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1.
Environ Health Prev Med ; 24(1): 36, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101002

RESUMO

BACKGROUND: Melanin is detectable in various sense organs including the skin in animals. It has been reported that melanin adsorbs toxic elements such as mercury, cadmium, and lead. In this study, we investigated the adsorption of molybdenum, which is widely recognized as a toxic element, by melanin. METHODS: Molybdenum level of the mouse skin was measured by inductively coupled plasma mass spectrometry. The pigmentation level of murine skin was digitalized as the L* value by using a reflectance spectrophotometer. An in vitro adsorption assay was performed to confirm the interaction between molybdenum and melanin. RESULTS: Our analysis of hairless mice with different levels of skin pigmentation showed that the level of molybdenum increased with an increase in the level of skin pigmentation (L* value). Moreover, our analysis by Spearman's correlation coefficient test showed a strong correlation (r = - 0.9441, p < 0.0001) between L* value and molybdenum level. Our cell-free experiment using the Langmuir isotherm provided evidence for the adsorption of molybdenum by melanin. The maximum adsorption capacity of 1 mg of synthetic melanin for molybdenum was 131 µg in theory. CONCLUSION: Our in vivo and in vitro results showed a new aspect of melanin as an adsorbent of molybdenum.


Assuntos
Melaninas/química , Molibdênio/química , Poluentes Químicos da Água/química , Adsorção , Animais , Melaninas/metabolismo , Camundongos , Camundongos Pelados , Camundongos Transgênicos , Molibdênio/metabolismo , Molibdênio/farmacologia , Pele/química , Pele/efeitos dos fármacos , Pigmentação da Pele/efeitos dos fármacos , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/farmacologia
2.
Hum Mol Genet ; 25(9): 1814-23, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26976849

RESUMO

Riboflavin, also known as vitamin B2, is essential for cellular reduction-oxidation reactions, but is not readily synthesized by mammalian cells. It has been proposed that riboflavin absorption occurs through solute carrier family 52 members (SLC52) A1, A2 and A3. These transporters are also candidate genes for the childhood onset-neural degenerative syndrome Brown-Vialetto-Van Laere (BVVL). Although riboflavin is an essential nutrient, why mutations in its transporters result in a neural cell-specific disorder remains unclear. Here, we provide evidence that Slc52a3 is the mouse ortholog of SLC52A3 and show that Slc52a3 deficiency results in early embryonic lethality. Loss of mutant embryos was associated with both defects in placental formation and increased rates of apoptosis in embryonic cells. In contrast, Slc52a3 -/- embryonic stem cell lines could be readily established and differentiated into motor neurons, suggesting that this transporter is dispensable for neural differentiation and short-term maintenance. Consistent with this finding, examination of Slc52a3 gene products in adult tissues revealed expression in the testis and intestine but little or none in the brain and spinal cord. Our results suggest that BVVL patients with SCL52A3 mutations may be good candidates for riboflavin replacement therapy and suggests that either the mutations these individuals carry are hypomorphic, or that in these cases alternative transporters act during human embryogenesis to allow full-term development.


Assuntos
Paralisia Bulbar Progressiva/genética , Paralisia Bulbar Progressiva/patologia , Diferenciação Celular , Embrião de Mamíferos/citologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Proteínas de Membrana Transportadoras/metabolismo , Neurônios Motores/citologia , Mutação/genética , Animais , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Neurogênese
3.
J Med Invest ; 62(3-4): 130-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26399336

RESUMO

Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells.


Assuntos
Células-Tronco Embrionárias/química , Células-Tronco Pluripotentes/química , Proteômica/métodos , Animais , Diferenciação Celular , Células Cultivadas , Eletroforese em Gel Bidimensional , Fator Inibidor de Leucemia/análise , Camundongos
4.
Stem Cells ; 32(12): 3099-111, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25187421

RESUMO

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells.


Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Nucléolo Celular/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , RNA Ribossômico/biossíntese , Animais , Diferenciação Celular/genética , Células Cultivadas , Fator Inibidor de Leucemia/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/fisiologia
5.
PLoS One ; 9(4): e81552, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24709813

RESUMO

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.


Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Células-Tronco Embrionárias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica/fisiologia , Complexo Mediador/biossíntese , Complexo Mediador/genética , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Repressoras/genética
6.
Nat Neurosci ; 16(12): 1725-7, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24185425

RESUMO

Using transgenic mice harboring a targeted LacZ insertion, we studied the expression pattern of the C9ORF72 mouse ortholog (3110043O21Rik). Unlike most genes that are mutated in amyotrophic lateral sclerosis (ALS), which are ubiquitously expressed, the C9ORF72 ortholog was most highly transcribed in the neuronal populations that are sensitive to degeneration in ALS and frontotemporal dementia. Thus, our results provide a potential explanation for the cell type specificity of neuronal degeneration caused by C9ORF72 mutations.


Assuntos
Esclerose Amiotrófica Lateral/genética , Encéfalo/patologia , Demência Frontotemporal/genética , Regulação da Expressão Gênica/genética , Mutação/genética , Neurônios/patologia , Proteínas/genética , Acetilcolinesterase/metabolismo , Idoso , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Recém-Nascidos , Proteína C9orf72 , Células Cultivadas , Embrião de Mamíferos , Demência Frontotemporal/patologia , Genótipo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transfecção
7.
Hum Cell ; 23(1): 1-14, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20590914

RESUMO

Abstract The establishment of efficient methods for promoting stem cell differentiation into target cells is important not only in regenerative medicine, but also in drug discovery. In addition to embryonic stem (ES) cells and various somatic stem cells, such as mesenchymal stem cells derived from bone marrow, adipose tissue, and umbilical cord blood, a novel dedifferentiation technology that allows the generation of induced pluripotent stem (iPS) cells has been recently developed. Although an increasing number of stem cell populations are being described, there remains a lack of protocols for driving the differentiation of these cells. Regeneration of organs from stem cells in vitro requires precise blueprints for each differentiation step. To date, studies using various model organisms, such as zebrafish, Xenopus laevis, and gene-targeted mice, have uncovered several factors that are critical for the development of organs. We have been using X. laevis, the African clawed frog, which has developmental patterns similar to those seen in humans. Moreover, Xenopus embryos are excellent research tools for the development of differentiation protocols, since they are available in high numbers and are sufficiently large and robust for culturing after simple microsurgery. In addition, Xenopus eggs are fertilized externally, and all stages of the embryo are easily accessible, making it relatively easy to study the functions of individual gene products during organogenesis using microinjection into embryonic cells. In the present review, we provide examples of methods for in vitro organ formation that use undifferentiated Xenopus cells. We also describe the application of amphibian differentiation protocols to mammalian stem cells, so as to facilitate the development of efficient methodologies for in vitro differentiation.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Coração/embriologia , Rim/embriologia , Células-Tronco Multipotentes/citologia , Organogênese , Medicina Regenerativa/métodos , Xenopus laevis/embriologia , Animais , Humanos , Camundongos , Miócitos Cardíacos/citologia , Pâncreas/citologia , Pâncreas/embriologia
8.
Biochim Biophys Acta ; 1804(6): 1272-84, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20152940

RESUMO

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.


Assuntos
Dissulfetos , Regulação para Baixo/fisiologia , Precursores Enzimáticos , Muramidase/química , Isomerases de Dissulfetos de Proteínas , Dobramento de Proteína , Espermatogênese/fisiologia , Espermatozoides/enzimologia , Animais , Sequência de Bases , Clonagem Molecular , Dissulfetos/química , Dissulfetos/metabolismo , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Epididimo/enzimologia , Fertilização/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Masculino , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Muramidase/metabolismo , Oxirredução , Desnaturação Proteica , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade da Espécie , Suínos
9.
Biomed Chromatogr ; 23(6): 607-14, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19280682

RESUMO

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses.


Assuntos
Cromatografia por Troca Iônica/métodos , Peptídeos/análise , Albumina Sérica/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Linhagem Celular , Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/citologia , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/isolamento & purificação , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Proteômica/métodos , Albumina Sérica/química , Albumina Sérica/metabolismo , Tripsina/metabolismo
10.
Proteomics ; 9(1): 126-37, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19053146

RESUMO

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self-renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2-D PAGE-based analysis using fluorescently labeled proteins and shotgun-based analysis with isotope-labeled peptides identified 338 proteins, including transmembrane, membrane-binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.


Assuntos
Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/citologia , Proteínas de Membrana/análise , Células-Tronco Pluripotentes/química , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Eletroforese em Gel Bidimensional , Células-Tronco Embrionárias/efeitos dos fármacos , Expressão Gênica , Marcação por Isótopo , Fator Inibidor de Leucemia/metabolismo , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos
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