RESUMO
Per- and polyfluoroalkyl substances (PFAS) are a group of chemicals that have gained interest because some PFAS have been shown to have negative health effects and prolonged environmental and biological persistence. Chemicals classified as PFAS have a wide range of chemical moieties that impart widely variable properties, leading to a range of water treatment process efficacies. The Polanyi Potential Adsorption Theory was used to estimate Freundlich isotherm parameters to predict the efficacy of granular activated carbon (GAC) treatment for 428 PFAS chemicals for which the vast majority had no previously published treatment data. This method accounts for the physical/chemical characteristics of the individual PFAS beyond molecular weight or chain length that have previously been employed. From a statistical analysis of available data and model results, many of the 428 PFAS were predicted to be effectively treatable by GAC. Although not directly applicable to full-scale design, the approach demonstrates a systematic method for predicting the effectiveness of GAC where isotherm or column data are not available. This then can be used to prioritize future research.
RESUMO
Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .
Assuntos
Músculo Esquelético/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Biomarcadores , Separação Celular/métodos , Citometria de Fluxo/métodos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/transplante , Regeneração , Células Satélites de Músculo Esquelético/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismoRESUMO
Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.
Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Imunofluorescência , Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Camundongos , Células Satélites de Músculo Esquelético/transplante , TransfecçãoRESUMO
Following skeletal muscle injury, muscle stem cells (satellite cells) are activated, proliferate, and differentiate to form myofibers. We show that mRNA-decay protein AUF1 regulates satellite cell function through targeted degradation of specific mRNAs containing 3' AU-rich elements (AREs). auf1(-/-) mice undergo accelerated skeletal muscle wasting with age and impaired skeletal muscle repair following injury. Satellite cell mRNA analysis and regeneration studies demonstrate that auf1(-/-) satellite cell self-renewal is impaired due to increased stability and overexpression of ARE-mRNAs, including cell-autonomous overexpression of matrix metalloprotease MMP9. Secreted MMP9 degrades the skeletal muscle matrix, preventing satellite-cell-mediated regeneration and return to quiescence. Blocking MMP9 activity in auf1(-/-) mice restores skeletal muscle repair and maintenance of the satellite cell population. Control of ARE-mRNA decay by AUF1 represents a mechanism for adult stem cell regulation and is implicated in human skeletal muscle wasting diseases.
Assuntos
Células-Tronco Adultas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Feminino , Ribonucleoproteína Nuclear Heterogênea D0 , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Regeneração/fisiologiaRESUMO
Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38α/ß MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis.
Assuntos
Músculo Esquelético/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Células Satélites de Músculo Esquelético/metabolismo , Tristetraprolina/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Músculo Esquelético/lesões , Proteína MyoD/genética , Proteína MyoD/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/patologia , Transdução de Sinais , Tristetraprolina/antagonistas & inibidores , Tristetraprolina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
As cells integrate molecular signals from their environment, cell surface receptors require modified proteoglycans for the robust activation of signaling pathways. Heparan sulfate proteoglycans (HSPGs) have long unbranched chains of repetitive disaccharide units that can be sulfated at specific positions by heparan sulfate O-sulfotransferase (OST) families. Here, we show that two members of the 3-OST family are required in distinct signaling pathways to control left-right (LR) patterning through control of Kupffer's vesicle (KV) cilia length and motility. 3-OST-5 functions in the fibroblast growth factor pathway to control cilia length via the ciliogenic transcription factors FoxJ1a and Rfx2. By contrast, a second 3-OST family member, 3-OST-6, does not regulate cilia length, but regulates cilia motility via kinesin motor molecule (Kif3b) expression and cilia arm dynein assembly. Thus, two 3-OST family members cell-autonomously control LR patterning through distinct pathways that regulate KV fluid flow. We propose that individual 3-OST isozymes create distinct modified domains or 'glycocodes' on cell surface proteoglycans, which in turn regulate the response to diverse cell signaling pathways.
Assuntos
Cílios/enzimologia , Sulfotransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Estruturas Animais/efeitos dos fármacos , Estruturas Animais/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/ultraestrutura , Dineínas/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Fatores de Crescimento de Fibroblastos/metabolismo , Cinesinas/metabolismo , Modelos Biológicos , Morfolinos/farmacologia , Movimento/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologiaRESUMO
In response to muscle injury, satellite cells activate the p38α/ß MAPK pathway to exit quiescence, then proliferate, repair skeletal muscle, and self-renew, replenishing the quiescent satellite cell pool. Although satellite cells are capable of asymmetric division, the mechanisms regulating satellite cell self-renewal are not understood. We found that satellite cells, once activated, enter the cell cycle and a subset undergoes asymmetric division, renewing the satellite cell pool. Asymmetric localization of the Par complex activates p38α/ß MAPK in only one daughter cell, inducing MyoD, which permits cell cycle entry and generates a proliferating myoblast. The absence of p38α/ß MAPK signaling in the other daughter cell prevents MyoD induction, renewing the quiescent satellite cell. Thus, satellite cells employ a mechanism to generate distinct daughter cells, coupling the Par complex and p38α/ß MAPK signaling to link the response to muscle injury with satellite cell self-renewal.
