RESUMEN
Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases (HACDs) HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs However, we observed only an â¼40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1-KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.
Asunto(s)
Ácidos Grasos/metabolismo , Hidroliasas/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Esquelético/enzimología , Mioblastos Esqueléticos/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Sistemas CRISPR-Cas , Dominio Catalítico , Línea Celular Tumoral , Células Cultivadas , Ácidos Grasos/química , Regulación Enzimológica de la Expresión Génica , Humanos , Hidroliasas/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones Noqueados , Estructura Molecular , Peso Molecular , Músculo Esquelético/citología , Músculo Esquelético/patología , Enfermedades Musculares/enzimología , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/patología , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
The representative DNA-labeling agent 5-ethynyl-2'-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxymethyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro. PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.
Asunto(s)
ADN/análisis , Desoxiuridina/análogos & derivados , Células 3T3 , Animales , ADN/metabolismo , Desoxiuridina/administración & dosificación , Desoxiuridina/química , Desoxiuridina/metabolismo , Células HeLa , Humanos , Ratones , Fosforilación , Coloración y Etiquetado/métodosRESUMEN
The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies.
Asunto(s)
Conducta Animal , Gangliósido G(M3)/fisiología , Actividad Motora , Animales , Trastorno por Déficit de Atención con Hiperactividad/genética , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Emociones , Femenino , Gangliósido G(M3)/genética , Hipercinesia/genética , Hipercinesia/fisiopatología , Masculino , Aprendizaje por Laberinto , Metilfenidato/administración & dosificación , Ratones , Ratones NoqueadosRESUMEN
We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.
RESUMEN
Primary cardiac sarcoma is rare, and there have been only a few reports on its cytologic findings. Myxofibrosarcoma, a variant of fibrosarcoma of the heart, is an extremely rare entity. We present a case of primary cardiac myxofibrosarcoma in a 63-year-old woman. Pleural fluid cytology and imprint cytology of the resected tumor at operation and autopsy were obtained. Cytologic evaluation with immunocytochemical staining utilizing a cell transfer technique revealed that tumor cells of the resected tumor and autopsy specimen and pleural effusion demonstrated large and pleomorphic cells with irregular, hyperchromatic nuclei and were positive for vimentin. Combination of morphology and immunoprofile of the cells of pleural effusion was compatible with the diagnosis of metastatic myxofibrosarcoma. Diagn. Cytopathol. 2016;44:1112-1116. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Fibrosarcoma/patología , Neoplasias Cardíacas/patología , Mixoma/patología , Derrame Pleural Maligno/patología , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana EdadRESUMEN
OBJECTIVE: We focused on a small New World monkey, the common marmoset (Callithrix jacchus), to establish a nonhuman primate model of the treatment of hematological disorders. In this study, we developed the first monoclonal antibodies (MAbs) against marmoset CD34 and tested the in vitro and in vivo hemopoietic activity of cell populations isolated using one of these MAbs. METHODS AND RESULTS: Marmoset cDNA encoding a human CD34 homologue was cloned from bone marrow (BM)-derived RNA using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The amino acid sequence of the marmoset CD34 had 81% homology with the human sequence. Five mouse MAbs were raised against marmoset CD34 transfectant. One representative MAb, MA24 (IgM), reacted with approximately 0.5 to 1% of BM mononuclear cells (MNCs), where the colony-forming unit granulocyte/macrophage (CFU-GM) was enriched approximately 11- to 75-fold as compared with the whole BM MNCs. Multilineage differentiation of marmoset CD34+ cells in NOD/SCID mice was confirmed by flow cytometry 1 month after xenotransplantation. CONCLUSION: These results demonstrated that MA24 is useful for the analysis and enrichment of hematopoietic progenitor cells in the marmoset model for preclinical experiments.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD34/análisis , Callithrix/sangre , Separación Celular/métodos , Hematopoyesis , Secuencia de Aminoácidos , Animales , Antígenos CD34/genética , Antígenos CD34/inmunología , Clonación Molecular , Macaca fascicularis , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Datos de Secuencia MolecularRESUMEN
Conventional culture of human induced pluripotent (hiPS) cells requires feeder cell preparation that is time consuming and labour intensive. Alternatively, feeder-free culture of hiPS cells requires high cell seeding densities, specialized culture medium, and growth supplements, which raise the cost. Furthermore, recombinant systems require a long time for colony formation. Here, we employed chemically fixed autologous feeder cells for hiPS cell culture without additional time needed for colony formation. Treatment of (2.5% glutaraldehyde or formaldehyde for 10 min) autologous human dermal fibroblasts allowed hiPS cells to adhere and grow as undifferentiated colonies characterized by the expression of pluripotency markers such as alkaline phosphatase, Oct-3/4, and stage-specific embryonic antigen-4. Furthermore, hiPS cells cultured on chemically fixed feeders formed teratomas in vivo, characterized by all three germ layers. The chemically fixed autologous feeders may be used as a substitute for large scale culture of hiPS cells as a convenient in-house and a cost-effective method.
