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1.
J Immunol ; 192(3): 940-7, 2014 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-24363428

RESUMEN

We document human T lymphotropic virus type 1 (HTLV-1) bZIP factor (HBZ)-specific CD4 T cell responses in an adult T cell leukemia/lymphoma (ATL) patient after allogeneic hematopoietic stem cell transplantation (HCT) and identified a novel HLA-DRB1*15:01-restricted HBZ-derived naturally presented minimum epitope sequence, RRRAEKKAADVA (HBZ114-125). This peptide was also presented on HLA-DRB1*15:02, recognized by CD4 T cells. Notably, HBZ-specific CD4 T cell responses were only observed in ATL patients after allogeneic HCT (4 of 9 patients) and not in nontransplanted ATL patients (0 of 10 patients) or in asymptomatic HTLV-1 carriers (0 of 10 carriers). In addition, in one acute-type patient, HBZ-specific CD4 T cell responses were absent in complete remission before HCT, but they became detectable after allogeneic HCT. We surmise that HTLV-1 transmission from mothers to infants through breast milk in early life induces tolerance to HBZ and results in insufficient HBZ-specific T cell responses in HTLV-1 asymptomatic carriers or ATL patients. In contrast, after allogeneic HCT, the reconstituted immune system from donor-derived cells can recognize virus protein HBZ as foreign, and HBZ-specific immune responses are provoked that contribute to the graft-versus-HTLV-1 effect.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos HTLV-I/inmunología , Trasplante de Células Madre Hematopoyéticas , Virus Linfotrópico T Tipo 1 Humano/inmunología , Leucemia-Linfoma de Células T del Adulto/inmunología , Proteínas Virales/inmunología , Aloinjertos , Secuencia de Aminoácidos , Enfermedades Asintomáticas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Linfocitos T CD8-positivos/inmunología , Portador Sano/inmunología , Línea Celular , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Femenino , Genotipo , Efecto Injerto vs Leucemia/inmunología , Antígenos HLA-D/genética , Antígenos HLA-D/inmunología , Humanos , Interferón gamma/biosíntesis , Leucemia-Linfoma de Células T del Adulto/cirugía , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Proteínas de los Retroviridae , Factor de Necrosis Tumoral alfa/biosíntesis , Proteínas Virales/química
2.
J Immunol ; 191(1): 135-44, 2013 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23733874

RESUMEN

We expanded human T-lymphotropic virus type 1 Tax-specific CTL in vitro from PBMC of three individual adult T cell leukemia/lymphoma (ATL) patients and assessed their therapeutic potential in an in vivo model using NOG mice bearing primary ATL cells from the respective three patients (ATL/NOG). In these mice established with cells from a chronic-type patient, treatment by i.p. injection of autologous Tax-CTL resulted in greater infiltration of CD8-positive T cells into each ATL lesion. This was associated with a significant decrease of ATL cell infiltration into blood, spleen, and liver. Tax-CTL treatment also significantly decreased human soluble IL-2R concentrations in the sera. In another group of ATL/NOG mice, Tax-CTL treatment led to a significant prolongation of survival time. These findings show that Tax-CTL can infiltrate the tumor site, recognize, and kill autologous ATL cells in mice in vivo. In ATL/NOG mice with cells from an acute-type patient, whose postchemotherapeutic remission continued for >18 mo, antitumor efficacy of adoptive Tax-CTL therapy was also observed. However, in ATL/NOG mice from a different acute-type patient, whose ATL relapsed after 6 mo of remission, no efficacy was observed. Thus, although the therapeutic effects were different for different ATL patients, to the best of our knowledge, this is the first report that adoptive therapy with Ag-specific CTL expanded from a cancer patient confers antitumor effects, leading to significant survival benefit for autologous primary cancer cell-bearing mice in vivo. The present study contributes to research on adoptive CTL therapy, which should be applicable to several types of cancer.


