RESUMEN
PURPOSE: The internet has resulted in huge efficiency gains in health care, the ability to deal with massive data accumulation and better manage patient data. However, potential and real pitfalls exist, including breeches in security of data and patient confidentiality, data storage issues, errors, and user interface issues. METHODS: A MEDLINE review was performed using MeSH terms "health care" and "information technology." Cross-referencing was used to explore the different opportunities and challenges the internet has to offer. RESULTS: As health professionals, we are fast adopting technologies at our fingertips, such as WhatsApp and video capabilities, into our clinical practice to increase productivity and improve patient care. However, the potential security breaches are significant for the health professional and health service. Further, electronic medical records have theoretical advantages to improve patient care, reduce medication errors, and expedite referrals. The downside is a less personalized approach to patient care, as well as the potential for these systems to be even more cumbersome. In regard to the acquisition of knowledge, there is no doubt the internet is our friend. Health care professionals as well as patients have unlimited resources for learning, including podcasts videos, apps, simulators, and wearable devices. Unfortunately, this comes with a risk of misinformation and poorly referenced data with little to no regulation of content. CONCLUSION: In this increasing digital world, it is our task as health care providers to embrace these new technologies but develop guidelines and control systems to minimize the pitfalls.
Asunto(s)
Atención a la Salud , Internet , Informática Médica , Seguridad del Paciente , Procedimientos Quirúrgicos Operativos/normas , Educación en Salud , HumanosRESUMEN
The Akt/protein kinase B (PKB) serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoinositide 3-kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase-9 (MMP-9) production, in a manner highly dependent on its kinase activity and membrane-translocating ability. The increase in MMP-9 production was mediated by activation of nuclear factor-kappaB transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell-cell or cell-matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.
Asunto(s)
Movimiento Celular/fisiología , Metaloproteinasa 9 de la Matriz/biosíntesis , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Adhesión Celular/fisiología , Activación Enzimática , Humanos , FN-kappa B/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Transcripción Genética , Células Tumorales Cultivadas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.
Asunto(s)
Anticarcinógenos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacología , Células HL-60/efectos de los fármacos , Compuestos de Organoselenio/farmacología , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Antioxidantes/farmacología , Western Blotting , Supervivencia Celular/efectos de los fármacos , Grupo Citocromo c/metabolismo , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Células HL-60/enzimología , Células HeLa/efectos de los fármacos , Células HeLa/enzimología , Humanos , Potenciales de la Membrana , Selenocisteína/análogos & derivadosRESUMEN
Intracoronary stent implantation resulted in the complete or near complete dilatation of high grade occlusions of the left anterior descending coronary arteries in the four patients in whom it was undertaken. Intracoronary stent implantation is a useful adjunct to Percutaneous Transluminal Angioplasty (PTCA) and is applicable in selected patients with symptomatic ischaemic heart disease in a developing country with limited health resources like Jamaica. This is so since financial data presented here document the significant savings this technique (when appropriately utilised) could realise compared to the use of balloon angioplasty alone.
Asunto(s)
Angioplastia de Balón/métodos , Enfermedad Coronaria/terapia , Stents , Anciano , Angioplastia de Balón/economía , Enfermedad Coronaria/diagnóstico , Ahorro de Costo , Electrocardiografía , Humanos , Jamaica , Masculino , Persona de Mediana Edad , Stents/economíaRESUMEN
Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.
Asunto(s)
Metilación de ADN , Embrión de Mamíferos/fisiología , Fertilización In Vitro , Animales , Secuencia de Bases , Blastocisto , Bovinos , Clonación de Organismos , Islas de CpG , Fibroblastos , Masculino , Datos de Secuencia MolecularRESUMEN
Apoptosis, a programmed process of cell suicide, has been proposed as the most plausible mechanism for the chemopreventive activities of selenocompounds. In our study, we found that Se-methylselenocysteine (MSC) induced apoptosis through caspase activation in human promyelocytic leukemia (HL-60) cells. Measurements of cytotoxicity, DNA fragmentation and apoptotic morphology revealed that MSC was more efficient at inducing apoptosis than selenite, but was less toxic. Moreover, MSC increased both the apoptotic cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 activity, whereas selenite did not. We next examined whether caspases and serine proteases are required for the apoptotic induction by MSC. A general caspase inhibitor, z-VAD-fmk, dramatically decreased cytotoxicity in MSC-treated HL-60 cells and several other apoptotic features, such as, caspase-3 activation, the apoptotic DNA ladder, TUNEL-positive staining and the DNA double-strand break. Interestingly, a general serine protease inhibitor, AAPV-cmk, also effectively inhibited MSC-mediated cytotoxicity and apoptosis. These results demonstrate that MSC is a selenocompound that efficiently induces apoptosis in leukemia cells and that proteolytic machinery, in particular caspase-3, is necessary for MSC-induced apoptosis. On the other hand, selenite-induced cell death could be derived from necrosis rather than apoptosis, since selenite did not significantly induce several apoptotic phenomena, including the activation of caspase-3.
