Polycomb-group (PcG) proteins are essential regulators of hematopoietic stem cells (HSCs). In contrast to Bmi1, a component of Polycomb repressive complex 1 (PRC1), the role of PRC2 and its components in hematopoiesis remains elusive. Here we show that Ezh2, a core component of PRC2, is essential for fetal, but not adult, HSCs. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells and defective erythropoiesis in fetal liver. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis, except for lymphopoiesis. Of note, Ezh2-deficient fetal liver cells showed a drastic reduction in trimethylation of histone H3 at lysine 27 (H3K27me3) accompanied by derepression of a large cohort of genes, whereas on homing to BM, they acquired a high level of H3K27me3 and long-term repopulating capacity. Quantitative RT-PCR revealed that Ezh1, the gene encoding a backup enzyme, is highly expressed in HSCs/progenitor cells in BM compared with those in fetal liver, whereas Ezh2 is ubiquitously expressed. These findings suggest that Ezh1 complements Ezh2 in the BM, but not in the fetal liver, and reveal that the reinforcement of PcG-mediated gene silencing occurs during the transition from proliferative fetal HSCs to quiescent adult HSCs.
Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of “off-the-shelf” T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce “off-the-shelf” and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.
The amino groups on the inner surface of the nanotube that was self-assembled from unsymmetrical bolaamphiphile, N-(2-aminoethyl)-N‘-(β-d-glucopyranosyl)-icosanediamide 1, were modified covalently with a fluorescence donor dye. This functionalization of the nanotube inner surfaces has allowed us to achieve the construction of an optical recognition system for the encapsulation of guest molecules. Fluorescence resonance energy transfer (FRET) from the fluorescence donor located on the inner surface to the ferritin labeled with fluorescence acceptor enabled us to visualize the encapsulation and nanofluidic features of the ferritin in the nanochannel shaped by the hollow cylinder structure. By using this system, we were able to estimate the diffusion constants for ferritin and gold nanoparticles in the organic nanochannels on the basis of lipid nanotubes. We have also demonstrated that the size and surface charge of the nanochannel strongly affect the encapsulation behavior toward the biomacromolecules such as DNA and spherical proteins.
A gram-negative bacterium, Sphingomonas sp. strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface. The pit is produced only when the cells have to incorporate and assimilate alginate. An alginate uptake-deficient mutant was derived from cells of strain A1. One open reading frame, algS (1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation. algS was followed by two open reading frames, algM1 (972 bp) and algM2 (879 bp), which exhibit homology with the transmembrane permeases of ABC transporters. Disruption of algS of strain A1 resulted in the failure to incorporate alginate and to form a pit. Hexahistidine-tagged AlgS protein (AlgS His6 ) overexpressed in Escherichia coli and purified by Ni 2؉ affinity column chromatography showed ATPase activity. Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.Alginate is a heteropolysaccharide consisting of ␣-L-mannuronate and its 5Ј-epimer, ␣-L-guluronate, and is produced by brown seaweeds and certain bacteria. Other than its utilization in the medical, chemical, and food areas, this biopolymer has been widely associated with chronic Pseudomonas aeruginosa infection in the lungs of cystic fibrosis patients (5). A bacterium, Sphingomonas sp. strain A1, was isolated from soil as a potent producer of alginate lyase catalyzing the depolymerization of alginate (24). The cells of strain A1 are covered by many large plaits (Fig. 1a), and a mouth-like pit (0.02 to 0.1 m in diameter) is formed on their surface when they are grown in a medium containing alginate as the sole carbon source (10, 11) ( Fig. 1b and c). The pit formed in the presence of alginate disappears when the cells are transferred to a medium without alginate (10, 11). In the presence of alginate, a specific region of the cell surface and its neighborhood is intensely stained with an agent that interacts with mucopolysaccarides (alginate), and thin sections of cells show an irregular site where the cell membrane sinks deeply into the cytosol (10, 11).For the utilization of polysaccharides and other macromolecules, microbes usually degrade them by means of extracellular enzymes and then take the depolymerized low-molecular-weight (LMW) products up through their membranes. However, alginate lyase is exclusively localized in the cytoplasm, and the periplasmic and membrane fractions contain no alginate lyase activity (25). Furthermore, no extracellular alginate-depolymerizing activities have been detected in concentrated culture fluid of strain A1 (24, 25), when assayed by measuring the changes in viscosity and absorbance at 235 nm (for alginate lyase) or analysis of the depolymerization profile by thin-layer chromatography (data not shown). The amino acid sequence of alginate lyase purified from strain A1 was 53 amino acids shorter in the N-terminal region than t...
Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. Here, we report that GNBP3 N-terminal domain binds to fungi upon identifying long chains of -1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for -glucan recognition.
Cadmium sulfide (CdS) nanoparticles (NPs) were synthesized in the cavity of the recombinant Dps cage-shaped protein from Listeria innocua (rLiDps) modifying the slow chemical reaction synthesis (SCRY) and two-step synthesis protocol (TSSP). The SCRY is realized by stabilizing cadmium ions as tetraamminecadmium and employing thioacetic acid (which is added in the final stage of the TSSP). The optimized condition produced cubic CdS NPs in the rLiDps cavity with an average diameter of 4.2 nm and small size dispersion. These CdS NPs showed photoluminescence. It was also found that the pH of the reaction solution affects the elemental composition CdS NPs. At pH 4.5 and 6.5, Cd x S y (x < y) NPs were obtained, and at pH 8.5, cubic CdS NPs were synthesized at pH 8.5.
A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.
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