In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide. INTRODUCTIONSince its discovery in 1962 (Ritossa, 1962), the heat shock response has been the focus of intensive investigation, leading to significant insights into protein folding and global gene regulation. In virtually all organisms, the heat shock response is manifested as the stress-induced, rapid, and dramatic increase in synthesis rates of a small number of protein chaperones. The chaperones bind to partially unfolded proteins and act to prevent their aggregation and to facilitate their refolding. Thus, this highly conserved system serves as an intricate means to protect cells against damage resulting from environmental stress.The most common stresses that induce the heat shock response are elevated temperature and oxidative stress. The latter is particularly important medically, because it typically results from the production of superoxide anion that occurs during partial oxygen deprivation or during the recovery from anoxia that occurs upon reperfusion after ischemia. Induction of the heat shock system upon recovery from anoxia may be universal; it has been described not only in mammalian systems but in Drosophila (Ritossa, 1964) and yeast (Brazzell and Ingolia, 1984). It has been unclear, however, how reperfusion triggers the heat shock response. Superoxide is rapidly converted to hydrogen peroxide, which can stimulate the production of the highly reactive hydroxyl radical, which, in turn, causes considerable "reperfusion damage." Thus, the heat shock response could be induced directly via one or more of these reactive oxygen species, or it could be induced by the protein damage caused by the hydroxyl radical.In eukaryotes, the heat shock response depends on modulating the activity (rather than the concentration) of a transcription factor. The heat shock transcription factor (HSF) is synthesized constitutively; its activity is regulated posttranslationally. Despite this commonality, different species show remarkably dis...

Mass spectra of single-stranded DNA oligonucleotides were acquired using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Resolution enhancement using space-velocity correlation focusing allows for facile observation of different oligomers, the direct observation of individual DNA-metal adducts, and investigation of counter ion structure.

A novel time-of-flight mass calibration method has been developed. In contrast to conventional methods, where the relationship between ion flight time and mass is an arbitrary polynomial equation, this method is based on the physics of ion motion. Parameters needed to describe the physics are numerically optimized using a simplex algorithm. Once these parameters are established, unknown masses can be determined from their times-of-flight. This calibration method gives intrinsically well-behaved results, since nonlinearities (due to extraction delay, desorption velocity, etc.) are properly taken into account in the time-of-flight calculation. The simplex method is compared to curve fitting for the analysis of time-of-flight data, and some significant advantages are demonstrated. Salient features of the method include greatly improved mass extrapolation accuracy, no loss of interpolated calibration accuracy, the ability to obtain an accurate calibration with a minimal number of calibrants, and the ability to extract unknown parameters such as desorption velocities.

To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base.