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N-acetyl cysteine (NAC) is a versatile drug used in various conditions, but the limitations and toxicities are not clear. The acute toxicity and toxicological mechanisms of an intraperitoneal injection of NAC in normal mice were deciphered. The LD50 for male and female BALB/cByJNarl mice were 800 mg/kg and 933 mg/kg. The toxicological mechanisms of 800 mg/kg NAC (N800) were investigated. The serum biomarkers of hepatic and renal indices dramatically increased, followed by hepatic microvesicular steatosis, renal tubular injury and necrosis, and splenic red pulp atrophy and loss. Thus, N800 resulted in mouse mortality mainly due to acute liver, kidney, and spleen damages. The safe dose (275 mg/kg) of NAC (N275) increased hepatic antioxidant capacity by increasing glutathione levels and catalase activity. N275 elevated the hepatic gene expressions of lipid transporter, lipid synthesis, ß-oxidation, and ketogenesis, suggesting a balance between lipid production and consumption, and finally, increased ATP production. In contrast, N800 increased hepatic oxidative stress by decreasing glutathione levels through suppressing Gclc, and reducing catalase activity. N800 decreased the hepatic gene expressions of lipid transporter, lipid synthesis, and interferred ß-oxidation, leading to lipid accumulation and increasing Cyp2E1 expression, and finally, decreased ATP production. Therefore, NAC doses are limited for normal individuals, especially via intraperitoneal injection or similar means.
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After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.
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Citoplasma , Herpesvirus Humano 4 , Proteínas Serina-Treonina Quinasas , Proteínas Virales , Virión , Ensamble de Virus , Liberación del Virus , Proteínas Activadoras de ras GTPasa , Humanos , Proteínas de la Cápside/metabolismo , Citoplasma/metabolismo , Citoplasma/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crecimiento & desarrollo , Herpesvirus Humano 4/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Virales/metabolismo , Virión/química , Virión/crecimiento & desarrollo , Virión/metabolismo , Ensamble de Virus/fisiología , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismoRESUMEN
Transthyretin (TTR)-related amyloidosis (ATTR) is a syndrome of diseases characterized by the extracellular deposition of fibrillar materials containing TTR variants. Ala97Ser (A97S) is the major mutation reported in Taiwanese ATTR patients. Here, we combine atomic resolution structural information together with the biochemical data to demonstrate that substitution of polar Ser for a small hydrophobic side chain of Ala at residue 97 of TTR largely influences the local packing density of the FG-loop, thus leading to the conformational instability of native tetramer, the increased monomeric species, and thus the enhanced amyloidogenicity of apo-A97S. Based on calorimetric studies, the tetramer destabilization of A97S can be substantially altered by interacting with native stabilizers via similarly energetic patterns compared to that of wild-type (WT) TTR; however, stabilizer binding partially rearranges the networks of hydrogen bonding in TTR variants while FG-loops of tetrameric A97S still remain relatively flexible. Moreover, TTR in complexed with holo-retinol binding protein 4 is slightly influenced by the structural and dynamic changes of FG-loop caused by A97S substitution with an approximately five-fold difference in binding affinity. Collectively, our findings suggest that the amyloidogenic A97S mutation destabilizes TTR by increasing the flexibility of the FG-loop in the monomer, thus modulating the rate of amyloid fibrillization.
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Amiloide , Prealbúmina , Humanos , Amiloide/química , Proteínas Amiloidogénicas/genética , Calorimetría , Mutación , Prealbúmina/genética , Prealbúmina/químicaRESUMEN
A thermally stable vaccine platform is considered the missing piece of vaccine technology. In this article, we reported the creation of a novel protein nanoparticle and assessed its ability to withstand extended high temperature incubation while stimulating a long-lasting humoral immune response. This protein nanoparticle was assembled from a fusion protein composed of an amphipathic helical peptide derived from the M2 protein of the H5N1 influenza virus (AH3) and a superfolder green fluorescent protein (sfGFP). Its proposed structure was modeled according to transmission electronic microscope (TEM) images of protein nanoparticles. From this proposed protein model, we created a mutant with two gain-of-function mutations that work synergistically on particle stability. A protein nanoparticle assembled from this gain-of-function mutant is able to remove a hydrophobic patch from its surface. This gain-of-function mutant also contributes to the higher thermostability of protein nanoparticles and stimulates a long lasting humoral immune response after a single immunization. This assembled nanoparticle showed increasing particle stability at higher temperatures and salt concentrations. This novel protein nanoparticle may serve as a thermally-stable platform for vaccine development.
