Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight.

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

Discovery of a Locally and Orally Active CXCL12 Neutraligand (LIT-927) with Anti-inflammatory Effect in a Murine Model of Allergic Airway Hypereosinophilia.

We previously reported Chalcone-4 (1) that binds the chemokine CXCL12, not its cognate receptors CXCR4 or CXCR7, and neutralizes its biological activity. However, this neutraligand suffers from limitations such as poor chemical stability, solubility, and oral activity. Herein, we report on the discovery of pyrimidinone 57 (LIT-927), a novel neutraligand of CXCL12 which displays a higher solubility than 1 and is no longer a Michael acceptor. While both 1 and 57 reduce eosinophil recruitment in a murine model of allergic airway hypereosinophilia, 57 is the only one to display inhibitory activity following oral administration. Thereby, we here describe 57 as the first orally active CXCL12 neutraligand with anti-inflammatory properties. Combined with a high binding selectivity for CXCL12 over other chemokines, 57 represents a powerful pharmacological tool to investigate CXCL12 physiology in vivo and to explore the activity of chemokine neutralization in inflammatory and related diseases.

From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor.

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

Differential regulation of toxoflavin production and its role in the enhanced virulence of Burkholderia gladioli.

Burkholderia gladioli is a causal agent of bacterial panicle blight and sheath/grain browning in rice in many countries. Many strains produce the yellow pigment toxoflavin, which is highly toxic to plants, fungi, animals and microorganisms. Although there have been several studies on the toxoflavin biosynthesis system of B. glumae, it is still unclear how B. gladioli activates toxoflavin biosynthesis. In this study, we explored the genomic organization of the toxoflavin system of B. gladioli and its biological functions using comparative genomic analysis between toxoflavin-producing strains (B. glumae BGR1 and B. gladioli BSR3) and a strain not producing toxoflavin (B. gladioli KACC11889). The latter exhibits normal physiological characteristics similar to other B. gladioli strains. Burkholderia gladioli KACC11889 possesses all the genes involved in toxoflavin biosynthesis, but lacks the quorum-sensing (QS) system that functions as an on/off switch for toxoflavin biosynthesis. These data suggest that B. gladioli has evolved to use the QS signalling cascade of toxoflavin production (TofI/TofR of QS → ToxJ or ToxR → tox operons) similar to that in B. glumae. However, some strains may have evolved to eliminate toxoflavin production through deletion of the QS genes. In addition, we demonstrate that the toxoflavin biosynthetic system enhances the virulence of B. gladioli. These findings provide another line of evidence supporting the differential regulation of the toxoflavin system in Burkholderia strains.

Identification of potential genetic components involved in the deviant quorum-sensing signaling pathways of Burkholderia glumae through a functional genomics approach.

Burkholderia glumae is the chief causal agent for bacterial panicle blight of rice. The acyl-homoserine lactone (AHL)-mediated quorum-sensing (QS) system dependent on a pair of luxI and luxR homologs, tofI and tofR, is the primary cell-to-cell signaling mechanism determining the virulence of this bacterium. Production of toxoflavin, a major virulence factor of B. glumae, is known to be dependent on the tofI/tofR QS system. In our previous study, however, it was observed that B. glumae mutants defective in tofI or tofR produced toxoflavin if they grew on the surface of a solid medium, suggesting that alternative signaling pathways independent of tofI or tofR are activated in that growth condition for the production of toxoflavin. In this study, potential genetic components involved in the tofI- and tofR-independent signaling pathways for toxoflavin production were sought through screening random mini-Tn5 mutants of B. glumae to better understand the intercellular signaling pathways of this pathogen. Fifteen and three genes were initially identified as the potential genetic elements of the tofI- and tofR-independent pathways, respectively. Especially, the ORF (bglu_2g06320) divergently transcribed from toxJ, which encodes an orphan LuxR protein and controls toxoflavin biosynthesis, was newly identified in this study as a gene required for the tofR-independent toxoflavin production and named as toxK. Among those genes, flhD, dgcB, and wzyB were further studied to validate their functions in the tofI-independent toxoflavin production, and similar studies were also conducted with qsmR and toxK for their functions in the tofR-independent toxoflavin production. This work provides a foundation for future comprehensive studies of the intercellular signaling systems of B. glumae and other related pathogenic bacteria.

The roles of the shikimate pathway genes, aroA and aroB, in virulence, growth and UV tolerance of Burkholderia glumae strain 411gr-6.

