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1.
Int J Mol Sci ; 19(4)2018 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-29601474

RESUMEN

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) are markers of fast-cycling and quiescent intestinal stem cells, respectively. To determine the functions of these proteins in large animals, we investigated their effects on the proliferation of intestinal epithelial cells from pigs. Our results indicated that LGR5 and BMI1 are highly conserved proteins and that the pig proteins have greater homology with the human proteins than do mouse proteins. Overexpression of either LGR5 or BMI1 promoted cell proliferation and WNT/ß-catenin signaling in pig intestinal epithelial cells (IPEC-J2). Moreover, the activation of WNT/ß-catenin signaling by recombinant human WNT3A protein increased cell proliferation and LGR5 and BMI1 protein levels. Conversely, inhibition of WNT/ß-catenin signaling using XAV939 reduced cell proliferation and LGR5 and BMI1 protein levels. This is the first report that LGR5 and BMI1 can increase proliferation of pig intestinal epithelial cells by activating WNT/ß-catenin signaling.


Asunto(s)
Proliferación Celular/fisiología , Complejo Represivo Polycomb 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Intestinos/citología , Complejo Represivo Polycomb 1/genética , Receptores Acoplados a Proteínas G/genética , Porcinos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo
2.
Oncotarget ; 8(26): 43192-43200, 2017 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-28574820

RESUMEN

We isolated and cultured satellite cells (SCs) from the longissimus dorsi muscles of 1-day-old male Landrace and Lantang piglets to compare the SC differentiation capacity in the two breeds. Lantang piglets yielded more (P < 0.05) SCs per gram of muscle than Landrace piglets (5.2 ± 0.9×104 vs. 2.4 ± 0.2×104). Transcription of the differentiation markers myogenin and myosin heavy chain I (MyHC I) in the longissimus dorsi muscle was higher in Lantang than Landrace piglets (P < 0.05). Protein levels of myogenin (P < 0.05), MyHC I (P < 0.05), and myogenic regulatory factor 4 (P = 0.07) were higher in Lantang than Landrace piglet SCs after 72 h of differentiation. Creatine kinase activity was higher in Lantang than Landrace piglet SCs after 24, 48, and 72 h of differentiation (P < 0.05), and there was a greater fusion index in Landrace piglet SCs after 72 h of differentiation. In addition, phosphorylation of Akt, mTOR, S6K1, S6, and 4EBP1 was lower in Lantang than Landrace piglet SCs (P < 0.05). Thus differentiation was more extensive in Lantang than Landrace piglet SCs, but expression of the mTOR signaling pathway was lower in Lantang piglet SCs, suggesting mTOR signaling may inhibit myogenic differentiation. These findings reveal that mTOR signaling is a factor in myogenesis and imply that mTOR could potentially serve as an activator of myoblast differentiation and muscle regeneration.


Asunto(s)
Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Diferenciación Celular/fisiología , Masculino , Transducción de Señal , Porcinos
3.
Virology ; 476: 72-84, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25528440

RESUMEN

Marek's disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-ß binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-ß1, with suppression of TGF-ß signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.


Asunto(s)
Proteínas de Unión a TGF-beta Latente/metabolismo , Enfermedad de Marek/metabolismo , MicroARNs/metabolismo , Proteína Oncogénica p55(v-myc)/metabolismo , ARN Viral/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Secuencia de Bases , Pollos , Regulación hacia Abajo , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Proteínas de Unión a TGF-beta Latente/genética , Enfermedad de Marek/genética , Enfermedad de Marek/virología , MicroARNs/genética , Datos de Secuencia Molecular , Proteína Oncogénica p55(v-myc)/genética , ARN Viral/genética
4.
Indian J Virol ; 23(3): 344-8, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24293822

RESUMEN

Japanese encephalitis virus (JEV) causes severe viral encephalitis in humans and some other mammalian animals. Highly efficient culture of the virus is critical for antigen preparation, vaccine production and other basic researches. We have investigated the influence of a number of variables such as the strain virulence, the state of the host cells, medium composition, infection method and others on the proliferation of distinct JEV strains in BHK-21 cells. The results showed that two distinct strategies are needed for the propagation of virulent or attenuated JEV strains. The most critical variables were the method of infection, and especially the density of the host cell. Our studies indicate the general approaches to the in vitro culture of other JEV strains using BHK-21 cell line.

