Clotrimazole binds to heme and enhances heme-dependent hemolysis: proposed antimalarial mechanism of clotrimazole.

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K(a) = 6.54 x 10(8) m(-2)). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = 1/2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite.     

Roles of cottony hairs in directed seed dispersal in riparian willows

Willows usually establish on wet substrates with fine sediments at sites that are created by large disturbances, but suitable microsites are spatially and temporally limited. Thus, we hypothesized that willow seeds are selectively dispersed to suitable microsites, such as those with a wet substrate, rather than unsuitable microsites, such as those with a dry substrate, with seedling establishment mediated by the cottony hairs attached to seeds (directed dispersal). To test our hypothesis, we compared several recruitment-related traits, including buoyancy, germination, and trapping at favorable microsites, in seeds of the riparian willows Salix sachalinensis and S. integra with and without cottony hairs in laboratory and field experiments. In both field and laboratory experiments, more seeds with cottony hairs were trapped in water and wet sand than in dry sand, in which no seeds of either species germinated. These results indicate that cottony hairs facilitate the recruitment of seeds to microsites favorable for seed germination and help seeds avoid unfavorable microsites. On the water surface, 17.6% of S. sachalinensis seeds and 68.0% S. integra seeds with cottony hairs floated for more than 6 days, whereas all seeds without cottony hairs sank immediately after being placed on the water surface. These results suggest that cottony hairs facilitate long-distance dispersal via flowing water and also help avoid germination under water, where willow seedlings fail to establish. Seeds of the two willow species were released from the cottony hairs and germinated immediately after the seeds were placed on wet sand, but not after placement on water or dry sand. These results suggest that the seeds are released from the cottony hairs when the hairs become wet and the seeds are striking to a suitable microsite for seedling establishment, such as wet sand. In riparian willows, the cottony hairs promote directed dispersal by moving seeds to discrete and predictable microsites where the seedling establishment is disproportionately high.     

Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.     

The bacterial stringent response, conserved in chloroplasts, controls plant fertilization

The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.