Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 371(Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway.     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.     

Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S¹¹³–R²⁵⁰) and 60 kDa (I²⁵¹–S⁷⁷⁷) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.     

Landslide-facilitated species diversity in a beech-dominant forest

To evaluate the extent to which landslides affect community dynamics and consequent species diversity in a beech-dominated forest, differences in the composition and size structure of tree species were compared between landslide and adjacent stable (control) stands. Demography and changes in size were compared between the two stands over a 5-year period about 60 years after a landslide. In the control stand, replacement occurred even amongst late-successional species, with beech (Fagus crenata)—the most dominant species—increasing in relative abundance. In the landslide stand, very few large individuals of late-successional species occurred, whereas large individuals of early-successional species occurred only in the landslide stand. The traits indicate that the landslide strongly facilitated species diversity, not only by reducing the dominance of late-successional species, but also by promoting recruitment of early-successional species. However, new recruitment of early-successional species was inhibited in the landslide stand, although we observed succeeding regeneration and subsequent population growth of late-successional species there. As a result, the relative dominance of late-successional species increased with succession after the landslide, thus decreasing future species diversity. In beech-dominant forest landscapes in Japan that include communities with different developmental stages, the mosaic of serial stages may facilitate species diversity after a landslide.     

A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.     

Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser ²²⁵ and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A ₂ and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.     

Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells.

BCR/ABL tyrosine kinase generated from the chromosomal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To examine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (Delta p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or Delta p85; that of cyclin E by N17; and that of cyclin A by Delta p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Delta p85 were hardly effective. In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or Delta p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-alpha- and dexamethasone-induced apoptosis, disruption of one pathway by N17, 694F, or Delta p85 sensitized K562 to these reagents. These results suggested that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.     

Synanceia quinque,a new species of stonefish (Synanceiidae) from Borneo and Flores

A new species of stonefish, Synanceia quinque (Synanceiidae) is described on the basis of two specimens (61.5–84.4 mm standard length) collected off Sabah (Borneo), Malaysia and Flores, Indonesia. The new species is characterized by 12 pectoral-fin rays, and is most similar to Synanceia alula Eschmeyer and Rama-Rao 1973 (11 pectoral-fin rays in the latter vs. 14 or more in other congeners). Other characters distinguishing S. quinque from S. alula include numbers of pelvic-fin rays [I, 5 in the former vs. I, 3 or 4 (usually I, 4) in the latter], gill rakers (0 + 4 or 5 vs. 0 or 1 + 7), and five preopercular spines/skin flaps (upper three spines relatively well developed, lower two rudimentary) (vs. four preopercle spines, fifth spine or skin flap absent). An updated key to species of Synanceia is provided.