Regulation of Development of CD56brightCD11c+ NK-like Cells with Helper Function by IL-18

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56^brightCD11c⁺ cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56^brightCD11c⁺ cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56^brightCD11c⁺ cells. CD14⁺ monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56^intCD11c⁺ cells to promote their differentiation to CD56^brightCD11c⁺ cells with helper function. The development of CD56^brightCD11c⁺ cells was suppressed in an IFN-α dependent manner. These results indicate that CD14⁺ monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56^brightCD11c⁺ cells, which in turn modulate the expansion of γδ T cells. CD56^brightCD11c⁺ NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.     

Species richness of marine macrophytes is correlated to a wave exposure gradient

Wave exposure is one of the fundamental variables of the coastal environment and many coastal processes are affected by waves and wave‐induced water motion. Directly or indirectly, waves affect the lives of many coastal macroalgae and seagrasses. Although the idea that waves can affect macrophyte distribution and biodiversity is not new, it remains poorly examined. We looked at how a gradient of wave exposure, determined using the surf similarity number, influences the species richness of macrophytes among phyla and functional form groups. We suggest that wave exposure is a biologically and statistically significant variable and that the species richness of most phyla and groups decrease with increasing exposure. More interestingly, the analysis suggests that the Phaeophyta and the thick‐leathery functional form group, which are associated with important coastal habitats (i.e. submarine forests), increases in species richness as exposure increases. It appears that the effect of wave exposure varies with phyla and functional form group. Consequently, we reinforce the belief that studies that address the biodiversity and distribution of macrophytes must include wave exposure as a measured variable, to provide any meaningful synthesis of the ecology of these organisms.     

Effect of Vascular Cadherin Knockdown on Zebrafish Vasculature during Development

Background

Vascular endothelial cadherin (VE-cad) is essential for endothelial barrier integrity and vascular sprouting. However, the role of this important protein in cardiovascular development is only recently becoming apparent.

Methodology/Principal Findings

To characterize the role of VE-cadherin in cardiovascular development, we analyzed cardiovascular development in a zebrafish VE-cad knockdown model. Embryos deficient in VE-cad show profoundly impaired cardiac development despite having apparently normal peripheral vasculature. Initial formation of the heart proceeds normally in knockdown embryos, but subsequent looping morphogenesis is impaired. Consistent with these results, VE-cad knockdown embryos demonstrate impaired cardiac function and early circulatory arrest. Histologic examination of knockdown embryos shows persistent, abnormal separation of the endocardial and myocardial layers. Using transmission electron microscopy, we demonstrate that endocardial junctions form poorly in VE-cad knockdown embryos, with resulting leak across the endothelial layer and reduction in the density of the cardiac jelly.

Conclusions

Our results demonstrate a significant role for VE-cadherin in cardiac development independent of its effects on the formation of the peripheral vasculature.     

Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca²⁺. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca²⁺-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca²⁺-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca²⁺ and valinomycin on the inner and outer mitochondrial membranes are discussed.Mitochondria are well known as the organelle for energy conversion in all eukaryotes. This energy conversion, i.e. ATP synthesis, is performed by using the electrochemical gradient of H⁺ across the inner mitochondrial membrane. To enable effective energy conversion, the mitochondrial inner membrane is highly resistant to the permeation of solutes and ions. However, under certain conditions, such as in the presence of Ca²⁺ and inorganic phosphate, the permeability of this inner membrane is known to be markedly increased. This phenomenon is referred to as the permeability transition (PT)¹ and is believed to result from the formation of a proteinaceous pore, referred to as the PT pore, which makes the inner membrane permeable to various solutes and ions smaller than 1.5 kDa (1–3). The physiological importance of the PT has long been uncertain; however, recent studies have revealed that the changes in the permeability of the inner mitochondrial membrane due to the induction of PT cause the release of cytochrome c into the cytosol and that the released cytochrome c then triggers subsequent steps of programmed cell death, which is known as apoptosis (4–6). Thus, the PT is considered to be one of the major regulatory steps of apoptosis. However, the questions as to how the PT is induced and how cytochrome c is released accompanied by the induction of PT have remained unanswered.To characterize the features of the mitochondrial PT and to understand the mechanism underlying the release of cytochrome c from mitochondria, investigators have studied the effects of various agents on this organelle. As a result, the PT and the release of cytochrome c were found to be induced not only by Ca²⁺ but also by other agents (7–9). We also found that copper-o-phenanthroline (10), metal ions (11), and cyanine dyes (12, 13) induced this PT and the release of cytochrome c from mitochondria. Furthermore we reported that valinomycin, known as a potassium-selective ionophore, also induces the release of cytochrome c from mitochondria but without the induction of PT (14). This finding indicated that cytochrome c could be released from mitochondria in two different manners: one with the induction of PT and the other without it. To understand how cytochrome c is released from mitochondria, it is very important to know what protein species are released from mitochondria concomitant with the release of cytochrome c. To address these questions, in the present study we used a mass spectrometry (LC-MS/MS system)-based proteome analysis approach, which allowed us to identify the protein species present in a limited amount of protein samples. Using proteomics techniques, we examined the protein species released from mitochondria treated with valinomycin or with Ca²⁺, and we discuss our findings on the status of inner and outer mitochondrial membranes treated with these agents.     