Asunto(s)
Productos del Gen tax/inmunología , Productos del Gen tax/uso terapéutico , Virus Linfotrópico T Tipo 1 Humano/inmunología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Leucemia-Linfoma de Células T del Adulto/inmunología , Leucemia-Linfoma de Células T del Adulto/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular Transformada , Enfermedad Crónica , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Productos del Gen tax/administración & dosificación , Humanos , Inmunoterapia Adoptiva/métodos , Inyecciones Intraperitoneales , Subunidad gamma Común de Receptores de Interleucina/genética , Células K562 , Leucemia-Linfoma de Células T del Adulto/virología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Cultivo Primario de Células , Linfocitos T Citotóxicos/trasplante , Linfocitos T Citotóxicos/virología
3.
Eur J Haematol ; 92(3): 219-28, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24188416

RESUMEN

OBJECTIVE: The objective of this study was to evaluate the therapeutic potential of bevacizumab with or without systemic chemotherapy for adult T-cell leukemia/lymphoma (ATL) and clarify the significance of angiogenesis for ATL pathogenesis. METHODS: NOD/Shi-scid, IL-2Rγ(null) (NOG) mice were used as recipients of tumor cells from a patient with ATL, which engraft and proliferate in a microenvironment-dependent manner. The ATL cells could be serially transplanted in NOG mice, but could not be maintained in in vitro cultures. RESULTS: Injection of bevacizumab alone significantly increased necrosis and decreased vascularization in the tumor tissue. Levels of human soluble interleukin two receptor in the serum (reflecting the ATL tumor burden) of bevacizumab-treated mice were significantly lower than in untreated mice. Although bevacizumab monotherapy showed these clear anti-angiogenesis effects, it did not prolong survival. In contrast, injection of bevacizumab together with cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP) led to a significant prolongation of survival of the ATL mice relative to CHOP alone. CONCLUSIONS: This is the first report to evaluate the efficacy of bevacizumab for ATL in a tumor microenvironment-dependent model. Bevacizumab therapy combined with chemotherapy could be a valuable treatment strategy for that subgroup of ATL probably depending to a large extent on angiogenesis via vascular endothelial growth factor.


Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Antineoplásicos/química , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bevacizumab , Línea Celular Tumoral , Proliferación Celular , Ciclofosfamida/uso terapéutico , Doxorrubicina/uso terapéutico , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Neovascularización Patológica , Prednisolona/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Vincristina/uso terapéutico
4.
J Biol Chem ; 285(7): 4870-82, 2010 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19940141

RESUMEN

ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs)-like (ADAMTSL) proteins, a subgroup of the ADAMTS superfamily, share several domains with ADAMTS proteinases, including thrombospondin type I repeats, a cysteine-rich domain, and an ADAMTS spacer, but lack a catalytic domain. We identified two new members of ADAMTSL proteins, ADAMTSL-6alpha and -6beta, that differ in their N-terminal amino acid sequences but have common C-terminal regions. When transfected into MG63 osteosarcoma cells, both isoforms were secreted and deposited into pericellular matrices, although ADAMTSL-6alpha, in contrast to -6beta, was barely detectable in the conditioned medium. Immunolabeling at the light and electron microscopic levels showed their close association with fibrillin-1-rich microfibrils in elastic connective tissues. Surface plasmon resonance analyses demonstrated that ADAMTSL-6beta binds to the N-terminal half of fibrillin-1 with a dissociation constant of approximately 80 nm. When MG63 cells were transfected or exogenously supplemented with ADAMTSL-6, fibrillin-1 matrix assembly was promoted in the early but not the late stage of the assembly process. Furthermore, ADAMTSL-6 transgenic mice exhibited excessive fibrillin-1 fibril formation in tissues where ADAMTSL-6 was overexpressed. All together, these results indicated that ADAMTSL-6 is a novel microfibril-associated protein that binds directly to fibrillin-1 and promotes fibrillin-1 matrix assembly.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Southern Blotting , Cartílago/metabolismo , Cartílago/ultraestructura , Línea Celular , Embrión de Mamíferos/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Fibrilina-1 , Fibrilinas , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/fisiología , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas de Microfilamentos/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie
5.
Proc Natl Acad Sci U S A ; 105(35): 12849-54, 2008 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-18757743

RESUMEN

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.