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Adaptive mechanisms that facilitate intestinal colonization by the human microbiota, including Escherichia coli, may be better understood by analyzing the physiology and gene expression of bacteria in low-oxygen environments. We used high-throughput transcriptomics and proteomics to compare the expression profiles of E. coli grown under aerobic versus microaerobic conditions. Clustering of high-abundance transcripts under microaerobiosis highlighted genes controlling acid-stress adaptation (gadAXW, gadAB, hdeAB-yhiD and hdeD operons), cell adhesion/biofilm formation (pgaABCD and csgDEFG operons), electron transport (cydAB), oligopeptide transport (oppABCDF), and anaerobic respiration/fermentation (hyaABCDEF and hycABCDEFGHI operons). In contrast, downregulated genes were involved in iron transport (fhuABCD, feoABC and fepA-entD operons), iron-sulfur cluster assembly (iscRSUA and sufABCDSE operons), aerobic respiration (sdhDAB and sucABCDSE operons), and de novo nucleotide synthesis (nrdHIEF). Additionally, quantitative proteomics showed that the products (proteins) of these high- or low-abundance transcripts were expressed consistently. Our findings highlight interrelationships among energy production, carbon metabolism, and iron homeostasis. Moreover, we have identified and validated a subset of differentially expressed noncoding small RNAs (i.e., CsrC, RyhB, RprA and GcvB), and we discuss their regulatory functions during microaerobic growth. Collectively, we reveal key changes in gene expression at the transcriptional and post-transcriptional levels that sustain E. coli growth when oxygen levels are low.
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Proteínas de Escherichia coli , Escherichia coli , Anaerobiosis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Humanos , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Oxígeno/metabolismo , Proteómica , ARN no Traducido/metabolismo , TranscriptomaRESUMEN
The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFß1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFß1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, clustering at the nuclear periphery and reintegrating into the nucleoplasm. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFß1-induced compositional changes in the chromatin and nuclear lamina.
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Histonas , Membrana Nuclear , Línea Celular Tumoral , Núcleo Celular , Humanos , Lámina Nuclear , Isoformas de ProteínasRESUMEN
Acetaminophen (APAP) overdose induces acute liver damage and even death. The standard therapeutic dose of N-acetyl cysteine (NAC) cannot be applied to every patient, especially those with high-dose APAP poisoning. There is insufficient evidence to prove that increasing NAC dose can treat patients who failed in standard treatment. This study explores the toxicity of NAC overdose in both APAP poisoning and normal mice. Two inbred mouse strains with different sensitivities to propacetamol-induced hepatotoxicity (PIH) were treated with different NAC doses. NAC therapy decreased PIH by reducing lipid oxidation, protein nitration and inflammation, and increasing glutathione (GSH) levels and antioxidative enzyme activities. However, the therapeutic effects of NAC on PIH were dose-dependent from 125 (N125) to 275 mg/kg (N275). Elevated doses of NAC (400 and 800 mg/kg, N400 and N800) caused additional deaths in both propacetamol-treated and normal mice. N800 treatments significantly decreased hepatic GSH levels and induced inflammatory cytokines and hepatic microvesicular steatosis in both propacetamol-treated and normal mice. Furthermore, both N275 and N400 treatments decreased serum triglyceride (TG) and induced hepatic TG, whereas N800 treatment significantly increased interleukin-6, hepatic TG, and total cholesterol levels. In conclusion, NAC overdose induces hepatic and systemic inflammations and interferes with fatty acid metabolism.
