Trimethoprim binds in a bacterial mode to the wild-type and E30D mutant of mouse dihydrofolate reductase

Previous crystallographic studies of the antibacterial trimethoprim in complexes with bacterial and avian dihydrofolate reductases have shown substantial differences in the mode of binding, providing plausible explanations for the origin of the remarkable species selectivity of this inhibitor (Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., Kaufman, B. T., Beddell, C. R., Champness, J. N., Stammers, D. K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391; Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., and Kraut, J. (1985) J. Biol. Chem. 260, 392-399). A major species difference between the active sites is that the only carboxylate present is always Glu in vertebrates and Asp in bacteria. Crystallographic studies of the wild-type and E30D mutant of the enzyme from mouse now reveal that in both cases trimethoprim is bound in an identical fashion to that observed with the bacterial enzyme, and there is no obvious single explanation for the origin of the 10(5)-fold selectivity of trimethoprim binding. In an earlier study of a mouse wild-type enzyme using more limited data it was proposed that trimethoprim bound in the avian mode (Stammers, D. K., Champness, J. N., Beddell, C. R., Dann, J. G., Eliopoulos, E. E., Geddes, A. J., Ogg, D., and North, A. C. T. (1987) FEBS Lett. 218, 178-184), but a re-examination indicates that the occupancy of the active site by trimethoprim is less than had been thought, and we are currently unable to make an unambiguous interpretation of the electron density maps and cannot confirm the avian mode of binding in those crystals.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

The characteristics of cysteine proteinases of parasitic protozoa.

Cysteine proteinases have now been detected in most of the important species of parasitic protozoa. Characterization of the enzymes and sequence determinations have revealed that the enzymes are related to papain and the mammalian cathepsins. All of the protozoan enzymes analyzed to date are members of the cathepsin L/cathepsin H/papain branch of the papain superfamily and are more distantly related to cathepsin B. They thus share some characteristics with the cysteine proteinases of their hosts. Individual enzymes, however, are likely to have sufficient novel features to be potential targets for specific antiprotozoal drugs, and a number of proteinase inhibitors and substrates are currently being tested as possible chemotherapeutic agents.     

Toxicity of sewage-contaminated sediment cores to Macrocystis pyrifera (Laminariales,Phaeophyta) gametophytes determined by digital image analysis

Macrocystis pyrifera gametophytes were exposed in batch culture to varying mass concentrations of buried, sewage-contaminated, historically discharged sediment that had been sampled from two sites off Palos Verdes Peninsula, California. Significant gametophytic vegetative growth inhibition was detected in six days, using digital image analysis at sediment loadings ranging from 0.15 to 14.5 g in 500 mL nutrient-enriched seawater. Inhibition declined at low sediment loadings and increased at high loadings as cultures aged. Sediments corresponding to the historic emissions peak taken 2 km from the Joint Water Pollution Control Plant outfall inhibited vegetative growth more than did sediments sampled 13 km distant. Analysis showed elevated aqueous Cd(II), Cr(II) and p,p-DDE concentrations in high sediment-loading culture medium. Inhibition by Zn(II) alone was observed at similar concentrations in other experiments, but synergism or antagonism by other toxicants remains possible.     

The ascidian sperm reaction: Ca2+ uptake in relation to H+ efflux

During the ascidian sperm reaction the single large cylindrical mitochondrion which lies next to the nucleus in the head swells, becomes spherical, and migrates along the tail to be lost when it reaches the end. This sequence is initiated by eggs, egg water, high pH, low Na⁺, or the ionophore X537A. Accompanying the sperm reaction induced by low Na⁺ are H⁺ efflux and Ca²⁺ influx in a ratio of near 100:1 as determined by ⁴⁵Ca²⁺ and atomic absorption analysis. Simultaneous pH and Ca²⁺ electrode measurements suggest that the movement of H⁺ begins 10–13 sec before the movement of Ca²⁺. Ca²⁺ uptake can be inhibited by verapamil without affecting H⁺ efflux or the sperm reaction. Acid release and Ca²⁺ uptake are proportional to the initial pH of the medium when the reaction is triggered by high pH. Acid release initiated by low Na⁺ is proportional to Ca²⁺ concentrations above 2 mM. H⁺ and Ca²⁺ movements differ in magnitude, kinetics, and inhibition by verapamil, thus suggesting that H⁺ is probably not exchanged for Ca²⁺. Instead we propose that loss of H⁺ triggers the uptake of Ca²⁺, which initiates the sperm reaction.     

Characterization of cloned murine cytolytic T cell lines

Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.