ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.     

Inhibitory effect of tea polyphenols on histamine and leukotriene B4 release from rat peritoneal exudate cells

Summary The effect of tea polyphenols on the release of chemical mediators, histamine and leukotriene B₄ (LTB₄), from rat peritoneal exudate cells (PEC) was studied. Among polyphenols, (−)-epigallocatechin gallate (EGCG) most strongly inhibited the histamine release from the cells stimulated with a calcium ionophore, A23187 or compound 48/80. Though (+)-catechin (C) and (−)-epicatechin (EC) had no effect, (−)-epigallocatechin (EGC) and (−)-epicatechin gallate (ECG) moderately inhibited the histamine release. Similarly, EGCG, ECG, and EGC inhibited LTB₄ release from PEC, whereas C and EC were not effective. The magnitude of the inhibitory effect on the release of these mediators of tea polyphenols was in the order of EGCG>ECG>EGC. These results indicated an important role of the triphenol structure in the inhibitory activity. Therefore, the possible antiallergic effect of tea polyphenols can be expected.     

Protective effect of antiganglioside antibodies against experimental Trypanosoma brucei infection in mice

Liposome-associated ganglioside antigens (ganglioside GM1 or bovine brain gangliosides) were prepared to facilitate the potential protective efficacy for Trypanosoma brucei. Mice were immunized with liposome-associated ganglioside GM1 or bovine brain gangliosides intraperitoneally (i.p.). After immunization, significantly higher antigen-specific IgG and IgM antibodies were detected in sera than in the nonimmunized control group. When sera from immunized mice were analyzed for isotype distribution, antigen-specific IgG1, IgG2a, and IgG3 antibody responses were also noted. After immunization, mice were challenged i.p. with 1 x 10(2) cells of T. brucei. Sixty percentage of liposome-associated ganglioside GM1-immunized mice survived the infection, and all the mice immunized with bovine brain gangliosides-containing liposomes survived. However, all control mice died within 7 days after infection. These data demonstrate that liposomes containing ganglioside antigens have the potential usefulness for the induction of a protective immune response against T. brucei infection and suggest the possibility of developing vaccines that may ultimately be used for the prevention of trypanosomiasis.     

Selective expansion of foxp3-positive regulatory T cells and immunosuppression by suppressors of cytokine signaling 3-deficient dendritic cells

Dendritic cells (DCs) induce immunity and immunological tolerance as APCs. It has been shown that DCs secreting IL-10 induce IL-10(+) Tr1-type regulatory T (Treg) cells, whereas Foxp3-positive Treg cells are expanded from naive CD4(+) T cells by coculturing with mature DCs. However, the regulatory mechanism of expansion of Foxp3(+) Treg cells by DCs has not been clarified. In this study, we demonstrated that suppressors of cytokine signaling (SOCS)-3-deficient DCs have a strong potential as Foxp3(+) T cell-inducing tolerogenic DCs. SOCS3(-/-) DCs expressed lower levels of class II MHC, CD40, CD86, and IL-12 than wild-type (WT)-DCs both in vitro and in vivo, and showed constitutive activation of STAT3. Foxp3(-) effector T cells were predominantly expanded by the priming with WT-DCs, whereas Foxp3(+) Treg cells were selectively expanded by SOCS3(-/-) DCs. Adoptive transfer of SOCS3(-/-) DCs reduced the severity of experimental autoimmune encephalomyelitis. Foxp3(+) T cell expansion was blocked by anti-TGF-beta Ab, and SOCS3(-/-) DCs produced higher levels of TGF-beta than WT-DCs, suggesting that TGF-beta plays an essential role in the expansion of Foxp3(+) Treg cells. These results indicate an important role of SOCS3 in determining on immunity or tolerance by DCs.     

Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca²⁺ and Cl⁻, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca²⁺ and Cl⁻ retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.     

Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation

Soluble endoplasmic reticulum (ER)-resident proteins have very long lives because of their ER residency. This residency depends largely on ER-retrieval signals at their C-terminus. We examined the long-term destiny of endogenous ER-resident proteins, a lumenal binding protein (BiP) and a protein disulfide isomerase (PDI), with cultured cells of Arabidopsis. ER residents, in contrast to vacuolar proteinases, were considerably degraded in cells at the stationary phase. A subcellular fractionation analysis suggested that ER residents were transported into the vacuoles, which accumulated the residents lacking the ER-retrieval signals. We showed that the PDI located in the vacuoles had high mannose glycans, but not complex glycans, which suggested that the ER resident was transported to the vacuoles independent of the medial/trans-Golgi complex. To visualize the pathway of transport of ER-resident proteins, tobacco BY-2 cells were transformed with a chimeric gene encoding an ER-targeted green fluorescent protein (30 kDa GFP-HDEL). In the transformed cells at the stationary phase, GFP fluorescence was observed in the vacuoles. A subcellular fractionation revealed that a trimmed form of 27 kDa GFP was localized in the vacuoles. Treatment with E-64d, an inhibitor of papain-type cysteine proteinases that inhibits the degradation of GFP in the vacuoles, resulted in a stable accumulation of 27 kDa GFP in the vacuoles, even in the logarithmic phase. Our results suggest that endogenous ER residents are transported constitutively to the vacuoles by bypassing the Golgi complex and are then degraded.     

pH-Dependent Assembly and Segregation of the Coiled-Coil Segments of Yeast Putative Cargo Receptors Emp46p and Emp47p

Emp46p and Emp47p are yeast putative cargo receptors that recycle between the endoplasmic reticulum and the Golgi apparatus. These receptors can form complexes in a pH-dependent manner, but their molecular mechanisms remain unclear. Here, we successfully reproduced their interactions in vitro solely with their coiled-coil segments, which form stable heterotetramers in the neutral condition but segregate at lower pH. Mutational data identified a key glutamate residue of Emp46p that serves as the pH-sensing switch of their oligomer formation. Our findings elucidate the mechanisms of the dynamic cargo receptor interactions in the secretory pathway and the design framework of the environment-responsive molecular assembly and disassembly systems.     

Diversity of γ- glutamyl peptides and oligosaccharides,the “kokumi” taste enhancers,in seeds from soybean mini core collections

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the “kokumi” taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the “kokumi” taste components in soybean by performing a genetic analysis.     

O‐Acyl isopeptide method: development of an O‐acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1‐42 isopeptide

The O‐acyl isopeptide method was developed for the efficient preparation of difficult sequence‐containing peptide. Furthermore, development of the O‐acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid‐phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.