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Neuromuscular junctions (NMJs) govern efficient neuronal communication with muscle cells, relying on proper architecture of specialized postsynaptic compartments. However, the intrinsic mechanism in muscle cells contributing to NMJ development remains unclear. In this study, we reveal that dynamin-2 (Dyn2) is involved in postsynaptic development of NMJs. Mutations of Dyn2 have been linked to human muscular disorder and centronuclear myopathy (CNM), as well as featured with muscle atrophy and defective NMJs, yet the function of Dyn2 at the postsynaptic membrane is largely unknown. We demonstrate that Dyn2 is enriched at the postsynaptic membrane and regulates NMJ development via actin remodeling. Dyn2 functions as an actin-bundling GTPase to regulate podosome turnover and cytoskeletal organization of the postsynaptic apparatus, and CNM-Dyn2 mutations display abnormal actin remodeling and electrophysiological activity of fly NMJs. Altogether, Dyn2 primarily regulates actin cytoskeleton remodeling and NMJ morphogenesis at the postsynaptic membrane, which is distinct from its endocytosis regulatory role at the presynaptic membrane.
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Citoesqueleto/fisiología , Dinamina II/metabolismo , Unión Neuromuscular/crecimiento & desarrollo , HumanosRESUMEN
Dynamic fission and fusion events regulate mitochondrial shape, distribution, and rejuvenation, and proper control of these processes is essential for neuronal homeostasis. Here, we report that Gas7, a known cytoskeleton regulator, controls mitochondrial dynamics within neurons of the central nervous system. In this study, we generated an improved Gas7-knockout mouse and evaluated its mitochondrial phenotype. We first identified Gas7 in mitochondrial fractions from wild-type brain tissue, and observed Gas7 colocalization with mitochondria in primary cortical neurons. In Gas7-deficient brain tissue and neuronal cultures mitochondria were elongated with perinuclear clustering. These morphological abnormalities were associated with increased levels mitochondrial fusion proteins and increased PKA-dependent phosphorylation of Drp-1 in brain tissues, suggesting an imbalance of mitochondrial fusion and fission. Moreover, expression of mitochondrial quality control kinase, PINK1, and PINK1-specific phosphorylation of Mfn-2 (S442), Parkin (S65), and ubiquitin (S65) were all reduced in the knockout cells. Ectopic expression of Gas7 restored mitochondrial morphology and distribution, as well as PINK1 expression in Gas7-null cortical neurons. Collectively, our results introduce a novel role of mouse Gas7 in determining the dynamics, morphology, and intracellular distribution of neuronal mitochondria, which are expected to be required for normal neuronal function.
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Skin depigmentation diseases, such as vitiligo, are pigmentation disorders that often destroy melanocytes. However, their pathological mechanisms remain unclear, and therefore, promising treatments or prevention has been lacking. Here, we demonstrate that a zebrafish insertional mutant showing a significant reduction of nicastrin transcript possesses melanosome maturation defect, Tyrosinase-dependent mitochondrial swelling, and melanophore cell death. The depigmentation phenotypes are proven to be a result of γ-secretase inactivation. Furthermore, live imaging demonstrates that macrophages are recruited to and can phagocytose melanophore debris. Thus, we characterize a potential zebrafish depigmentation disease model, a nicastrinhi1384 mutant, which can be used for further treatment or drug development of diseases related to skin depigmentation and/or inflammation.
