No abstract

Experiments using purified recombinant human NAD(P)H:quinone oxidoreductase 1 (NQO1) revealed that the auto-oxidation of fully reduced protein resulted in a 1:1 stoichiometry of oxygen consumption to NADH oxidation with the production of hydrogen peroxide. The rate of auto-oxidation of fully reduced NQO1 was markedly accelerated in the presence of superoxide (O 2 . ), whereas the addition of superoxide dismutase greatly inhibited the rate of auto-oxidation.

No abstract

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

No abstract

HAMLET (human ␣-lactalbumin made lethal to tumor cells) is a complex of human ␣-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from ␣-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of ␣-lactalbumin is sufficient to induce cell death. We used the bovine ␣-lactalbumin Ca 2+ site mutant D87A, which is unable to bind Ca 2+ , and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine ␣-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca 2+ site, as HAMLET maintained a high affinity for Ca 2+ but D87A-BAMLET was active with no Ca 2+ bound. We conclude that partial unfolding of ␣-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca 2+ -binding site is not required for conversion of ␣-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca 2+ site.

Electron paramagnetic resonance imaging was demonstrated on two plant species, Apium graveolens and Coleus blumei. This was accomplished by soaking stems of these plants in the paramagnetic nitroxide imaging agent 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. The experiments were accomplished at L-band frequency (1.4 to 1.9 gigahertz) with single-turn, flat-loop surface coils. One-dimensional imaging spectra were diagnostic of capillary structure and long-term stability.

beta-Lactoglobulin (Big) binds 1 mol of a fatty acid spin-label analog, 5-doxylstearic acid (5-DSA), per mole of protein with a dissociation constant Kd = 0.8 microM for the strongest binding site. There are also several weaker sites for this ligand. Blg saturated with either retinol or retinoic acid binds 5-DSA with essentially equal affinity (Kd = 0.6 and 1 microM, respectively). Palmitic acid and SDS displace bound 5-DSA from Blg. However, unlike palmitic acid, 5-DSA binding does not enhance the structural stability of Blg to urea denaturation. The spin-labeled fatty acid also binds to the protein at low pH, presumably at secondary fatty acid binding sites. These results suggest that Blg binds at least two different types of hydrophobic ligands simultaneously.