Asunto(s)
Proteínas de la Matriz Extracelular/análisis , Perfilación de la Expresión Génica , Animales , Membrana Basal/citología , Membrana Basal/metabolismo , Línea Celular , Biología Computacional , Epitelio/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Transporte de Proteínas , Diente/citología , Diente/embriología
6.
Endocrinology ; 160(7): 1701-1718, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31135891

RESUMEN

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.


Asunto(s)
Linaje de la Célula/fisiología , Células Madre Embrionarias/metabolismo , Células Ependimogliales/metabolismo , Hipotálamo/metabolismo , Células-Madre Neurales/metabolismo , Animales , Células Cultivadas , Células Madre Embrionarias/citología , Células Ependimogliales/citología , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hipotálamo/citología , Proteínas con Homeodominio LIM/metabolismo , Ratones , Células-Madre Neurales/citología , Factores de Transcripción/metabolismo
7.
Stem Cell Res ; 40: 101572, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31539858

RESUMEN

High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies.


Asunto(s)
Diferenciación Celular , Línea Celular/citología , Hipotálamo/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/citología , Animales , Línea Celular/metabolismo , Células Cultivadas , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Hipotálamo/metabolismo , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Vasopresinas/metabolismo
8.
Sci Rep ; 8(1): 3615, 2018 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-29483626

RESUMEN

Arginine-vasopressin (AVP) neurons exist in the hypothalamus, a major region of the diencephalon, and play an essential role in water balance. Here, we established the differentiation method for AVP-secreting neurons from human embryonic stem cells (hESCs) by recapitulating in vitro the in vivo embryonic developmental processes of AVP neurons. At first, the differentiation efficiency was improved. That was achieved through the optimization of the culture condition for obtaining dorsal hypothalamic progenitors. Secondly, the induced AVP neurons were identified by immunohistochemistry and these neurons secreted AVP after potassium chloride stimulation. Additionally, other hypothalamic neuropeptides were also detected, such as oxytocin, corticotropin-releasing hormone, thyrotropin-releasing hormone, pro-opiomelanocortin, agouti-related peptide, orexin, and melanin-concentrating hormone. This is the first report describing the generation of secretory AVP neurons derived from hESCs. This method will be applicable to research using disease models and, potentially, for regenerative medicine of the hypothalamus.


Asunto(s)
Arginina Vasopresina/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteína Relacionada con Agouti/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Humanos , Hormonas Hipotalámicas/metabolismo , Hipotálamo/citología , Hipotálamo/metabolismo , Inmunohistoquímica , Melaninas/metabolismo , Neurofisinas/metabolismo , Orexinas/metabolismo , Oxitocina/metabolismo , Hormonas Hipofisarias/metabolismo , Precursores de Proteínas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Vasopresinas/metabolismo
9.
Neurosci Lett ; 396(3): 241-6, 2006 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-16368189

RESUMEN

Accumulated evidence suggests that actin and microtubule regulating proteins contribute to neuronal structural dynamics, which subsequently affect neuronal plasticity. SCG10 is a neuronal-specific stathmin protein with microtubule destabilizing activity that is affected by multiple phosphorylation, at least in vitro. SCG10 has four major phosphorylation sites: Ser50 and Ser97 targeted by protein kinase A (PKA), and Ser62 and Ser73 targeted by mitogen-activated protein kinase (MAPK). To explore the potential roles of site-specific phosphorylation in physiological models, we developed phosphorylation site-specific antibodies and examined the SCG10 status in primary cultured hippocampal neurons and tissues. Although SCG10 is concentrated in growth cones and the Golgi apparatus in primary cultured neurons, the phosphorylated form was also detected in both regions, suggesting that MT dynamics within the growth cone may be regulated by protein phosphorylation. In the adult hippocampus, an intense stimulus such as kainate treatment induced a rapid phosphorylation of Ser73 within 15 min that was sustained for at least 60 min. This response was mediated through the N-methyl D-aspartic acid (NMDA) receptor and was ablated by the antagonist MK-801. The MAPK enzyme Erk2 was simultaneously activated along a similar time course to SCG10, suggesting that Erk2 may directly phosphorylate Ser73. These results demonstrate that changes in the phosphorylation status of SCG10 in vivo, dependent upon neural activity and/or plasticity, could affect the microtubule dynamics in neuronal dendrites.


