Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
J Assist Reprod Genet ; 31(3): 295-306, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24408183

RESUMEN

PURPOSE: We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. METHODS: Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. RESULTS: No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0-3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21% vs 48%; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. CONCLUSIONS: This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.


Asunto(s)
Células del Cúmulo/citología , Desarrollo Embrionario/genética , Oocitos/citología , Folículo Ovárico/citología , Animales , Blastocisto/citología , Proteína Morfogenética Ósea 15/genética , Técnicas de Cocultivo , Células del Cúmulo/metabolismo , Femenino , Factor 9 de Diferenciación de Crecimiento/genética , Humanos , Ratones , Oocitos/metabolismo
2.
Biochem Biophys Res Commun ; 275(1): 84-90, 2000 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-10944446

RESUMEN

Viral vectors displaying specific ligand binding moities such as scFv fragments or intact antibodies hold promise for the development of targeted gene therapy vectors. In this report we describe baculoviral vectors displaying either functional scFv fragments or the synthetic Z/ZZ IgG binding domain derived from protein A. Display on the baculovirus surface was achieved via fusion of the scFv fragment or Z/ZZ domain to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus, AcNPV. As examples of scFv fragments we have used a murine scFv specific for the hapten 2-phenyloxazolone and a human scFv specific for carcinoembryonic antigen. In principle, the Z/ZZ IgG binding domain displaying baculoviruses could be targeted to specific cell types via the binding of an appropriate antibody. We envisage applications for scFv and Z/ZZ domain displaying baculoviral vectors in the gene therapy field.


Asunto(s)
Baculoviridae/genética , Sitios de Unión de Anticuerpos/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Biblioteca de Péptidos , Animales , Especificidad de Anticuerpos , Baculoviridae/metabolismo , Sitios de Unión de Anticuerpos/genética , Western Blotting , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/inmunología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Haptenos/genética , Haptenos/inmunología , Humanos , Fragmentos de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Ratones , Oxazolona/análogos & derivados , Oxazolona/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/virología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo
3.
Biochem Biophys Res Commun ; 151(1): 207-14, 1988 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-2450535

RESUMEN

The insulin-like growth factor binding protein (BP) secreted by bovine kidney (MDBK) cells has been purified by affinity chromatography on a rat IGF-2 Sepharose column. Purified BP migrated as a single band of Mr 40,000 upon SDS polyacrylamide gel electrophoresis. An N-terminal sequence of 53 residues was obtained which was very similar up to residue 21 to the corresponding rat BRL-3A BP sequence. In competitive binding experiments with bovine IGF-1 and IGF-2, and recombinant human IGF-1, BP had a similar affinity for these ligand when IGF-1 tracer was used, but a higher affinity for IGF-2 with IGF-2 as radioligand. The N-terminal destripeptide truncated form of bovine IGF-1, which has enhanced biological activity, was found to have a markedly reduced affinity for BP compared to intact IGF-1. The increased bioactivity of destripeptide IGF-1 can be explained by this reduced affinity for BP.


Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos/metabolismo , Somatomedinas/metabolismo , Animales , Unión Competitiva , Proteínas Portadoras/metabolismo , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor II del Crecimiento Similar a la Insulina/metabolismo , Mapeo Peptídico
4.
J Cell Biochem ; 61(3): 325-37, 1996 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-8761938

RESUMEN

The effects of 1 alpha, 25(OH)2 vitamin D3 on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR-ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal-c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10-20 mg/l of culture. Both forms of the recombinant receptor bound 1 alpha, 25(OH)2 vitamin D3 with high affinity; estimated Kd values from Scatchard analysis for the purified full-length receptor and the ligand binding domain were 0.16 +/- 0.07 nM and 0.04 +/- 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and delta 22-1, 25S, 26 (OH)3 vitamin D3, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1 alpha, 25(OH)2 vitamin D3. In addition, the full-length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXR alpha. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis.


Asunto(s)
Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Unión Competitiva , Cromatografía de Afinidad , Regulación de la Expresión Génica , Humanos , Ligandos , Plásmidos , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Recombinación Genética
5.
Biochem Biophys Res Commun ; 284(3): 777-84, 2001 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-11396970

RESUMEN

Viral vectors displaying specific ligand binding moieties have raised an increasing interest in the area of targeted gene therapy. In this report, we describe baculovirus vectors displaying either a functional single chain antibody fragment (scFv) specific for the carcinoembryonic antigen (CEA) or the synthetic IgG binding domains (ZZ) derived from protein A of Staphylococcus aureus. In addition, the vectors were engineered to incorporate a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the transcriptional regulation of the cytomegalovirus (CMV) IE promoter. Display of the targeting moieties on the viral surface was achieved through fusion to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus (AcNPV). Specific binding of the gp64 fusion viruses to mammalian target cells was demonstrated by using monoclonal anti-gp64 antibodies followed by fluorescence and/or confocal microscopy. The anti-CEA scFv displaying baculovirus was shown to bind specifically to CEA expressing cells (PC-3). Similarly, the virus displaying the ZZ domains of protein A was targeted to BHK cells via binding of an appropriate IgG antibody. In all cases, the reporter gene was expressed in the transduced mammalian cells as shown by fluorescence microscopy and flow cytometric analyses.


Asunto(s)
Baculoviridae/genética , Proteínas Virales de Fusión/genética , Animales , Anticuerpos Monoclonales/inmunología , Antígeno Carcinoembrionario/inmunología , Línea Celular , Cricetinae , Genes Reporteros , Terapia Genética , Vectores Genéticos , Proteínas Fluorescentes Verdes , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/genética , Proteína Estafilocócica A/inmunología , Transducción Genética , Transfección , Células Tumorales Cultivadas , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/metabolismo
6.
Immunity ; 2(2): 149-54, 1995 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7895171

RESUMEN

The superantigen encoded by the mouse mammary tumor virus (MMTV) is a potent stimulator of T cells when bound to MHC class II molecules. Recent data from this laboratory have shown that the Mtv7 superantigen, Mls-1, elicits a strong T cell response when presented by HLA-DR. To expand these observations further, we have produced the 28 kDa extracellular domain and the 18 kDa carboxy-terminal subfragment of the Mls-1 protein in E. coli and studied their interaction with human MHC class II molecules in vitro. In this report, we demonstrate direct binding of these recombinant forms of the Mls-1 protein to soluble HLA-DR1 and HLA-DR4, but not to HLA-A2. Our data imply a unique class II interaction site of retroviral superantigens that is not shared with bacterial superantigens.


Asunto(s)
Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos Estimulantes de Linfocito Menor/metabolismo , Animales , Antígenos Virales/metabolismo , Secuencia de Bases , Sitios de Unión , Escherichia coli/genética , Humanos , Ratones , Antígenos Estimulantes de Linfocito Menor/genética , Antígenos Estimulantes de Linfocito Menor/inmunología , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/metabolismo
7.
Biol Reprod ; 71(3): 732-9, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15128595

RESUMEN

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFbeta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0-160 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mitógenos/metabolismo , Oocitos/citología , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 15 , Femenino , Factor 9 de Diferenciación de Crecimiento , Péptidos y Proteínas de Señalización Intercelular/química , Ratones , Mitógenos/química , Mitógenos/inmunología , Datos de Secuencia Molecular , Oocitos/metabolismo , Estructura Terciaria de Proteína , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA