RESUMEN
The objective of this research were to investigate the effect of a conjugated linoleic acid (CLA)-enriched diet on Isa Brown laying hen health status and to provide a comprehensive analysis of changes in blood parameters, liver morphology and selected hepatic gene expression. Hens were allocated to the control and experimental group (diet enriched with 0.75% CLA) for a total period of 4 m. At the end of the experiment half of the hens from each group were slaughtered for analyses. The remaining hens were transferred to an organic farm for the next 5 m and fed on the diet without CLA supplementation. The CLA-enriched diet resulted in significant changes in blood and serum parameters; specifically, haematocrit (HCT), mean corpuscular volume (MCV) and white blood cells (WBC) count were decreased compared to the control. The total cholesterol (TC) was not significantly affected while the triacylglycerol's (TG) concentration was elevated. The activity of alanine aminotransferase (ALT) was significantly increased in the CLA-supplemented group, while aspartate aminotransferase (AST) showed an increasing tendency. Liver biopsies showed pathological changes classified as non-alcoholic fatty liver disease (NAFLD). Additionally, the expression of hepatic genes involved in fatty acids synthesis (ME1, ACLY, ACC, FASN, SCD1), oxidation (CPT1α, PPARA), detoxification processes (Cytochrome P450, CYP, Flavin-containing monooxygenase, FMO3), oxidative stress (NOX4, XbP1) and inflammation (IL6, TNFα) were elevated. Cessation of CLA supplementation for 5 m of organic farming resulted in normalisation of blood and hepatic parameters to the levels observed in control hens. The results of this study indicate that dietary CLA triggers an integrated stress response in laying hens and activates mechanisms involved in liver detoxification.
Asunto(s)
Pollos/genética , Dieta/veterinaria , Regulación de la Expresión Génica , Ácidos Linoleicos Conjugados/metabolismo , Alimentación Animal/análisis , Animales , Análisis Químico de la Sangre/veterinaria , Pollos/sangre , Pollos/metabolismo , Suplementos Dietéticos/análisis , Femenino , Ácidos Linoleicos Conjugados/administración & dosificación , Hígado/anatomía & histología , Hígado/metabolismo , Distribución AleatoriaRESUMEN
The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasion-promoting role of microglia are unknown. Transforming growth factor-beta (TGF-beta) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-beta activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-beta signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-beta type II receptor (TbetaIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbetaIIR abolished TGF-beta-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbetaIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbetaRII, indicating a crucial role of microglia-derived TGF-beta in tumor-host interactions. Our results demonstrate a successful targeting of TGF-beta-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.
Asunto(s)
Movimiento Celular/fisiología , Silenciador del Gen/fisiología , Glioma/patología , Microglía/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Técnicas de Cocultivo , Colágeno/metabolismo , Combinación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Glioma/metabolismo , Humanos , Laminina/metabolismo , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
Toxoplasmosis is an important parasitic infection of man and animals. It is well-known that the progression and severity of disease depend on the immunological status of the host, but recent studies suggest that the genetics of the parasite can also play a role. Diagnosis based on clinical appearance and serology is not always easy. However, molecular methods do not depend on an immune response, and allow direct detection of the parasite in biological samples. Thus they can be used to establish a diagnosis when serological tests are not definitive. Multicopy sequences specific for Toxoplasma gondii, e.g., the B1 gene or the 529-bp sequence, are especially useful in molecular tests. Real-time PCR is very sensitive and is a promising technique that is capable of providing a quantitative result. Molecular methods are also used for genotypic characterisation of T. gondii isolates. Analysis of polymorphic sequences determines the precise strain. The choice of sequence is critical when undertaking studies on the correlation between clinical signs and symptoms of disease and the T. gondii genotype. Further studies involving direct genotyping of T. gondii from clinical samples are needed.
Asunto(s)
Toxoplasmosis/diagnóstico , Animales , Genotipo , Humanos , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Toxoplasma/patogenicidad , VirulenciaRESUMEN
The escape of malignant cells from primary tumour and their active migration to the surrounding tissues are among the most important steps in the metastatic process. During migration, tumour cells interact with neighbouring neoplastic and normal cells and such interactions may affect their motile activity. We investigated the effect of extracellular calcium ions on migration of mouse melanoma B16 cells stimulated by homotypic cell-to-cell contacts. It was found that the decreasing of extracellular Ca2+ influx into B16 cells by lowering Ca2+ concentration in culture medium, or by the application of 0.5 mM La3+ (non-selective inorganic Ca2+ channels blocker), reduced the contact-mediated acceleration of migration of melanoma cells but only slightly affected the basal motile activity of non-stimulated single, separated cells moving without contacts with neighbouring ones in sparse culture. Since it was suggested that contact-mediated acceleration of migration of melanoma B16 cells may be controlled by mechanosensitive and/or voltage-gated ion channels, the presented data support the concept that these channels may affect cell migration by regulation of extracellular Ca2+ influx into stimulated cell.
Asunto(s)
Canales de Calcio/fisiología , Calcio/farmacología , Movimiento Celular , Melanoma/patología , Metástasis de la Neoplasia/fisiopatología , Calcio/química , Comunicación Celular , Humanos , Células Tumorales CultivadasRESUMEN
We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain, termed SOS gp140 (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variable-loop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.
Asunto(s)
Disulfuros/metabolismo , Productos del Gen env/metabolismo , Variación Genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Antígenos CD4/metabolismo , Epítopos de Linfocito B , Productos del Gen env/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis , Procesamiento Proteico-Postraduccional , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.