Assuntos
Divisão Celular Assimétrica , Moléculas de Adesão Celular/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Complexos Multiproteicos/metabolismo , Desenvolvimento Muscular , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
The vertebrate body plan features a consistent left-right (LR) asymmetry of internal organs. In several vertebrate embryos, motile cilia generate an asymmetric fluid flow that is necessary for normal LR development. However, the mechanisms involved in orienting LR asymmetric flow with previously established anteroposterior (AP) and dorsoventral (DV) axes remain poorly understood. In zebrafish, asymmetric flow is generated in Kupffer's vesicle (KV). The cellular architecture of KV is asymmetric along the AP axis, with more ciliated cells densely packed into the anterior region. Here, we identify a Rho kinase gene, rock2b, which is required for normal AP patterning of KV and subsequent LR development in the embryo. Antisense depletion of rock2b in the whole embryo or specifically in the KV cell lineage perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ LR asymmetries. Analyses of KV architecture demonstrated that rock2b knockdown altered the AP placement of ciliated cells without affecting cilia number or length. In control embryos, leftward flow across the anterior pole of KV was stronger than rightward flow at the posterior end, correlating with the normal AP asymmetric distribution of ciliated cells. By contrast, rock2b knockdown embryos with AP patterning defects in KV exhibited randomized flow direction and equal flow velocities in the anterior and posterior regions. Live imaging of Tg(dusp6:memGFP)(pt19) transgenic embryos that express GFP in KV cells revealed that rock2b regulates KV cell morphology. Our results suggest a link between AP patterning of the ciliated Kupffer's vesicle and LR patterning of the zebrafish embryo.
Assuntos
Cílios/metabolismo , Embrião não Mamífero/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Cílios/fisiologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Quinases Associadas a rho/genéticaRESUMO
Streptomyces coelicolor A3(2) ftsI- and ftsW-null mutants produced aerial hyphae with no evidence of septation when grown on a traditional osmotically enhanced medium. This phenotype was partially suppressed when cultures were grown on media prepared without sucrose. We infer that functional FtsZ rings can form in ftsI- and ftsW-null mutants under certain growth conditions.
Assuntos
Proteínas de Bactérias/genética , Meios de Cultura/metabolismo , Proteínas de Membrana/genética , Mutação , Proteínas de Ligação às Penicilinas/genética , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/metabolismo , Divisão Celular , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fenótipo , Streptomyces coelicolor/citologia , Streptomyces coelicolor/genéticaRESUMO
Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains, termed the HS fine structure, which give HS specific binding affinities for extracellular ligands. HS 2-O-sulfotransferase (2-OST) catalyzes the transfer of sulfate groups to the 2-O position of uronic acid residues of HS. We report here the characterization and developmental expression patterns of 2-OST in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin. The 2-OST gene has spatially and temporally distinct expression, which is a surprise given the essential role of 2-OST in HS fine structure formation. Furthermore, although 2-OST and C5-epimerase are predicted to be interdependent for protein translocation from the endoplasmic reticulum to the Golgi, their expression is not coordinately regulated during zebrafish development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Racemases e Epimerases/metabolismo , Sulfotransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Análise por Conglomerados , Hibridização In Situ , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da Espécie , Sulfotransferases/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 6-O-sulfotransferases (6-OST) catalyze the transfer of sulfate groups to the 6-O position of glucosamine residues of HS. We report here the characterization and developmental expression analysis of the 6-OST gene family in the zebrafish. The zebrafish 6-OST gene family consists of four conserved vertebrate orthologues, including a gene duplication specific to zebrafish. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin development. Members of the 6-OST gene family have spatially and temporally distinct restricted expression, suggesting in vivo functional differences exist between members of this family.
Assuntos
Sulfotransferases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Dados de Sequência Molecular , Família Multigênica , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Sulfotransferases/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 3-O-sulfotransferases (3-OST) catalyze the transfer of sulfate groups to the 3-O position of glucosamine residues of HS, a rare, but essential HS chain modification required for HS fine structure. We report here the first characterization and developmental expression analysis of the 3-OST gene family in a vertebrate. There are eight 3-OST genes in zebrafish: seven genes with homology to known 3-OST genes in mouse and human, as well as a novel, 3-OST-7. A phylogenetic comparison of human, mouse, and zebrafish indicates the 3-OST family can be subdivided into two distinct subgroups. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, somites, brain, internal body organ primordial, and pectoral fin development. The 3-OST gene family has both specifically expressed and ubiquitously expressed genes, suggesting in vivo functional differences exist between members of this family.
Assuntos
Sulfotransferases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Família Multigênica , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Sulfotransferases/química , Sulfotransferases/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) activates the aryl hydrocarbon receptor (AhR) to mediate transcriptional activity of dioxin-responsive genes. The transactivation domain (TAD) of human AhR (hAhR) has potentially distinct acidic, glutamine-rich, and proline/serine/threonine-rich subdomains. Cotransfection of exogenous hAhR into BP8 cells with isolated subdomains of hAhR TAD fused to glutathione S-transferase exhibited squelching of TCDD-dependent dioxin-response element (DRE)-driven luciferase reporter-gene activity with each subdomain. To study the potential cross talk between AhR- and estrogen receptor (ER)-mediated activities, BP8 cells were cotransfected with hAhR TAD subdomain constructs and ERalpha. The three hAhR TAD subdomains inhibited the 17beta-estradiol-induced estrogen-response element-mediated reporter-gene transactivation. Cotransfection of hAhR with the ligand-binding domain (LBD) of ERalpha also squelched TCDD-dependent DRE-driven reporter-gene activity in the presence of 17beta-estradiol. Similar results were observed in T47D cells that express functional AhR and ERalpha. These results indicate that the isolated subdomains of hAhR's TAD and LBD of ERalpha are capable of squelching ligand-dependent transactivation of either the AhR or the ER, by titrating crucial proteins from an existing common pool of cofactors.