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow-green and purple) pigment compounds in casein-peptone-glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment-deficient mutant derivatives of the virulent and pigment-proficient strain 411gr-6, having mini-Tn5gus insertions in aroA encoding 3-phosphoshikimate 1-carboxyvinyltransferase and aroB encoding 3-dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.

Discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-ones as novel PKM2 activators.

The M2 isoform of pyruvate kinase is an emerging target for antitumor therapy. In this letter, we describe the discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-ones as potent and selective PKM2 activators which were found to have a novel binding mode. The original lead identified from high throughput screening was optimized into an efficient series via computer-aided structure-based drug design. Both a representative compound from this series and an activator described in the literature were used as molecular tools to probe the biological effects of PKM2 activation on cancer cells. Our results suggested that PKM2 activation alone is not sufficient to alter cancer cell metabolism.

Toxoflavin lyase enzyme as a marker for selecting potato plant transformants.

This study established a new system for potato transformation using toxoflavin as selection agent and toxoflavin lyase (tflA) as selectable marker gene. Potato plants expressing tflA was successfully transformed on toxoflavin medium with 27% efficiency, similar to that for the hygromycin/hpt selection system. The transgenic potato expressing tflA also showed resistance to Burkholderia glumea infection.

Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.

Discovery tactics to mitigate toxicity risks due to reactive metabolite formation with 2-(2-hydroxyaryl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3h)-one derivatives, potent calcium-sensing receptor antagonists and clinical candidate(s) for the treatment of osteoporosis.

The synthesis and structure-activity relationship studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones as antagonists of the human calcium receptor (CaSR) have been recently disclosed [ Didiuk et al. ( 2009 ) Bioorg. Med. Chem. Lett. 19 , 4555 - 4559 ). On the basis of its pharmacology and disposition attributes, (R)-2-(2-hydroxyphenyl)-3-(1-phenylpropan-2-yl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one (1) was considered for rapid advancement to first-in-human (FIH) trials to mitigate uncertainty surrounding the pharmacokinetic/pharmacodynamic (PK/PD) predictions for a short-acting bone anabolic agent. During the course of metabolic profiling, however, glutathione (GSH) conjugates of 1 were detected in human liver microsomes in an NADPH-dependent fashion. Characterization of the GSH conjugate structures allowed insight(s) into the bioactivation pathway, which involved CYP3A4-mediated phenol ring oxidation to the catechol, followed by further oxidation to the electrophilic ortho-quinone species. While the reactive metabolite (RM) liability raised concerns around the likelihood of a potential toxicological outcome, a more immediate program goal was establishing confidence in human PK predictions in the FIH study. Furthermore, the availability of a clinical biomarker (serum parathyroid hormone) meant that PD could be assessed side by side with PK, an ideal scenario for a relatively unprecedented pharmacologic target. Consequently, progressing 1 into the clinic was given a high priority, provided the compound demonstrated an adequate safety profile to support FIH studies. Despite forming identical RMs in rat liver microsomes, no clinical or histopathological signs prototypical of target organ toxicity were observed with 1 in in vivo safety assessments in rats. Compound 1 was also devoid of metabolism-based mutagenicity in in vitro (e.g., Salmonella Ames) and in vivo assessments (micronuclei induction in bone marrow) in rats. Likewise, metabolism-based studies (e.g., evaluation of detoxicating routes of clearance and exhaustive PK/PD studies in animals to prospectively predict the likelihood of a low human efficacious dose) were also conducted, which mitigated the risks of idiosyncratic toxicity to a large degree. In parallel, medicinal chemistry efforts were initiated to identify additional compounds with a complementary range of human PK predictions, which would maximize the likelihood of achieving the desired PD effect in the clinic. The back-up strategy also incorporated an overarching goal of reducing/eliminating reactive metabolite formation observed with 1. Herein, the collective findings from our discovery efforts in the CaSR program, which include the incorporation of appropriate derisking steps when dealing with RM issues are summarized.

Synthesis and biological evaluation of novel 2-(substituted phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones as potent HIV-1 NNRTIs.

A series of novel 2-(phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones have been designed and synthesized. All of the new compounds were evaluated for their anti-HIV activities in MT-4 cells. Most of these new compounds showed moderate to potent activities against wild-type HIV-1 with an EC(50) ranging from 4.48microM to 0.18microM. Among them, 2-[(4-bromophenylamino)carbonylmethylthio]-6-(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one 4b3 was identified as the most promising compound (EC(50) = 0.18+/-0.06microM, CC(50) >243.56microM, SI >1326). The structure-activity relationship (SAR) of these new congeners is discussed.