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Secretasas de la Proteína Precursora del Amiloide/genética , Hipopigmentación/genética , Glicoproteínas de Membrana/genética , Monofenol Monooxigenasa/metabolismo , Piel/inmunología , Proteínas de Pez Cebra/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Embrión no Mamífero , Humanos , Hipopigmentación/inmunología , Hipopigmentación/patología , Melanosomas/inmunología , Melanosomas/metabolismo , Melanosomas/ultraestructura , Glicoproteínas de Membrana/metabolismo , Microscopía Electrónica de Transmisión , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/genética , Mutación , Piel/patología , Pigmentación de la Piel/efectos de los fármacos , Pigmentación de la Piel/genética , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
O-acetyl-ADP-ribose (AAR) is a metabolic small molecule relevant in epigenetics that is generated by NAD-dependent histone deacetylases, such as Sir2. The formation of silent heterochromatin in yeast requires histone deacetylation by Sir2, structural rearrangement of SIR complexes, spreading of SIR complexes along the chromatin, and additional maturation processing. AAR affects the interactions of the SIR-nucleosome in vitro and enhances the chromatin epigenetic silencing effect in vivo. In this study, using isothermal titration calorimetry (ITC) and dot blotting methods, we showed the direct interaction of AAR with Sir3. Furthermore, through chromatin immunoprecipitation (ChIP)-on-chip and chromatin affinity purification (ChAP)-on chip assays, we discovered that AAR is capable of increasing the extended spreading of Sir3 along telomeres, but not Sir2. In addition, the findings of a quantitative real-time polymerase chain reaction (qRT-PCR) and examinations of an in vitro assembly system of SIR-nucleosome heterochromatin filament were consistent with these results. This study provides evidence indicating another important effect of AAR in vivo. AAR may play a specific modulating role in the formation of silent SIR-nucleosome heterochromatin in yeast.
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Cromatina/genética , O-Acetil-ADP-Ribosa/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Telómero/genética , Epigénesis Genética , Regulación Fúngica de la Expresión Génica , Código de Histonas , Unión Proteica , Saccharomyces cerevisiaeRESUMEN
In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.
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Heterocromatina/metabolismo , Nucleosomas/metabolismo , O-Acetil-ADP-Ribosa/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Epigénesis Genética , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , Sirtuina 2/metabolismoRESUMEN
To study the epigenetic gene silencing, yeast is an excellent model organism. Sir proteins are required for the formation of silent heterochromatin. Sir2 couples histone deacetylation and NAD hydrolysis to generate an endogenous epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). AAR is involved in the conformational change of SIR complexes, modulates the formation of SIR-nucleosome preheterochromatin and contributes to the spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin regions. Here, we show that AAR is capable of enhancing the chromatin silencing effect under either an extra exogenous AAR or a defect AAR metabolic enzyme situation, but decreasing the chromatin silencing effect under a defect AAR synthetic enzyme state. Our results provide an evidence of biological function importance of AAR. It is indicated that AAR does not only function in vitro but also play a role in vivo to increase the effect of heterochromatin epigenetic gene silencing. However, further investigations of AAR are warranted to expand our knowledge of epigenetics and associated small molecules.
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Cromatina/genética , O-Acetil-ADP-Ribosa/genética , O-Acetil-ADP-Ribosa/metabolismo , Cromatina/fisiología , Epigénesis Genética/genética , Epigenómica/métodos , Silenciador del Gen/fisiología , Heterocromatina/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , O-Acetil-ADP-Ribosa/fisiología , Procesamiento Proteico-Postraduccional/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/genética , Sirtuina 2/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography-tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, ß3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not ß1, ß4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.
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Integrina alfa3/metabolismo , Laminina/metabolismo , Linfangiogénesis , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Células Endoteliales/patología , Vesículas Extracelulares/patología , Técnicas de Silenciamiento del Gen , Humanos , Laminina/genética , Ganglios Linfáticos/patología , Metástasis Linfática/diagnóstico , Metástasis Linfática/patología , Vasos Linfáticos/citología , Masculino , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Yeast silent heterochromatin provides an excellent model with which to study epigenetic inheritance. Previously we developed an in vitro assembly system to demonstrate the formation of filament structures with requirements that mirror yeast epigenetic gene silencing in vivo. However, the properties of these filaments were not investigated in detail. Here we show that the assembly system requires Sir2, Sir3, Sir4, nucleosomes, and O-acetyl-ADP-ribose. We also demonstrate that all Sir proteins and nucleosomes are components of these filaments to prove that they are SIR-nucleosome filaments. Furthermore, we show that the individual localization patterns of Sir proteins on the SIR-nucleosome filament reflect those patterns on telomeres in vivo. In addition, we reveal that magnesium exists in the SIR-nucleosome filament, with a role similar to that for chromatin condensation. These results suggest that a small number of proteins and molecules are sufficient to mediate the formation of a minimal yeast silent pre-heterochromatin in vitro.
