RESUMO
Human induced pluripotent stem cells (hiPSCs) are useful starting materials for the generation of cell therapy products, due to their pluripotency and ability to self-renew. Quality control of hiPSCs is extremely important in creating a stable supply of hPSC-derived products. Previously we identified an hiPSC-specific lectin probe, rBC2LCN, which binds specifically to α1,2-fucosylated glycan and recognizes podocalyxin (PODXL) as a glycoprotein ligand. In this study, we produced monoclonal antibodies (mAbs) specific for α1,2-fucosylated PODXL expressed on hiPSCs. PODXL was recombinantly expressed in fucosyltransferase 1 (FUT1)-transfected HEK293, followed by immunization into mice. Monoclonal antibodies, which bind to PODXL/FUT1-transfected cells, but not to cells transfected with only one of PODXL or FUT1, were screened by flow cytometry. The two mAbs generated (179-6B8C9 and 179-7E12E10), termed α1,2-fucosylated PODXL-specific mAbs (FpMabs), showed binding specificity to PODXL/FUT1-transfected cells. The FpMabs bound to hiPSCs but never to human adipose-derived mesenchymal stem cells, human dermal fibroblasts, or hiPSC-derived mesoderm. Altogether, FpMabs are highly specific probes for hiPSCs, which might be a powerful tool for the characterization of hiPSCs used in regenerative medicine.
Assuntos
Anticorpos Monoclonais , Células-Tronco Pluripotentes Induzidas/imunologia , Sialoglicoproteínas/imunologia , Animais , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Fluorescência , Fucosiltransferases/genética , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Polissacarídeos/análise , Polissacarídeos/imunologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , TransfecçãoRESUMO
Subseafloor pelagic sediments with high concentrations of organic matter form habitats for diverse microorganisms. Here, we determined depth profiles of genes for SSU rRNA, mcrA, dsrA and amoA from just beneath the seafloor to 363.3 m below the seafloor (mbsf) using core samples obtained from the forearc basin off the Shimokita Peninsula. The molecular profiles were combined with data on lithostratigraphy, depositional age, sedimentation rate and pore-water chemistry. The SSU rRNA gene tag structure and diversity changed at around the sulfate-methane transition zone (SMTZ), whereas the profiles varied further with depth below the SMTZ, probably in connection with the variation in pore-water chemistry. The depth profiles of diversity and abundance of dsrA, a key gene for sulfate reduction, suggested the possible niche separations of sulfate-reducing populations, even below the SMTZ. The diversity and abundance patterns of mcrA, a key gene for methanogenesis/anaerobic methanotrophy, suggested a stratified distribution and separation of anaerobic methanotrophy and hydrogenotrophic or methylotrophic methanogensis below the SMTZ. This study provides novel insights into the relationships between the composition and function of microbial communities and the chemical environment in the nutrient-rich continental margin subseafloor sediments, which may result in niche separation and variability in subseafloor microbial populations.
Assuntos
Bactérias/isolamento & purificação , Sedimentos Geológicos/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Ecossistema , Sedimentos Geológicos/química , Japão , Metano/análise , Metano/metabolismo , Filogenia , Sulfatos/análise , Sulfatos/metabolismoRESUMO
The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.
Assuntos
Proteínas Arqueais/genética , Genoma Arqueal , Ubiquitinas/genética , Sequência de Aminoácidos , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/química , Sequência de Bases , Ciclo Celular/genética , Reparo do DNA , Replicação do DNA , Metabolismo Energético/genética , Evolução Molecular , Genes Arqueais , Biblioteca Genômica , Proteínas de Choque Térmico/genética , Metagenoma , Dados de Sequência Molecular , Filogenia , Biossíntese de Proteínas , Alinhamento de Sequência , Transcrição Gênica , Ubiquitinas/químicaRESUMO
OBJECTIVE: To elucidate the role of the fractalkine (FKN)/CX3 CR1 pathway in joint destruction in rheumatoid arthritis. METHODS: We examined the effect of treatment with anti-mouse FKN (anti-mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen-induced arthritis (CIA) model. DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti-mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x-ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme-linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow-derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed. RESULTS: Both prophylactic and therapeutic treatment with anti-mFKN mAb significantly decreased the arthritis and soft x-ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti-mFKN mAb. Histologic analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. Anti-mFKN mAb strongly inhibited the migration of bone marrow-derived OCPs into the affected synovium. CONCLUSION: Anti-mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX3 CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Movimento Celular/efeitos dos fármacos , Quimiocina CX3CL1/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Proteína de Matriz Oligomérica de Cartilagem/efeitos dos fármacos , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Quimiocina CX3CL1/imunologia , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Sinovite/patologia , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas/metabolismo , Animais , Diferenciação Celular/genética , Separação Celular/métodos , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Feminino , Humanos , Macaca fascicularis , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos Endogâmicos C57BL , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Proteínas/genética , Ratos Sprague-Dawley , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transplante de Células-Tronco/métodos , Transplante HeterólogoRESUMO
Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4+ T helper 2 cells play critical roles in the pathogenesis of allergic airway inflammation, and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention through inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and Myl12 are new functional ligands for CD69. Blockade of CD69-Myl9/12 interaction ameliorates allergic airway inflammation in ovalbumin-induced and house dust mite-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of Myl9/12 was increased and that platelet-derived Myl9/12 localized to the luminal surface of blood vessels and formed intravascular net-like structures. Analysis of nasal polyps of eosinophilic chronic rhinosinusitis patients revealed that Myl9/12 expression was increased in inflammatory lesions and was distributed within net-like structures in the intravascular space. In addition, we detected Myl9/12 in perivascular spaces where many CD69+ cells were positioned within Myl9/12 structures. Thus, CD69-Myl9/12 interaction is a key event in the recruitment of activated CD69+ T cells to inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases.
RESUMO
Strain Hiromi 1, a sulfur-oxidizing gammaproteobacterium was isolated from a hydrothermal vent chimney in the Okinawa Trough and represents a novel genus that may include a phylogenetic group found as endosymbionts of deep-sea gastropods. The SSU rRNA gene sequence similarity between strain Hiromi 1 and the gastropod endosymbionts was approximately 97%. The strain was shown to grow both chemolithoautotrophically and chemolithoheterotrophically with an energy metabolism of sulfur oxidation and O2 or nitrate reduction. Under chemolithoheterotrophic growth conditions, the strain utilized organic acids and proteinaceous compounds as the carbon and/or nitrogen sources but not the energy source. Various sugars did not support growth as a sole carbon source. The observation of chemolithoheterotrophy in this strain is in line with metagenomic analyses of endosymbionts suggesting the occurrence of chemolithoheterotrophy in gammaproteobacterial symbionts. Chemolithoheterotrophy and the presence of homologous genes for virulence- and quorum sensing-related functions suggest that the sulfur-oxidizing chomolithotrophic microbes seek animal bodies and microbial biofilm formation to obtain supplemental organic carbons in hydrothermal ecosystems.
Assuntos
Gammaproteobacteria/genética , Fontes Hidrotermais/microbiologia , Microbiologia da Água , Sistemas de Secreção Bacterianos/genética , Quimiotaxia , Metabolismo Energético , Gammaproteobacteria/isolamento & purificação , Gammaproteobacteria/ultraestrutura , Genoma Bacteriano , Redes e Vias Metabólicas , Dados de Sequência Molecular , Tipagem Molecular , Filogenia , Subunidades Ribossômicas Menores de Bactérias/genéticaAssuntos
Acetilcisteína/farmacologia , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/metabolismo , Animais , Quinase I-kappa B , Oxirredução , Células PC12 , Feocromocitoma , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA, and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 x 10(7) cells mL(-1) or copies g(-1) sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin.
Assuntos
Archaea/genética , Ecossistema , Sedimentos Geológicos/microbiologia , Microbiologia da Água , Archaea/classificação , Archaea/enzimologia , Biomassa , DNA Arqueal/genética , Genes Arqueais , Genes de RNAr , Hidrogenase/metabolismo , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.