Merocyanine 540-sensitized photokilling of leukemia cells: role of post-irradiation chain peroxidation of plasma membrane lipids as revealed by nitric oxide protection.

The lipophilic dye merocyanine 540 (MC540) localizes primarily in the plasma membrane (PM) of tumor cells, where it can sensitize lethal photoperoxidative damage of potential therapeutic importance. We postulated (i) that chain peroxidation triggered by iron-catalyzed turnover of nascent hydroperoxides (LOOHs) generated by singlet oxygen ((1)O(2)) attack on PM lipids contributes significantly to overall cytolethality, and (ii) that nitric oxide (NO), a known scavenger of organic free radicals, would suppress this and, thus, act cytoprotectively. In accordance, irradiation of MC540-sensitized L1210 cells produced 5alpha-OOH, a definitive (1)O(2) adduct of PM cholesterol, which decayed during subsequent dark incubation with appearance of other signature peroxides, viz. free-radical-derived 7alpha/beta-OOH. Whereas chemical donor (SPNO or SNAP)-derived NO had little or no effect on post-irradiation 5alpha-OOH disappearance, it dose-dependently inhibited 7alpha/beta-OOH accumulation, consistent with interception of chain-carrying radicals arising from one-electron reduction of primary LOOHs. Using [(14)C]cholesterol as an L1210 PM probe, we detected additional after-light products of chain peroxidation, including diols (7alpha-OH, 7beta-OH) and 5,6-epoxides, the yields of which were enhanced by iron supplementation, but strongly suppressed by NO. Correspondingly, photoinitiated cell killing was significantly inhibited by NO introduced either immediately before or after light exposure. These findings indicate that prooxidant LOOH turnover plays an important role in photokilling and that NO, by intercepting propagating radicals, can significantly enhance cellular resistance.

The fidelity of replication of the three-base-pair set adenine/thymine, hypoxanthine/cytosine and 6-thiopurine/5-methyl-2-pyrimidinone with T7 DNA polymerase.

With the goal of constructing a genetic alphabet consisting of a set of three base pairs, the fidelity of replication of the three base pairs T(H) (5-methyl-2-pyrimidinone)/H(S) (6-thiopurine; thiohypoxanthine), C/H (hypoxanthine) and T/A was evaluated using T7 DNA polymerase, a polymerase with a strong 3'-->5' exonuclease activity. An evaluation of the suitability of a new base pair for replication should include both the contribution of the fidelity of a polymerase activity and the contribution of proofreading by a 3'-->5' exonuclease activity. Using a steady-state kinetics method that included the contribution of the 3'-->5' exonuclease activity, the fidelity of replication was determined. The method determined the ratio of the apparent rate constant for the addition of a deoxynucleotide to the primer across from a template base by the polymerase activity and the rate constant for removal of the added deoxynucleotide from the primer by the 3'-->5' exonuclease activity. This ratio was designated the eni (efficiency of net incorporation). The eni of the base pair C/H was equal to or greater than the eni of T/A. The eni of the base pair T(H)/H(S) was 0.1 times that of A/T for T(H) in the template and 0.01 times that of A/T for H(S) in the template. The ratio of the eni of a mismatched deoxynucleotide to the eni of a matched deoxynucleotide was a measure of the error frequency. The error frequencies were as follows: thymine or T(H) opposite a template hypoxanthine, 2x10(-6); H(S) opposite a template cytosine, <3x10(-4). The remaining 24 mismatched combinations of bases gave no detectable net incorporation. Two mismatches, hypoxanthine opposite a template thymine or a template T(H), showed trace incorporation in the presence of a standard dNTP complementary to the next template base. T7 DNA polymerase extended the primer beyond each of the matched base pairs of the set. The level of fidelity of replication of the three base pairs with T7 DNA polymerase suggests that they are adequate for a three-base-pair alphabet for DNA replication.

Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers.

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones.

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 2. Approaches to eliminate opioid agonist metabolites via modification of linker and 4-methoxycarbonyl-4-phenylpiperidine moiety.