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Silenciador del Gen/fisiología , Nucleosomas/metabolismo , O-Acetil-ADP-Ribosa/metabolismo , Sitios de Unión , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Epigenómica/métodos , Heterocromatina/metabolismo , Histonas/metabolismo , Magnesio , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuinas/metabolismo , Telómero/metabolismoRESUMEN
Dengue fever is a mosquito-borne viral pandemic disease that is widespread in the tropical and subtropical areas. Dengue virus uses human mannose-binding receptor (MR) and DC-SIGN on macrophages as primary receptors, and CLEC5A as signaling receptor to sense the dengue virus invasion and then to signal and stimulate macrophages to secrete cytokines. But the interplay between MR/DC-SIGN and CLEC5A is unknown. Here we demonstrate a plausible mechanism for the interaction, i.e. MR/DC-SIGN first attracts the virus with high avidity, and the virus concurrently interacts with CLEC5A in close proximity to form a multivalent hetero-complex and facilitate CLEC5A-mediated signal transduction. Our study suggests that the cooperation between a high-avidity lectin-virus interaction and a nearby low-avidity signaling receptor provides a necessary connection between binding and signaling. Understanding this mechanism may lead to the development of a new antiviral strategy.
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Moléculas de Adhesión Celular/metabolismo , Virus del Dengue/fisiología , Dengue/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/virología , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Células Cultivadas , Humanos , Macrófagos/metabolismo , Receptor de Manosa , Unión ProteicaRESUMEN
Synthetic liposomes provide a biocompatible and biodegradable approach for delivering drugs and antigens. In addition, self-adjuvanting recombinant lipoproteins (rlipoproteins) can enhance Th1 anti-tumor immune responses via the TLR2 signaling pathway. To generate a liposomal rlipoprotein for a cancer immunotherapeutic vaccine, we assessed 3 types of synthetic liposomes for use with the rlipoproteins rlipoE7m and rlipoOVA. We determined that the cationic liposome DOTAP could stabilize anionic rlipoproteins and delay rlipoprotein release. Surprisingly, rlipoproteins and DOTAP could synergistically up-regulate CD83 expression in bone marrow-derived dendritic cells (BMDCs). Compared with other liposome formulations, the rlipoprotein/DOTAP formulation elicited higher cytotoxic T-lymphocyte (CTL) responses. To explore the mechanism of BMDC activation by rlipoprotein/DOTAP, we assessed the production of reactive oxygen species (ROS) and the TNF-α secretion of BMDCs. We observed that rlipoprotein/DOTAP induced ROS to the same extent as DOTAP did. In addition, TLR2 signaling was also required for the TNF-α secretion of rlipoprotein/DOTAP-treated BMDCs. Moreover, compared with rlipoOVA-treated BMDCs, rlipoOVA/DOTAP-treated BMDCs increased the levels of IFN-γ produced by OVA-specific T cells. We also observed that rlipoE7m/DOTAP treatment but not rlipoE7m treatment delayed tumor growth. These results indicate that the rlipoprotein/DOTAP formulation can synergistically activate BMDCs via ROS and the TLR2 signaling pathway. In summary, rlipoprotein/DOTAP is a novel and stable formulation for cancer immunotherapy.
