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1.
Sci Rep ; 12(1): 3216, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35217678

RESUMO

Sialic acid (SA) is present in glycoconjugates and important in cell-cell recognition, cell adhesion, and cell growth and as a receptor. Among the four mammalian sialidases, cytosolic NEU2 has a pivotal role in muscle and neuronal differentiation in vitro. However, its biological functions in vivo remain unclear due to its very low expression in humans. However, the presence of cytoplasmic glycoproteins, gangliosides, and lectins involved in cellular metabolism and glycan recognition has suggested the functional importance of cytosolic Neu2 sialidases. We generated a Neu2 knockout mouse model via CRISPR/Cas9-mediated genome engineering and analyzed the offspring littermates at different ages to investigate the in vivo function of cytosolic Neu2 sialidase. Surprisingly, knocking out the Neu2 gene in vivo abrogated overall lipid metabolism, impairing motor function and leading to diabetes. Consistent with these results, Neu2 knockout led to alterations in sialylated glycoproteins involved in lipid metabolism and muscle function, as shown by glycoproteomics analysis.


Assuntos
Metabolismo dos Lipídeos , Músculos , Neuraminidase , Animais , Citosol/metabolismo , Mamíferos/metabolismo , Camundongos , Músculos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo
2.
Cells ; 11(3)2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35159173

RESUMO

High-grade ovarian cancer (HGOC) is the most lethal gynecological cancer, with high metastasis and recurrence. Cancer stem cells (CSCs) are responsible for its apoptosis resistance, cancer metastasis, and recurrence. Thus, targeting CSCs would be a promising strategy for overcoming chemotherapy resistance and improving patient prognosis in HGOC. Among upregulated oncogenic proteins in HGOC, we found that transcription factor SOX9 showed a strong correlation with stemness-regulating ALDH1A1 and was localized predominantly in the cytoplasm of HGOC with lymph node metastasis. In order to address the role of unusual cytoplasmic SOX9 and to explore its underlying mechanism in HGOC malignancy, a Y2H assay was used to identify a necroptotic cell death-associated cytoplasmic protein, receptor-interacting serine/threonine protein kinase 1 (RIPK1), as a novel SOX9-interacting partner and further mapped their respective interacting domains. The C-terminal region containing the transactivation domain of SOX9 interacted with the death domain of R1PK1. Consistent with its stemness-promoting function, SOX9 knockdown in vitro resulted in changes in cell morphology, cell cycle, stem cell marker expression, cell invasion, and sphere formation. Furthermore, in vivo knockdown completely inhibited tumor growth in mouse xenograft model. We propose that cytoplasmic SOX9-mediated cell death suppression would contribute to cancer stem cell survival in HGOC.


Assuntos
Neoplasias , Proteína Serina-Treonina Quinases de Interação com Receptores , Fatores de Transcrição SOX9 , Animais , Morte Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
3.
Cells ; 9(8)2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32784646

RESUMO

Previously, we demonstrated that the homeoprotein Msx1 interaction with p53 inhibited tumor growth by inducing apoptosis. However, Msx1 can exert its tumor suppressive effect through the inhibition of angiogenesis since growth of the tumor relies on sufficient blood supply from the existing vessels to provide oxygen and nutrients for tumor growth. We hypothesized that the inhibition of tumor growth by Msx1 might be due to the inhibition of angiogenesis. Here, we explored the role of Msx1 in angiogenesis. Overexpression of Msx1 in HUVECs inhibited angiogenesis, and silencing of Msx1 by siRNA abrogated its anti-angiogenic effects. Furthermore, forced expression of Msx1 in mouse muscle tissue inhibited vessel sprouting, and application of an Ad-Msx1-transfected conditioned medium onto the chicken chorioallantoic membrane (CAM) led to a significant inhibition of new vessel formation. To explore the underlying mechanism of Msx1-mediated angiogenesis, yeast two-hybrid screening was performed, and we identified PIASy (protein inhibitor of activated STAT Y) as a novel Msx1-interacting protein. We mapped the homeodomain of Msx1 and the C-terminal domain of PIASy as respective interacting domains. Consistent with its anti-angiogenic function, overexpression of Msx1 suppressed the reporter activity of VEGF. Interestingly, PIASy stabilized Msx1 protein, whereas deletion of the Msx1-interacting domain in PIASy abrogated the inhibition of tube formation and the stabilization of Msx1 protein. Our findings suggest the functional importance of PIASy-Msx1 interaction in Msx1-mediated angiogenesis inhibition.


Assuntos
Fator de Transcrição MSX1/metabolismo , Neovascularização Fisiológica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Embrião de Galinha , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica
4.
RSC Adv ; 10(19): 11517-11523, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-35495314

RESUMO

For InP-based QDs, the current technology does not outperform CdSe-based QDs in many respects, one of which is stability. The optical stability of QDs is closely related to their surface properties, so QDs often use organic ligands for surface protection. These organic ligands are dynamically attached and detached on the QD surface; during detachment, their surfaces are easily damaged and oxidized, thereby deteriorating their optical characteristics. Therefore, we have synthesized a ligand 1,2-hexadecanedithiol with a bidentate form, inducing one ligand to bind to the QD surface strongly through the chelate effect, as a good way to improve the stability of the QDs; thus, the PL stability of the green-light-emitting InP-based QDs was greatly increased. To confirm the existence of the dithiol ligand, we used thermogravimetric analysis/simultaneous thermal analysis-mass spectroscopy (TGA/STA-MS). After that, we applied the ligand to blue-light-emitting ZnSe QDs and red-light-emitting InP QDs, and for those two types of QD we also confirmed that the stability was increased. Additionally, we tested dithiol exchanged QDs at a high temperature of 150 °C, and the increase of stability was effective even in a high temperature condition.

