Sugar Specificity in the Induction and on the Activity of Streptomyces α-d-Galactosidases

The α-d-galactosidases of six Streptomyces strains were examined on their inducer susceptibility, substate specificity, and inhibitor susceptibility. In all strains examined, α-d-galactosidase was induced by d-galactose, but neither by d-fucose nor by l-arabinose. α-d-Fucosidase activity was always induced accompanying with α-d-galactosedase activity. β-l-Arabinosidase activity, however, was never observed. These α-d-galactosidases were purified to electrophoretically pure degree by successive ammonium sulfate and ethanol precipitation, and ion exchange and gel filtration chromatography. The purified preparations from six strains were different from each other in their chromatographic behaviors and in some physical properties, but they all showed strong α-d-fucosidase activity as well. The α-d-galactosidase activities were strongly inhibited by d-galactose and l-arabinose, but scarcely by d-fucose. On the other hand, their α-d-fucosidase activities were inhibited by d-fucose as well as by d-galactose and l-arabinose.     

Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model

Recent studies have shown that low concentrations of H₂O₂ are produced endogenously by nonphagocytes after wounding. We observed that H₂O₂ at such concentrations can stimulate proliferation as well as migration of keratinocytes in a scratch-wound assay. Both wounding and H₂O₂ can induce phosphorylation of ERK1/2 via EGFR, but the activation of ERK1/2 by H₂O₂ is more sustained and can last more than 8 h. Sustained ERK1/2 activation is required for the increased proliferation and migration induced by H₂O₂. The p38 MAPK was also found to be phosphorylated upon treatment with H₂O₂ but it was not required for H₂O₂-induced migration or proliferation. Furthermore, it was observed that there is a cross talk between the ERK1/2 and the p38 pathways whereby inhibition of either pathway can lead to activation of the other. As a result, the motogenic effects of H₂O₂ were further enhanced when p38 was inhibited. Our data are consistent with the view that H₂O₂ may play an important signaling role in wound healing.     

14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

Seasonal modulation of free radical metabolism in estivating land snails Helix aspersa

We investigated the regulation of free radical metabolism in Helix aspersa snails during a cycle of 20-day estivation and 24-h arousal in summer in comparison with estivation/arousal in winter-snails. In winter-snails (J. Exp. Biol. 206, 675-685, 2003), we had already observed an increase in the selenium-dependent glutathione-peroxidase (Se-GPX) activity in foot muscle and hepatopancreas and in the contents of hepatopancreas GSH-equivalents (GSH-eq=GSH+2 GSSG) during estivation compared with 24-h aroused snails. Summer-estivation prompted a 3.6-fold increase in Se-GPX activity in hepatopancreas, though not in foot muscle. Total-superoxide dismutase and catalase activities in hepatopancreas decreased (by 30-40%) during summer-estivation; however, no changes occurred in the activities of glutathione reductase, glutathione S-transferase and glucose-6-phosphate dehydrogenase in the two organs. GSH-eq levels were increased (by 54%) in foot muscle during estivation, but were unchanged in hepatopancreas. In contrast with winter-snails, oxidative stress markers (lipid peroxidation, carbonyl protein, and the GSSG/GSH-eq ratio) were unaltered during estivation/arousal in summer. These results demonstrate that seasonality modulates not only the absolute activities/levels of antioxidants (enzymes and GSH-eq) in H. aspersa, but also the regulatory process that controls the snail's antioxidant capacity during estivation/arousal. These results suggest that H. aspersa has an "internal clock" controlling the regulation of free radical metabolism in the different seasons.     

CNI1/ATL31, a RING‐type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full‐length cDNAs, for individuals able to continue post‐germinative growth under severe C/N stress. One line, cni1‐D (carbon/nitrogen insensitive 1‐dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING‐type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1‐D phenotype, and a knock‐out of this gene resulted in hypersensitivity to C/N conditions during post‐germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

Hydogen peroxide-dependent photocytotoxicity by phloxine B,a xanthene-type food colorant

Background

Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.

Methods

We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.

Reuslts and conclusions

PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H₂O₂), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H₂O₂ in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H₂O₂-dependent mechanism.

General significance

Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.     

Application of the ferrous oxidation-xylenol orange assay for the screening of 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LO) is the key enzyme involved in leukotriene synthesis and its improper regulation is implicated in several inflammatory diseases. A rapid and sensitive assay for 5-LO activity suitable for high-throughput format is not yet available. In this study, we examined whether the ferrous oxidation-xylenol orange (FOX) assay could be applicable for the high-throughput screening of 5-LO inhibitors. Using insect cell lysates overexpressing rat 5-LO, the effects of cofactors of 5-LO such as ATP, Ca2+, and L-alpha-phosphatidylcholine (PC) on the color development of FOX reagents were investigated. ATP quenched substantially color development by hydroperoxide, an intermediate of 5-LO reaction, and an optimum concentration of ATP with little interference was determined as 20 microM. Ethylenediaminetetraacetate (0.4 mM) also affected the complex formation with FOX reagents. On the other hand, neither Ca2+ nor PC influenced complex formation with FOX reagents. Under optimized assay conditions, zileuton, a 5-LO-specific inhibitor, exhibited inhibitory potency (IC50 values of 0.1-0.2 microM) similar to that determined by the conventional spectrophotometric assay. Taken together, this study shows that the FOX assay with some modifications can be employed for high-throughput assay format for the measurement of 5-LO activity at the stage of primary screening.