Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BM-MCs IntroductionMast cells are hematopoietic-lineage cells that participate in immunoglobulin (Ig)E-associated immune responses, including allergic reactions and parasite resistance (see Galli 1 for a review). It was recently shown that mast cells also participate in the innate immunity to bacterial infection, in which IgE may not be involved. 2 Genetic evidence indicates that Kit ligand (KitL) and its receptor, c-Kit, play essential roles in mast cell development. Mutations in the mouse Kit ligand and c-Kit genes (Steel and White spotting) lead to defects in the development of melanocytes, germ cells, erythroid cells, basophils, and mast cells. 3-5 c-Kit is a receptor-type tyrosine kinase that displays some homology with platelet-derived growth factor receptors. The binding of KitL to c-Kit induces the dimerization and transphosphorylation of c-Kit. Tyrosyl-phosphorylated c-Kit recruits signaling molecules containing the Src homology 2 (SH2) domain, such as phosphatidyl inositol (PI)-3 kinase, 6 phospholipase C␥1, 7,8 Grb2, and the Src kinase, 9 to c-Kit and initiates cytoplasmic signaling. In addition to KitL/c-Kit signaling, interleukin (IL)-3 is also involved in mast cell development. IL-3-deficient mice maintain a basal level of mast cells, whereas mast cells fail to expand in response to infection by the nematode Stronglyoides venezuelensis. 10 Double-mutant Kit W /Kit W-v , IL-3 Ϫ/Ϫ mice display a more severe reduction in mast cell and basophil expansion elicited by the nematode infection than do single-mutant mice. 10 The result suggests that IL-3 is not essential for the generation of mast cells in a resting state but that it is required for the increase in mast cells in the immune response elicited by parasites. On the other hand, KitL-mediated signals are required for the development of the basal level of mast cells.Gab2 is a member of the Gab/DOS family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for the SH2 and SH3 domains. [11][12][13][14] Gab2 is tyrosine phosphorylated on stimulation by growth factors, cytokines, and Tand B-cell antigen receptors, including KitL and IL-3, and phosphorylated Gab2 binds SHP-2 and p85 PI-3 kinase. 11,15,16 Overexpression of Gab2 enhances the activation of cytokine-dependent ERK mitogen-activated protei...

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

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BackgroundThe ciliate Paramecium bursaria harbors several hundred cells of the green-alga Chlorella sp. in their cytoplasm. Irrespective of the mutual relation between P. bursaria and the symbiotic algae, both cells retain the ability to grow without the partner. They can easily reestablish endosymbiosis when put in contact with each other. Consequently, P. bursaria is an excellent model for studying cell–cell interaction and the evolution of eukaryotic cells through secondary endosymbiosis between different protists. Despite the importance of this organism, no genomic resources have been identified for P. bursaria to date. This investigation compared gene expressions through RNA-Seq analysis and de novo transcriptome assembly of symbiont-free and symbiont-bearing host cells.ResultsTo expedite the process of gene discovery related to the endosymbiosis, we have undertaken Illumina deep sequencing of mRNAs prepared from symbiont-bearing and symbiont-free P. bursaria cells. We assembled the reads de novo to build the transcriptome. Sequencing using Illumina HiSeq2000 platform yielded 232.3 million paired-end sequence reads. Clean reads filtered from the raw reads were assembled into 68,175 contig sequences. Of these, 10,557 representative sequences were retained after removing Chlorella sequences and lowly expressed sequences. Nearly 90% of these transcript sequences were annotated by similarity search against protein databases. We identified differentially expressed genes in the symbiont-bearing P. bursaria cells relative to the symbiont-free cells, including heat shock 70 kDa protein and glutathione S-transferase.ConclusionsThis is the first reported comprehensive sequence resource of Paramecium – Chlorella endosymbiosis. Results provide some keys for the elucidation of secondary endosymbiosis in P. bursaria. We identified P. bursaria genes that are differentially expressed in symbiont-bearing and symbiont-free conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-183) contains supplementary material, which is available to authorized users.

The bacterium Holospora obtusa is a macronuclear-specific symbiont of the ciliate Paramecium caudatum. H. obtusa-bearing paramecia could survive even after the cells were quickly heated from 25 degrees C to 35 degrees C. To determine whether infection with H. obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P. caudatum. The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes. Competitive PCR showed that H. obtusa-free paramecia expressed only trace amounts of hsp60 and hsp70 mRNA at 25 degrees C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35 degrees C. H. obtusa-bearing paramecia expressed high levels of hsp7O mRNA even at 25 degrees C and the level was further enhanced when the cells were incubated at 35 degrees C. In contrast, the expression pattern of hsp60 mRNA was the same in H. obtusa-bearing as in H. obtusa-free paramecia. These results indicate that infection with its endosymbiont can confer a heat-shock resistant nature on its host cells.

Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25+/-1 degrees C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host's digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis.

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