerization of melittin. When bound to vesicles of dimyristoyl-L-alpha-phosphatidylcholine, the extent of energy transfer was found to be equivalent to that of monomeric melittin, irrespective of the presence of monomeric or tetrameric melittin in the aqueous phase. We conclude that membrane-bound melittin is monomeric.     

Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry

We report the first anisotropy decays of protein fluorescence obtained using a frequency-domain fluorometer. The ultraviolet light source (300 nm) was a ring dye laser equipped with an intracavity frequency doubler, pumped by an argon ion laser. The data, measured at modulation frequencies from 2 to 200 MHz, reveal the presence of subnanosecond motions (0.1-0.2 ns) of the single tryptophan residues in melittin and monellin. For melittin the data also indicate the presence of slower motions near 1 ns, which may be the result of concerted motions of several peptide units. Smaller amplitude motions, on a similar timescale, were observed for the single tryptophan residue in staphylococcal nuclease. We demonstrate using N-acetyl-L-tryptophanamide in water that the method of frequency-domain fluorometry is capable of measuring correlation times as short as 50 ps. This method can provide data for the direct comparison of measured anisotropy decays with those predicted from molecular dynamics calculations.     

Biomass: Is it a useful tool in paleocommunity reconstruction?

In general, more of the biomass of the community is preserved than is its numerical abundance. Thus, the paleontologist, on the average, works with more of the community when biomass is used. Community characteristics such as taxon dominance and habitat proportions are at least as accurately derived from biomass as numerical abundance. The use of biomass is clearly more appropriate in describing energy flow and trophic proportions. Whenever possible, biomass should be used as a complement to numerical abundance in future paleoecologic reconstructions.     

On the Trail of Atmospheric Mutagens and Carcinogens: A Combined Chemical/Microbiological Approach

Major ecological problems of our polluted troposphere includeairborne toxic chemicals, acid rain and photochemical smog,all three of which are now recognized as being closely relatedchemical phenomena. We also recognize that inorder to developcost-effective strategies for their control, which protect publichealth and the environment, there must be close scientific interactionsbetween chemists and biological scientists. For example, ofrapidly emerging importance is the development of risk assessmentevaluations for specific aspects of each of these problem areas.In preparing such assessments, chemists must define the "exposure,"and biological scientists the "effects." In this paper, I discuss an example of how such close interactionsproved indispensible in our search for atmospheric mutagensand carcinogens. Thus, an integrated chemical/ microbiologicalprocedure for the isolation and identificationof particulatechemical mutagens in respirable diesel soot and ambient particlesis described. Emphasis is placed on our use of the short-term,Ames Salmonella typhimurium bacterial mutagenicity test as arapid, and relatively inexpensive, means of following the biologicalactivities of these environmental mutagens through the chemicalsteps of their separation, isolation and identification fromhighly complex environmental samples. Possible mechanisms offormation of these particulate mutagens are discussed. Theyinclude the reactions of polycyclic aromatic hydrocarbons presenton the surfaces of combustion-generated particles with gaseousco-pollutants such as nitrogen dioxide plus nitric acid, andozone. In discussing this research on a societally "relevant" problem,we illustrate the importance of "Science as a Way of Knowing."We further suggest that this integrated approach to scientificproblem solving by chemical and biological scientists mightserve as an example of a discussion topic on human ecology forundergraduate courses in the natural sciences.     

Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data.

Recently it has become possible to measure fluorescence phase-shift and modulation data over a wide range of modulation frequencies. In this paper we describe the analysis of these data by the method of nonlinear least squares to determine the values of the lifetimes and fractional intensities for a mixture of exponentially decaying fluorophores. Analyzing simulated data allowed us to determine those experimental factors that are most critical for successfully resolving the emissions from mixtures of fluorophores. The most critical factors are the accuracy of the experimental data, the relative difference of the individual decay times, and the inclusion of data measured at multiple emission wavelengths. After measuring at eight widely spaced modulation frequencies, additional measurements yielded only a modest increase in resolution. In particular, the uncertainty in the parameters decreased approximately as the reciprocal of the square root of the number of modulation frequencies. Our simulations showed that with presently available precision and data for one emission bandpass, two decay times could be accurately determined if their ratio were greater than or equal to 1.4. Three exponential decays could also be resolved, but only if the range of the lifetimes were fivefold or greater. To reliably determine closely-spaced decay times, the data were measured at multiple emission wavelengths so that the fractional intensities of the components could be varied. Also, independent knowledge of any of the parameters substantially increased the accuracy with which the remaining parameters could be determined. In the subsequent paper we present experimental results that broadly confirm the predicted resolving potential of variable-frequency phase-modulation fluorometry.