Crystal structure of neoculin: insights into its sweetness and taste-modifying activity

Although the majority of sweet compounds are of low molecular mass, several proteins are known to elicit sweet taste responses in humans. The fruit of Curculigo latifolia contains a heterodimeric protein, neoculin, which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Here, we report the crystal structure of neoculin at 2.76A resolution. This is the first well-defined tertiary structure of a taste-modifying protein of this kind. The overall structure is quite similar to those of monocot mannose-binding lectins. However, crucial topological differences are observed in the C-terminal regions of both subunits. In both subunits of neoculin, the C-terminal tails turn up to form loops fixed by inter-subunit disulfide bonds that are not observed in the lectins. Indeed, the corresponding regions of the lectins stretch straight over the surface of another subunit. Such a C-terminal structural feature as is observed in neoculin results in a decrease in subunit-subunit interactions. Moreover, distribution of electrostatic potential on the surface of neoculin is unique and significantly different from those of the lectins, particularly in the basic subunit (NBS). We have found that there is a large cluster composed of six basic residues on the surface of NBS, and speculate that it might be involved in the elicitation of sweetness and/or taste-modifying activity of neoculin. Molecular dynamics simulation based on the crystallography results suggests that neoculin may adopt a widely "open" conformation at acidic pH, while unprotonated neoculin at neutral pH is in a "closed" conformation. Based on these simulations and the generation of a docking model between neoculin and the sweet-taste receptor, T1R2-T1R3, we propose the hypothesis that neoculin is in dynamic equilibrium between open and closed states, and that the addition of an acid shifts the equilibrium to the open state, allowing ligand-receptor interaction.     

Specificity in reactive oxidant signaling: think globally, act locally

Although reactive oxidants have long been stigmatized as unwanted metabolic byproducts, the expression of oxidases specifically functioning to produce these same molecules in a regulated fashion is surprisingly pervasive throughout metazoan and plant evolution. Although the involvement of oxidants in many signaling pathways is well documented, the cellular strategies for conferring pathway specificity to such reactive molecules have remained more recondite. Recent studies now suggest that cells may spatially restrict oxidant production to allow microdomain-specific signaling.     

Three species of the genus Laboulbenia (Laboulbeniales) parasitic on Chydaeus bedeli (Coleoptera, Carabidae) from Asia

Three species of the genus Laboulbenia were recorded on Chydaeus bedeli (Coleoptera, Carabidae, Harpalini) from high-altitude localities in Asia. Laboulbenia obtusa was obtained from tarsi of the midlegs of the male hosts; Laboulbenia acrogeniodontis was on the margins of the elytra of the male and female hosts; and Laboulbenia polyphaga was on the elytra (near the apex) and the pronotum (at the base) of the male and female hosts. Each of the three species is distinct in the shape of the perithecia and the structure of the appendages.     

Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cyclic α-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.