We have previously described compound 1a as a high-affinity subtype selective alpha(1a) antagonist. In vitro and in vivo evaluation of compound 1a showed its major metabolite to be a mu-opioid agonist, 4-methoxycarbonyl-4-phenylpiperidine (3). Several dihydropyrimidinone analogues were synthesized with the goal of either minimizing the formation of 3 by modification of the linker or finding alternative piperidine moieties which when cleaved as a consequence of metabolism would not give rise to mu-opioid activity. Modification of the linker gave several compounds with good alpha(1a) binding affinity (K(i) = < 1 nM) and selectivity (>300-fold over alpha(1b) and alpha(1d)). In vitro analysis in the microsomal assay revealed these modifications did not significantly affect N-dealkylation and the formation of the piperidine 3. The second approach, however, yielded several piperidine replacements for 3, which did not show significant mu-opioid activity. Several of these compounds maintained good affinity at the alpha(1a) adrenoceptor and selectivity over alpha(1b) and alpha(1d). For example, the piperidine fragments of (+)-73 and (+)-83, viz. 4-cyano-4-phenylpiperidine and 4-methyl-4-phenylpiperidine, were essentially inactive at the mu-opioid receptor (IC(50) > 30 microM vs 3 microM for 3). Compounds (+)-73 and (+)-83 were subjected to detailed in vitro and in vivo characterization. Both these compounds, in addition to their excellent selectivity (>880-fold) over alpha(1b) and alpha(1d), also showed good selectivity over several other recombinant human G-protein coupled receptors. Compounds (+)-73 and (+)-83 showed good functional potency in isolated human prostate tissues, with K(b)s comparable to their in vitro alpha(1a) binding data. In addition, compound (+)-73 also exhibited good uroselectivity (DBP K(b)/IUP K(b) > 20-fold) in the in vivo experiments in dogs, similar to 1a.

Resistance of glucose-6-phosphate dehydrogenase deficiency to malaria: effects of fava bean hydroxypyrimidine glucosides on Plasmodium falciparum growth in culture and on the phagocytosis of infected cells.

The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malarial combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their beta-O-glucosidic bond, became protein pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of beta-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype.

Variation of favism-inducing factors (vicine, convicine and L-DOPA) during pod development in Vicia faba L.

Changes in the concentrations of vicine, convicine and L-DOPA in two cultivars of Vicia faba L. seeds in different stages of pod development were determined by high performance liquid chromatography (HPLC). The vicine and convicine content was highest in fresh green cotyledons (moisture content about 80%) and gradually declined until a constant level was reached when seed dry matter percentage was around 40%. A similar pattern of variation in glucoside concentration was observed for the seed coat. The pods contained neither vicine nor convicine but they were particularly rich in L-DOPA. These compounds were not homogeneously distributed in the seeds.

Bleaching of membrane-bound merocyanine 540 in conjunction with free radical-mediated lipid peroxidation.

The lipophilic dye merocyanine 540 (MC540) can photosensitize potentially lethal cell membrane damage as well as its own degradation (bleaching). Photobleaching in a test membrane, the human erythrocyte ghost has been examined. White light irradiation of MC540-sensitized ghosts resulted in lipid hydroperoxide (LOOH) formation, low-level thiobarbituric acid (TBA) reactivity, and dye bleaching (A568 decay). When the reaction was carried out in the presence of ascorbate (AH-), and added Fe3+, there was a large enhancement of TBA reactivity (indicative of free radical-mediated lipid peroxidation) and concomitant increase in the rate of photobleaching. Rapid bleaching also occurred when MC540 was incubated in the dark with ghosts that had been photoperoxidized with another dye (a phthalocyanine) and then exposed to AH-. The extent of bleaching in this system was found to be proportional to the starting level of LOOH. Like the wave of free radical lipid peroxidation that accompanied it, dye bleaching in AH(-)-treated, preperoxidized ghosts was stimulated by supplemental Fe3+, inhibited by desferrioxamine or butylated hydroxytoluene (BHT), but unaffected by catalase or superoxide dismutase. From this and related evidence, we deduce that: (1) in the absence of Fe3+/AH-, photoperoxidation and photobleaching occur independently and are nonradical, singlet oxygen-mediated processes; and (2) in the presence of Fe3+/AH-, 1-electron reduction of photogenerated LOOHs results in a surge of lipid peroxidation that amplifies dye loss via free radical processes. MC540 bleaching might be exploited as a relatively simple and sensitive indicator of lipid autoxidation in isolated membranes and cells.

Hydrolysis of vicine and convicine from fababeans by microbial beta-glucosidase enzymes.

The toxic glycosides vicine and convicine which are present in fababeans have been implicated in favism, an anaemic disease of humans. Vicine and convicine concentrations are reduced by growth of Lactobacillus plantarum on fababean suspensions. The glycosides are eliminated from the fababean substrate by the growth of the filamentous fungus Fusarium graminearum. Incubation of fababean suspension with concentrated culture filtrate of Aspergillus oryzae, induced for extracellular beta-glucosidase production, results in complete degradation of the glycosides. This study suggests a potential use of micro-organisms or microbial enzymes for detoxification of fababeans.