Significance of the Tryptophan Residue at the Low Affinity Saccharide-binding Site of Ricin E

Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc.     

Transcriptome analysis of soybean (Glycine max) root genes differentially expressed in rhizobial,arbuscular mycorrhizal,and dual symbiosis

Journal of Plant Research - Soybean (Glycine max) roots establish associations with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. Both rhizobia and AM fungi have been shown to...     

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.     

Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata

CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.     

Agrobacterium tumefaciens-mediated transformation of the vegetative dikaryotic mycelium of the cultivated mushroom Flammulina velutipes

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.     

Cytotoxicity of a GalNAc-specific C-type lectin CEL-I toward various cell lines

We found that CEL-I was a potent cytotoxic lectin. MDCK, HeLa, and XC cells were highly sensitive to CEL-I cytotoxicity and killed in a dose-dependent manner, whereas CHO, L929, and RAW264.7 cells were relatively resistant to CEL-I, and no significant toxicity was observed up to 10 microg/ml. Among these cell lines, MDCK cells showed the highest susceptibility to CEL-I cytotoxicity. A binding study using FITC-labeled CEL-I (F-CEL-I) revealed that the amounts of bound F-CEL-I on the sensitive cell lines were evidently greater than those on the resistant cell lines, suggesting that the different susceptibility of the cell lines to CEL-I cytotoxicity is partly explained by different efficiencies of binding of CEL-I to these cell lines. Interestingly, the cytotoxicity of CEL-I toward MDCK cells was more potent than those of other lectins such as WGA, PHA-L, and Con A, even though these lectins were capable of binding to MDCK cells at comparable levels to CEL-I. Since the cytotoxicity of CEL-I was strongly inhibited by GalNAc, the binding to cell surface specific carbohydrates is essential for the CEL-I cytotoxicity. The trypan blue dye exclusion test indicated that CEL-I caused a disorder of plasma membrane integrity as a relatively early event. CEL-I failed to induce the release of carboxyfluorescein (CF) from CF-loaded MDCK cells as seen for pore-forming hemolytic isolectin CEL-III, suggesting that the primary cellular target of CEL-I may be the plasma membrane, but its action mechanism differs from that of CEL-III. Although CEL-I induced dramatic cellular morphological changes in MDCK cells, neither typical apoptotic nuclear morphological changes nor DNA fragmentation was observed in CEL-I-treated MDCK cells even after such cellular changes. Our results demonstrated that CEL-I showed a potent cytotoxic effect, especially on MDCK cells, by causing plasma membrane disorder without induction of apoptosis.     

The effects of simulated rainfall on immature population dynamics of Aedes albopictus and female oviposition

Larvae of Aedes albopictus Skuse typically inhabit natural and artificial containers. Since these larval habitats are replenished by rainfall, Ae. albopictus may experience increased loss of immature stages in areas with high levels of rainfall. In this study, we investigated the effects of rainfall and container water level on population density, and oviposition activity of Ae. albopictus. In field and laboratory experiments, we found that rainfall resulted in the flushing of breeding habitats. Excess rain negatively impacted larval and pupal retention, especially in small habitats. When filled with water to overflowing, container habitats were significantly repellent to ovipositing females. Taken together, these data suggest that rainfall triggers population loss of Ae. albopictus and related species through a direct detrimental effect (flushing out) and an indirect effect (ovipositional repellency).     

Olfactory behavior and response of household ants (Hymenoptera) to different types of coffee odor: A coffee-based bait development prospect

Odor sensation is a sensory modality of considerable significance in the foraging behavior and interactional organization of ants. In the food bait technology, smell is the basis of attraction, which, in turn, is the line of bait use and a key parameter for judging efficacy. Yet, the currently available baits possess low attractiveness to many ant pests. Hence, strategies to produce ant bait with increased attractiveness are needed. Despite evidence that coffee has a diverse aroma complex that affects the behavior of honey bees and ants, its attraction to house-invading ants has yet to be investigated. In a series of Y-tube olfactometer bioassays, we examined the behavioral responses of Tapinoma indicum (TI), Monomorium pharaonis (MP) and Solenopsis geminata (SG) to various coffee-induced odor stimuli, comprised of extracts from Arabica, Robusta and Liberica. All coffee extracts showed an influence on the behavior of TI, MP and SG workers, with Arabica showed the most significant influence to the tested ants. The workers of TI, MP and SG were more attracted to the odor of 0.01% Arabica extract (ONE), in comparison with 0.05% Arabica extract (TWO) or 0.10% Arabica extract (THREE). Arabica extract mixed with sugar (S) elicited a significant attraction from workers of all three species in a balanced competition with either unsweetened Arabica extract or water. These results indicated that coffee, particularly Arabica, was attractive to the foragers of TI, MP and SG, thus, the use of coffee as a novel stimulus agent seems plausible in ant bait development.