RESUMEN
The biological activity of cell-derived substrates to maintain undifferentiated murine-induced pluripotent stem (iPS) cells was correlated to membrane fluidity as a new parameter of cell culture substrates. Murine embryonic fibroblasts (MEFs) were employed as feeder cells and their membrane fluidity was tuned by chemical fixation using formaldehyde (FA). Membrane fluidity was evaluated by real-time single-molecule observations of green fluorescent protein-labeled epidermal growth factor receptors on chemically fixed MEFs. Biological activity was monitored by colony formation of iPS cells. Treatment with a low concentration of FA sustained the membrane fluidity and biological activity, which were comparable to those of mitomycin C-treated MEFs. The biological activity was further confirmed by sustained expression of alkaline phosphatase, SSEA-1, and other pluripotency markers in iPS cells after 3-5 days of culture on FA-fixed MEFs. Chemical fixation of feeder cells has several advantages such as providing ready-to-use culture substrates without contamination by proliferating feeder cells. Therefore, our results provide an important basis for the development of chemically fixed culture substrates for pluripotent stem cell culture as an alternative to conventional treatment by mitomycin C or x-ray irradiation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Nutrientes/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Fluidez de la Membrana , Animales , Células Nutrientes/citología , Células Madre Pluripotentes Inducidas/citología , RatonesRESUMEN
We have reported that in ganglioside GM3-deficient (GM3(-/-)) mice, spontaneous alternation behavior assessed by a Y-maze task was significantly lower, and total arm entries were significantly higher than in wild-type mice. The objective of the present study was to examine the role of nicotinic acetylcholine receptor (nAChR) signaling in impairment of spontaneous alternation behavior of GM3(-/-) mice. Nicotine treatment (0.3, 1.0 mg/kg, s.c.) dose dependently improved the spontaneous alternation deficit without affecting total arm entries in GM3(-/-) mice. The nicotine-induced (1.0 mg/kg, s.c.) improvement was significantly abolished by the nAChR antagonist mecamylamine (1.0 mg/kg, i.p.). The α4ß2 nAChR antagonist dihydro-ß-erythroidine (2.5, 10.0 mg/kg, i.p.) dose dependently counteracted the nicotine-induced improvement of spontaneous alternation in GM3(-/-) mice, whereas the α7 nAChR antagonist methyllycaconitine (2.5, 10.0 mg/kg, i.p.) did not. In addition, the α4ß2 nAChR agonist RJR-2403 (5.0, 10.0 mg/kg, s.c.) dose dependently and significantly improved the spontaneous alternation deficit, whereas the α7 nAChR agonist PNU120596 (0.3, 1.0, 3.0 mg/kg, i.p.) did not. These findings revealed that nicotine improved spontaneous alternation behavior of GM3(-/-) mice via the activation of α4ß2, but not α7, nAChR. Thus, the ganglioside GM3 might be responsible for α4ß2 nAChR signaling in the spontaneous alternation behavior.
Asunto(s)
Conducta Animal , Gangliósido G(M3)/deficiencia , Receptores Nicotínicos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacologíaRESUMEN
The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain lengths are poorly understood. We generated mice deficient in the Elovl1 gene, which encodes the FA elongase responsible for producing C20 to C28 FAs. Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with ≥C26 FAs were decreased, while those of Cers with ≤C24 FAs were increased. In contrast, the levels of C24 sphingomyelin were reduced, accompanied by an increase in C20 sphingomyelin levels. Two ceramide synthases, CerS2 and CerS3, expressed in an epidermal layer-specific manner, regulate Elovl1 to produce acyl coenzyme As with different chain lengths. Elovl1 is a key determinant of epidermal Cer chain length and is essential for permeability barrier formation.
Asunto(s)
Acetiltransferasas/genética , Epidermis/enzimología , Ácidos Grasos/metabolismo , Acetiltransferasas/deficiencia , Animales , Ceramidas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Epidermis/anomalías , Epidermis/patología , Elongasas de Ácidos Grasos , Femenino , Genes Letales , Células HEK293 , Humanos , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Permeabilidad , Esfingosina N-Aciltransferasa/metabolismoRESUMEN
Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis.
Asunto(s)
Células Nutrientes/citología , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Fosfatasa Alcalina/metabolismo , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Separación Celular , Técnicas de Cocultivo , Matriz Extracelular/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Microscopía Fluorescente/métodos , Proteína Homeótica Nanog , Neuronas/metabolismo , Células Madre/citologíaRESUMEN
Bergmann glial cells are specialized astrocytes in the cerebellum. In the mature cerebellar molecular layer, Bergmann glial processes are closely associated with Purkinje cells, enclosing Purkinje cell dendritic synapses with a glial sheath. There is intensive gap junctional coupling between Bergmann glial processes, but their significance in cerebellar functions is not known. Connexin43 (Cx43), a major component of astrocytic gap junction channels, is abundantly expressed in Bergmann glial cells. To examine the role of Cx43-mediated gap junctions between Bergmann glial cells in cerebellar functions, we generated Cx43 conditional knockout mice with the S100b-Cre transgenic line (Cx43(fl/fl):S100b-Cre), which exhibited a significant loss of Cx43 in the Bergmann glial cells and astrocytes in the cerebellum with a postnatal onset. The Cx43(fl/fl):S100b-Cre mice had normal cerebellar architecture. Although gap junctional coupling between the Bergmann glial cells measured by spreading of microinjected Lucifer yellow was virtually abolished in Cx43(fl/fl):S100b-Cre mice, electrophysiologic analysis revealed that cerebellar long-term depression could be induced and maintained normally in their cerebellar slices. In addition, at the behavioral level, Cx43(fl/fl):S100b-Cre mice had normal motor coordination in the rotarod task and normal conditioned eyelid response. Our findings suggest that Cx43-mediated gap junctional coupling between Bergmann glial cells is not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning.