RESUMEN
Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.
Asunto(s)
Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Factor B de Crecimiento Endotelial Vascular/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Retina/metabolismoRESUMEN
This study was purposed to investigate the expression of heat shock protein 90 (HSP90) in peripheral blood plasma of patients with multipl myeloma (MM), and to explore its possible role in the pathogenesis of MM, and its relationship with treatment, prognosis and the outcome of patients. The peripheral blood samples from 58 patients with MM and 20 healthy volunteers were collected. The plasma concentration of HSP90 in patients and healthy volunteers was measured by ELISA. The results showed that the concentration of HSP90 in peripheral blood of patients with MM was significantly higher than that in the healthy volunteers [(32.398 ± 3.674) vs (25.762 ± 2.916) ng/ml] (P < 0.001). The concentration of HSP90 showed positively correlation with International Staging System(ISS) stage, therapeutic response, frequency of plasmocyte, globulin, immune globulin, M-protein, ß2 micro-globulin, and light chain of MM patients (P < 0.05) ; while it showed little correlation with sex, age and type of MM patients (P > 0.05) . It is concluded that the HSP90 may be involved in the occurrence and development of MM. Detection of HSP90 in plasma would contribute to judge the clinical course, therapeutic efficacy and prognosis of MM patients.
Asunto(s)
Proteínas HSP90 de Choque Térmico/sangre , Mieloma Múltiple/sangre , Humanos , Proteínas de Mieloma , PronósticoRESUMEN
Recombineering is a powerful method for DNA manipulation. It has advantages over restriction endonuclease-based methods and is usually rapid. Typically, recombineering uses long PCR primers (c. 65 bases), each of which contains a small region of target homology (c. 45 bases). We have developed a simple, albeit somewhat less rapid, strategy to create recombineering substrates that can use primers of ≤ 35 bases for all steps. The regions of homology can be several hundred base pairs in length to (1) increase the chance of obtaining the desired clone and/or (2) allow coliphage-based recombineering in some non-Escherichia coli bacteria. The method uses cloning techniques to construct a template for the generation of the recombineering substrate. Because the template is made from cloned DNA segments, the segments (including those for the homology regions) can be readily changed. During construction of the template plasmid, potential background transformants arising from the vector without insert are significantly reduced by cloning each segment with two restriction endonucleases that produce noncompatible ends. We have used this method to change the bla gene of pACYC177 to aadA, to add the MCS-lacZα region from pBBR1MCS to IncQ plasmid vectors, and to make an oriT(IncP) -aacC1 cassette and add it to a plasmid.
Asunto(s)
Cartilla de ADN/genética , Genética Microbiana/métodos , Recombinación Homóloga , Biología Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Bacteriófago lambda/enzimología , ADN Bacteriano/genética , Vectores Genéticos , PlásmidosRESUMEN
PURPOSE: To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. METHODS: Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. RESULTS: The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. CONCLUSIONS: Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes.
Asunto(s)
Envejecimiento/fisiología , Lámina Basal de la Coroides/fisiología , Matriz Extracelular/metabolismo , Epitelio Pigmentado de la Retina/citología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bestrofinas , Lámina Basal de la Coroides/ultraestructura , Proteínas Portadoras/genética , Bovinos , Recuento de Células , Técnicas de Cultivo de Célula , Supervivencia Celular/fisiología , Canales de Cloruro/genética , Técnicas de Cocultivo , Endotelio Corneal/citología , Endotelio Corneal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Ojo/genética , Femenino , Humanos , Masculino , Espectrometría de Masas , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , ARN Mensajero/genética , Epitelio Pigmentado de la Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , cis-trans-IsomerasasRESUMEN
OBJECTIVE: To investigate the expression of regulatory T cells and Th17 cells in idiopathic thrombocytopenic purpura (ITP) and its significance. METHODS: The quantity of CD4(+)CD25(+)T cells, CD4(+)CD25(high) T cells and CD4(+) IL-17(+)T cells in peripheral blood were measured with flow cytometry (FCM), the plasma level of IL-10 and IL-17 with ELISA and the expression of Foxp3 mRNA and RORc mRNA with RT-PCR in 29 newly diagnosed ITP patients and 28 healthy controls. RESULTS: The ratio of CD4(+)CD25(+)T cells/CD4(+) T cells in the peripheral blood of ITP patients was significantly higher than that of the normal controls (P < 0.05). And the ratio of CD4(+)CD25(high) T/CD4(+) T cells in the patients was remarkably lower (P < 0.05). There was no difference in the percentage of CD4(+) IL-17(+)T/CD4(+) T cells between ITP patients and controls (P > 0.05). The ratio of Treg/Th17 was lower in ITP patients (P < 0.05). The plasma level of IL-10 in ITP patients was significantly decreased (P < 0.05), whereas no statistical difference was observed in the level of IL-17 (P > 0.05). The Foxp3 mRNA expression levels were largely reduced (P < 0.05), but the RORc mRNA expression was increased compared with that in controls (P < 0.05). CONCLUSION: The imbalance of Treg/Th17 plays an important role in the pathogenesis of ITP.