Asunto(s)
Hipocampo/citología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras , Células Cultivadas , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroforesis en Gel de Poliacrilamida/métodos , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Técnica del Anticuerpo Fluorescente/métodos , Ácido Kaínico/farmacología , Masculino , Proteínas de la Membrana , Proteínas de Microtúbulos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Factores de Crecimiento Nervioso/química , Neuronas/efectos de los fármacos , Pentilenotetrazol/farmacología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Factores de Tiempo
10.
Biochem J ; 389(Pt 3): 675-84, 2005 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-15836428

RESUMEN

We screened more than 60000 RIKEN mouse cDNAs for novel ECM (extracellular matrix) proteins by extensive computational screening followed by recombinant expression and immunohistochemical characterization. We identified two novel olfactomedin-family proteins characterized by the presence of tandem CXCXCX9C motifs in the N-terminal region, a coiled-coil domain and an olfactomedin domain in the C-terminal region. These proteins, named photomedin-1 and photomedin-2, were secreted as disulphide-bonded dimers (photomedin-1) or oligomers/multimers (photomedin-2) with O-linked carbohydrate chains, although photomedin-1 was proteolytically processed in the middle of the molecule after secretion. In the retina, photomedin-1 was selectively expressed in the outer segment of photoreceptor cells and photomedin-2 was expressed in all retinal neurons. Among a panel of ECM components, including glycosaminoglycans, photomedins preferentially bound to chondroitin sulphate-E and heparin. These results, together, indicate that photomedins are novel olfactomedin-domain-containing extracellular proteins capable of binding to proteoglycans containing these glycosaminoglycan chains.


Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/química , Proteínas de la Matriz Extracelular/química , Proteínas del Ojo/química , Glicoproteínas/química , Retina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas del Ojo/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido
11.
Stem Cell Res ; 15(2): 290-8, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26209816

RESUMEN

During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES) cells. Generally, any lot differences in fetal bovine serum (FBS) and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9-36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP) 4 and fibroblast growth factor (FGF) treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78-90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells.


Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Animales , Ectodermo/citología , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factor de Transcripción Pit-1/genética , Factor de Transcripción Pit-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2
12.
J Biochem ; 147(4): 565-79, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19996152

RESUMEN

The C1q family is characterized by the C-terminally conserved globular C1q (gC1q) domain. Although more than 30 C1q family proteins have been identified in mammals, many of them remain ill-defined with respect to their molecular and biological properties. Here, we report on a novel C1q family protein specifically expressed in the central nervous system (CNS), which we designated neural C1q-like protein (nCLP) 2. nCLP2 was secreted as disulphide-bonded multimers comprising trimeric units. The multimers were stabilized by interchain disulphide bonds involving the cysteine residues in the N-terminal variable region and the C-terminal gC1q domain. The expression of nCLP2 was restricted to several brain regions and retina, including regions associated with memory formation (i.e. hippocampus, entorhinal cortex, anterodorsal thalamic nucleus). Immunoelectron microscopy revealed that nCLP2 was localized in the mossy fibre axons of hippocampal granule cells and their synaptic boutons and clefts, implying that nCLP2 was anterogradely transported in mossy fibres and secreted from the presynaptic termini. These results suggest that nCLP2 plays roles in synaptic function and maintenance in the CNS.


