The Arabidopsis genes FT and TERMINAL FLOWER1 (TFL1) encode related proteins with similarity to human Raf kinase inhibitor protein. FT, and likely also TFL1, is recruited to the promoters of floral genes through interaction with FD, a bZIP transcription factor. FT, however, induces flowering, while TFL1 represses flowering. Residues responsible for the opposite activities of FT and TFL1 were mapped by examining plants that overexpress chimeric proteins. A region important in vivo localizes to a 14-amino-acid segment that evolves very rapidly in TFL1 orthologs, but is almost invariant in FT orthologs. Crystal structures show that this segment forms an external loop of variable conformation. The only residue unambiguously distinguishing the FT and TFL1 loops makes a hydrogen bond with a residue near the entrance of a potential ligand-binding pocket in TFL1, but not in FT. This pocket is contacted by a C-terminal peptide, which also contributes to the opposite FT and TFL1 activities. In combination, these results identify a molecular surface likely to be recognized by FT-and/or TFL1-specific interactors.
A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BOD-IPY 581a591 . Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY 581a591 was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY 581a591 is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.z 1999 Federation of European Biochemical Societies.
Plasmodium falciparum, the causative agent of malaria, relies extensively on glycolysis coupled with homolactic fermentation during its blood-borne stages for energy production. Selective inhibitors of the parasite lactate dehydrogenase (LDH), central to NAD ؉ regeneration, therefore potentially provide a route to new antimalarial drugs directed against a novel molecular target. A series of heterocyclic, azole-based compounds are described that preferentially inhibit P. falciparum LDH at sub-micromolar concentrations, typically at concentrations about 100-fold lower than required for human lactate dehydrogenase inhibition. Crystal structures show these competitive inhibitors form a network of interactions with amino acids within the active site of the enzyme, stacking alongside the nicotinamide ring of the NAD ؉ cofactor. These compounds display modest activity against parasitized erythrocytes, including parasite strains with known resistance to existing anti-malarials and against Plasmodium berghei in BALB/c mice. Initial toxicity data suggest the azole derivatives have generally low cytotoxicity, and preliminary pharmocokinetic data show favorable bioavailability and circulation times. These encouraging results suggest that further enhancement of these structures may yield candidates suitable for consideration as new therapeutics for the treatment of malaria. In combination these studies also provide strong support for the validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH in the search for novel anti-malarials.Plasmodium parasites are believed to lack a functional Krebs (citric acid) cycle for at least part of their life cycle and hence rely extensively on ATP generation via the anaerobic fermentation of glucose (see Ref. 1 for review). The energy requirement of the parasitized erythrocyte is such that utilization of glucose is up to 100 times greater than in nonparasitized erythrocytes (2, 3), and virtually all glucose can be accounted for by production of lactate (2). Lactate dehydrogenase (LDH), 1 the last enzyme in the glycolytic pathway in Plasmodium falciparum, is a 2-hydroxy acid oxidoreductase that converts pyruvate to lactate and simultaneously the conversion of NADH to NAD ϩ . As a constant supply of NADH is a prerequisite for glycolysis, and LDH acts as the primary source in Plasmodium for the regeneration of NADH from NAD ϩ , inhibition of LDH is expected to stop production of ATP, with subsequent P. falciparum cell death. Any compound that blocks the LDH enzyme is a potentially potent antimalarial with a different mode of action to existing drugs. As such, P. falciparum lactate dehydrogenase (pfLDH) has been suggested as a drug target by several authors (4 -6). One well recognized difficulty is that the drug must potently inhibit pfLDH yet show much less activity against the three human LDH (hsLDH) isoforms.A comparison of the crystal structures of both P. falciparum and human LDH (7,8) shows the following two key differences: namely positioning of the NADH factor, re...
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