5.
Arch Virol ; 154(3): 513-7, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19225716

RESUMEN

In this paper, the infectivity and propagation of two attenuated infectious bursal disease virus (IBDV) strains in DT40 cells were investigated. The results showed that both of the tested strains, TAD and HN(3), directly infect and proliferate in DT40 cells, requiring no adaptive passages. Unexpectedly, IBDV can be rapidly propagated and continuously harvested at high titers for a long time, accompanied by the rapid growth of host cells and showing no increase in pathogenicity. Our results provide further support to suggest that DT40 cells can be used as an ideal model for studying IBDV pathogenesis. Additionally, the DT40 cell line could also serve as a potential system for commercial IBDV vaccine preparation.


Asunto(s)
Linfocitos B/virología , Virus de la Enfermedad Infecciosa de la Bolsa/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Datos de Secuencia Molecular , Alineación de Secuencia , Proteínas Estructurales Virales/genética , Cultivo de Virus/métodos
6.
Vet Parasitol ; 137(3-4): 286-93, 2006 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-16487659

RESUMEN

In the present study, an immunochromatographic strip was developed for the serological detection of trichinellosis in swine. In the strip, the excretory-secretory (ES) antigens of Trichinella labelled with colloidal gold was used as the detector, and the staphylococcal protein A (SPA) and goat anti-ES antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. The evaluation of the strip was performed by comparing 60 clinical positive blood samples detected by the artificial digestion method with 46 serum samples from pigs infected with parasites other than Trichinella and 30 serum samples of parasite-free healthy pigs. The strip was shown to be of high specificity and sensitivity that were closely correlated with those of ELISA. Furthermore, the dipstick assay based on the strip is rapid (10 min) and easy to perform with no requirement of special skill, reagent or equipment. This suggests the immunochromatographic strip is an acceptable alternative to be used in clinical laboratories lacking specialized equipment as well as for field diagnosis.


Asunto(s)
Antígenos Helmínticos/inmunología , Tiras Reactivas , Enfermedades de los Porcinos/diagnóstico , Trichinella/inmunología , Triquinelosis/veterinaria , Animales , Anticuerpos Antihelmínticos/sangre , Reacciones Antígeno-Anticuerpo , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Porcinos , Triquinelosis/diagnóstico , Triquinelosis/inmunología
7.
Protein Expr Purif ; 45(1): 168-74, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16125970

RESUMEN

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.


Asunto(s)
Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Pliegue de Proteína , Receptores de Citocinas/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Especificidad de Anticuerpos , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Cromatografía de Afinidad/métodos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Receptores de Citocinas/química , Receptores de Citocinas/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Células Tumorales Cultivadas
8.
Viral Immunol ; 18(3): 549-57, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16212534

RESUMEN

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.


Asunto(s)
Proteínas Estructurales Virales/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Pollos , Epítopos/genética , Ratones , Pruebas de Neutralización , Oligopéptidos/genética , Oligopéptidos/inmunología , Biblioteca de Péptidos , Mapeo Peptídico , Proteínas Estructurales Virales/genética
9.
Avian Dis ; 49(2): 177-81, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16094819

RESUMEN

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.


Asunto(s)
Anticuerpos Monoclonales , Antígenos Virales/aislamiento & purificación , Infecciones por Birnaviridae/veterinaria , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Enfermedades de las Aves de Corral/diagnóstico , Tiras Reactivas , Animales , Anticuerpos Monoclonales/inmunología , Infecciones por Birnaviridae/diagnóstico , Línea Celular , Técnicas y Procedimientos Diagnósticos/veterinaria , Ensayo de Inmunoadsorción Enzimática , Ratones , Conejos
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