Asunto(s)
Sistema Nervioso Central/metabolismo , Complemento C1q/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/citología , Complemento C1q/química , Complemento C1q/genética , Cisteína/química , Perfilación de la Expresión Génica , Biblioteca de Genes , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Fibras Musgosas del Hipocampo/metabolismo , Fibras Musgosas del Hipocampo/ultraestructura , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Retina/citología , Retina/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido
13.
Eur J Neurosci ; 23(3): 637-48, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16487145

RESUMEN

Neuronal growth-associated proteins, including superior cervical ganglia clone 10 (SCG10) family molecules, play roles in neurite outgrowth and network formation as well as structural and functional plasticity. The present ontogenetic study revealed that the expression of neuronal growth-associated proteins in the visual cortex (VC) exhibited a sharp peak in the early postnatal period when growing lateral geniculate nucleus (LGN) axon terminals segregate into the ocular dominance columns depending on retinal activity. We then hypothesized that SCG10 family molecules, known for catastrophic factors of microtubules, play important roles in the formation of ocular dominance columns. To test this hypothesis, we studied whether: (i) monocular blockade of retinal activity changed the SCG10 expression in LGN and VC and (ii) brain-derived neurotrophic factor (BDNF) cortical infusion modified the expression of SCG10 family molecules and the number of excitatory/inhibitory cortical synapses. Using northern blot and in situ hybridization, we revealed that: (i) silencing retinal activity with tetrodotoxin eye injections dynamically reduced the expression of SCG10 mRNA and (ii) it was enhanced by BDNF in VC and LGN of kittens but not adult cats. These findings suggest that cortical infusion of BDNF and retinal activity up-regulate the expression of SCG10 in the LGN and VC and that up-regulated SCG10 in turn initiates marked reorganization of the microtuble network, eventually resulting in increase in synapse formation in the VC.


Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Cuerpos Geniculados/efectos de los fármacos , Factores de Crecimiento Nervioso/metabolismo , Corteza Visual/efectos de los fármacos , Factores de Edad , Animales , Animales Recién Nacidos , Northern Blotting/métodos , Gatos , Femenino , Cuerpos Geniculados/crecimiento & desarrollo , Cuerpos Geniculados/ultraestructura , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Masculino , Microscopía Electrónica de Transmisión/métodos , Modelos Biológicos , Tetrodotoxina/farmacología , Corteza Visual/crecimiento & desarrollo , Corteza Visual/ultraestructura
14.
Mod Pathol ; 19(7): 974-85, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16648867

RESUMEN

D2-40 antibody is raised against an oncofetal antigen, the M2A antigen. It has been used as a marker for lymphatic endothelium as well as mesothelioma and cerebellar hemangioblastoma. We demonstrate here that positive D2-40 immunoreactivity was found in the developing cerebrum, particularly in the germinal matrix layer, immature ependyma, choroid plexus and meninges. In the developing cerebellum, positive D2-40 immunoreactivity was found in the external granular layer particularly of the outer portion and the Purkinje cell layer as well as meninges. Some brain tumors such as anaplastic ependymoma, some medulloblastomas, glioblastoma, pineal germinoma, craniopharyngioma, choroid plexus papilloma, choroid plexus carcinoma, and meningioma showed positive immunoreactivity with D2-40. Therefore, D2-40 antibody is considered a useful marker for research on developing brain and diagnosis of brain tumors, differentiation between choroid plexus carcinoma and metastatic carcinoma. In addition, on cultured human neural cells, D2-40 immunoreactivity was found in nestin-positive neural stem/progenitor cells and neuronal lineage cells. As D2-40 antibody recognizes cell surface antigen M2A, it might be a candidate cell surface marker for isolation of human neural stem cells/neuronal lineage cells in the fluorescence-activated cell sorting technique.


Asunto(s)
Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Neoplasias Encefálicas/inmunología , Cerebelo/inmunología , Telencéfalo/inmunología , Adulto , Anticuerpos Monoclonales de Origen Murino , Antígenos de Neoplasias/inmunología , Neoplasias Encefálicas/patología , Diferenciación Celular , Células Cultivadas , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Preescolar , Feto/inmunología , Edad Gestacional , Humanos , Inmunohistoquímica , Lactante , Persona de Mediana Edad , Neuronas/citología , Neuronas/inmunología , Prosencéfalo/citología , Células Madre/citología , Células Madre/inmunología , Telencéfalo/embriología , Telencéfalo/crecimiento & desarrollo
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