5.
Transgenic Res ; 27(5): 441-450, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30132177

RESUMO

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vß13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3' region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.


Assuntos
Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Transgenes/genética , Animais , Southern Blotting , Cromossomos , Expressão Gênica , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/metabolismo
6.
Arch Biochem Biophys ; 537(1): 21-30, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23810294

RESUMO

Celastrol (CEL) is known as a potent inducer of heat shock protein (HSP) in non-muscle cells and exhibits cytoprotective function and inhibitory effects on proteasome and glucocorticoid receptor activities. To investigate an anti-atrophic effect of CEL on skeletal muscle cells, C2C12 myotubes were treated with 150 µM dexamethasone (DEX) for 24h and 1.5 µM CEL was added for the last 6h during the 24h DEX treatment. Compared to the control, the myotube diameter was reduced by a factor of 0.30 by DEX, but CEL treatment almost abrogated the DEX-induced atrophy. CEL treatment also increased expression of HSP72 and phosphorylation of heat shock transcription factor 1 (p-HSF1) 11-fold and 3.4-fold, respectively, as well as accumulation of p-HSF1 in the nucleus. Furthermore, CEL treatment elevated activities of Akt1, p70/S6K and ERK1/2 2.0- to 4.4-fold whereas DEX had no effect on these signaling activities. Inhibition of Akt1 and ERK1/2 pathways by specific inhibitors confirmed CEL-induced anti-atrophic effect. Moreover, DEX-mediated downregulation of FoxO3 phosphorylation and upregulation of MuRF1 expression and proteasome activity were abrogated by CEL treatment. These results demonstrate a novel anti-atrophic function of CEL in muscle cells via both activation of protein anabolic signals and suppression of catabolic signaling activities.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Triterpenos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Triterpenos Pentacíclicos , Inibidores de Proteínas Quinases/administração & dosagem
7.
Oncol Rep ; 23(5): 1463-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20372865

RESUMO

Previously, we generated thymic lymphoma cell lines from EbetaR/Rp53-/- (EP) double mutant mice where the T cell receptor (TCR) beta enhancer (Ebeta) was deleted, and the p53 gene was inactivated. Here, we characterized the EP cell lines to study the roles of the Ebeta and p53 on TCRbeta rearrangements during lymphomagenesis. Recombination activation genes (RAGs) were expressed, while the TCRbeta chain was not expressed in the EP cell lines. Dbeta-Jbeta rearrangements were not detected at all, and Dbeta1 and Dbeta2 cleavages were also not detected in the EP cell lines. However, Jbeta cleavages suppressed in Ebeta mutant thymocytes were readily detected in the EP cell lines. The Jbeta cleavages appeared to be uncoupled, aberrant, RAG-dependent and Ebeta-independent and were not detected in a p53 or Ebeta single mutant background, suggesting that the Jbeta cleavages are selected in the Ebeta and p53 double mutant background. Sequence analysis showed that the cleavage occurred in the cryptic recombination signal sequences (RSSs) present throughout Jbeta gene segments. The results implicate that the uncoupled and aberrant V(D)J cleavages may contribute to double-strand break-mediated genome instability during lymphomagenesis in EP mice.


Assuntos
Elementos Facilitadores Genéticos , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Genes p53 , Linfoma/enzimologia , Linfoma/genética , Recombinação Genética , VDJ Recombinases/metabolismo , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Genes RAG-1 , Instabilidade Genômica , Camundongos , Camundongos Knockout
8.
Cell Tissue Res ; 333(2): 197-206, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18560898

RESUMO

Little is known about the cell-surface molecules that are related to the undifferentiated and pluripotent state of human embryonic stem cells (hESCs). Here, we generated a panel of murine monoclonal antibodies (MAb) against undifferentiated hESCs by a modification of a previously described decoy immunization strategy. H9 hESCs were differentiated in the presence of retinoic acid and used as a decoy immunogen. Twelve Balb/c mice were immunized in the right hind footpads with differentiated H9 cells and in the left hind footpads with undifferentiated H9 cells. After immunization, the left popliteal lymph node cells were collected and were fused with mouse myeloma cells. The fusion resulted in 79 hybridomas secreting MAbs that bound to the undifferentiated H9 cells as shown by flow cytometric analysis. Of these, 70 MAbs bound to the undifferentiated H9 cells, but only weakly or not at all to the differentiated H9 cells. We characterized 37 MAbs (32 IgGs, 5 IgMs) recognizing surface molecules that were down-regulated during embryoid body cell formation. One of the MAbs, L125-C2, was confirmed to immunoprecipitate CD9, previously known as a surface molecule on the undifferentiated hESCs. To investigate the relationship between the MAbs and hESC-specific antibodies, two representative MAbs, viz., L125-C2 and 291-D4, were selected and studied by multi-color flow cytometric analysis. This showed that more than 60% of L125-C2- and 291-D4-positive cells were also positive for the expression of hESC-specific surface molecules such as SSEA3, SSEA4, TRA-1-60, and TRA-1-81, indicating the close relationship between the two MAbs and the hESC-specific surface molecules. Our results suggest that the decoy immunization strategy is an efficient method for isolating a panel of MAbs against undifferentiated hESCs, and that the generated MAbs should be useful for studying the surface molecules on hESCs in the pluripotent and undifferentiated state.


Assuntos
Antígenos de Superfície/imunologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Imunização/métodos , Células-Tronco Pluripotentes/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/genética , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Pluripotentes/citologia
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