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Vacunas contra el Cáncer/administración & dosificación , Lipoproteínas/administración & dosificación , Neoplasias/terapia , Ovalbúmina/inmunología , Proteínas E7 de Papillomavirus/inmunología , Alérgenos/inmunología , Animales , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Dendríticas/inmunología , Ácidos Grasos Monoinsaturados/química , Lipoproteínas/inmunología , Lipoproteínas/uso terapéutico , Liposomas , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/patología , Compuestos de Amonio Cuaternario/química , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: The use of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to visualize cells has been applied clinically, showing the potential for monitoring cells in vivo with magnetic resonance imaging (MRI). USPIO conjugated with anti-CD133 antibodies (USPIO-CD133 Ab) that recognize the CD133 molecule, a cancer stem cell marker in a variety of cancers, was studied as a novel and potent agent for MRI contrast enhancement of tumor cells. MATERIALS AND METHODS: Anti-CD133 antibodies were used to conjugate with USPIO via interaction of streptavidin and biotin for in vivo labeling of CD133-positive cells in xenografted tumors and N-ethyl-N-nitrosourea (ENU)-induced brain tumors. The specific binding of USPIO-CD133 Ab to CD133-positive tumor cells was subsequently detected by Prussian blue staining and MRI with T2-weighted, gradient echo and multiple echo recombined gradient echo images. In addition, the cellular toxicity of USPIO-CD133 Ab was determined by analyzing cell proliferation, apoptosis, and reactive oxygen species production. RESULTS: USPIO-CD133 Ab specifically recognizes in vitro and labels CD133-positive cells, as validated using Prussian blue staining and MRI. The assays of cell proliferation, apoptosis, and reactive oxygen species production showed no significant differences in tumor cells with or without labeling of USPIO-CD133 Ab. In vivo imaging of CD133-positive cells was demonstrated by intravenous injection of USPIO-CD133 Ab in mice with HT29 xenografted tumors. The MRI of HT29 xenografts showed several clusters of hypotensive regions that correlated with CD133 expression and Prussian blue staining for iron. In rat, brain tumors induced by transplacental ENU mutagenesis, several clusters of hypointensive zones were observed in CD133-expressing brain tumors by MRI and intravenously administered USPIO-CD133 Ab. CONCLUSION: Combination of USPIO-CD133 Ab and MRI is valuable in recognizing CD133-expressing tumor cells in vitro, extracellularly labeling for cell tracking and detecting CD133-expressing tumors in xenografted tumors as well as ENU-induced rat brain tumors.
Asunto(s)
Antígenos CD/metabolismo , Dextranos/química , Glicoproteínas/metabolismo , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Imagen Molecular/métodos , Péptidos/metabolismo , Coloración y Etiquetado , Antígeno AC133 , Animales , Anticuerpos/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patología , Muerte Celular , Etilnitrosourea , Células HT29 , Humanos , Ratones , Unión Proteica , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PGC-1α is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1α might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1α directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1α-targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity. Bhlhe40 repressed PGC-1α activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1α intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1α target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1α-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1α-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1α activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Músculo Esquelético/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Línea Celular , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Músculo Esquelético/citología , Mioblastos/metabolismo , Oxidación-Reducción , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Receptor tyrosine kinases (RTKs) regulate many cellular processes, and Sprouty2 (Spry2) is known as an important regulator of RTK signaling pathways. Therefore, it is worth investigating the properties of Spry2 in more detail. In this study, we found that Spry2 is able to self-assemble into oligomers with a high-affinity KD value of approximately 16nM, as determined through BIAcore surface plasmon resonance analysis. The three-dimensional (3D) structure of Spry2 was resolved using an electron microscopy (EM) single-particle reconstruction approach, which revealed that Spry2 is donut-shaped with two lip-cover domains. Furthermore, the method of energy dispersive spectrum obtained through EM was analyzed to determine the elements carried by Spry2, and the results demonstrated that Spry2 is a silicon- and iron-containing protein. The silicon may contribute to the electroconductivity of Spry2, and this property exhibits a concentration-dependent feature. This study provides the first report of a silicon- and iron-containing protein, and its 3D structure may allow us (1) to study the potential mechanism through the signal transduction is controlled by switching the electronic transfer on or off and (2) to develop a new type of conductor or even semiconductor using biological or half-biological hybrid materials in the future.