Asunto(s)
Anticarcinógenos/farmacología , Apoptosis , Caspasas/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacología , Compuestos de Organoselenio/farmacología , Clorometilcetonas de Aminoácidos/metabolismo , Caspasa 3 , Daño del ADN , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática , Células HL-60 , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Poli(ADP-Ribosa) Polimerasas/metabolismo , Selenocisteína/análogos & derivados , Selenito de Sodio/farmacología , Factores de TiempoRESUMEN
Reactive oxygen species (ROS) have emerged as important signaling molecules in the regulation of various cellular processes. In our study, we investigated the effect of a wide range of ROS on Chinese hamster lung fibroblast (V79) cell proliferation. Treatment with H2O2 (100 microM), superoxide anion (generated by 1 mM xanthine and 1 mU/ml xanthine oxidase), menadione, and phenazine methosulfate increased the cell proliferation by approximately 50%. Moreover, a similar result was observed after partial inhibition of superoxide dismutase (SOD) and glutathione peroxidase. This upregulation of cell proliferation was suppressed by pretreatment with hydroxyl radical scavengers and iron chelating agents. In addition to ROS, treatment with exogenous catalase and SOD mimic (MnTMPyP) suppressed the normal cell proliferation. Short-term exposure of the cells to 100 microM H2O2 was sufficient to induce proliferation, which indicated that activation of the signaling pathway is important as an early event. Accordingly, we assessed the ability of H2O2 to activate mitogen-activated protein kinases (MAPK). Jun-N-terminal kinase (JNK) and p38 MAPK were both rapidly and transiently activated by 100 microM H2O2, with maximal activation 30 min after treatment. However, the activity of extracellular signal-regulated kinase (ERK) was not changed. Pretreatment with SB203580 and SB202190, specific inhibitors of p38 MAPK, reduced the cell proliferation induced by H2O2. The activation of both JNK and p38 MAPK was also suppressed by pretreatment with hydroxyl radical scavenger and iron chelating agents. Our results suggest that the trace metal-driven Fenton reaction is a central mechanism that underlies cell proliferation and MAPK activation.
Asunto(s)
División Celular/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos , Oxidantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Catalasa/metabolismo , Cricetinae , ADN/biosíntesis , Activación Enzimática/efectos de los fármacos , Fibroblastos , Depuradores de Radicales Libres/farmacología , Glutatión Peroxidasa/antagonistas & inhibidores , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Immunoblotting , Hierro/metabolismo , Quelantes del Hierro/farmacología , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metosulfato de Metilfenazonio/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Imitación Molecular , Oxidantes/antagonistas & inhibidores , Oxidantes/metabolismo , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Superóxidos/antagonistas & inhibidores , Superóxidos/farmacología , Factores de Tiempo , Vitamina K/antagonistas & inhibidores , Vitamina K/farmacología , Xantina Oxidasa/metabolismo , Xantina Oxidasa/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Selenium, an essential biological trace element, has been shown to reduce and prevent the incidence of cancer. Our previous studies have shown that selenite is involved in the chemoprevention of cancer and induction of apoptosis of cancer cells. In this study, we demonstrate that selenite also inhibits the invasion of tumor cells. Cancer cell invasion requires coordinated processes, such as changes in cell-cell and cell-matrix adhesion, degradation of the extracellular matrix, and cell migration. We found that selenite inhibited invasion of HT1080 human fibrosarcoma cells. Adhesion of HT1080 cells to the collagen matrix was also inhibited by treatment with selenite, but cell-cell interaction and cell motility were not affected by selenite. Moreover, selenite reduced expression of matrix metalloproteinase-2 and -9 and urokinase-type plasminogen activator, which are involved in matrix degradation, but increased a tissue inhibitor of metalloproteinase-1. This inhibitory effect of selenite on the protease expressions was mediated by the suppression of transcription factors, NF-kappaB and AP-1. However, selenate showed no remarkable effect on all the steps of cancer cell invasion.
Asunto(s)
Invasividad Neoplásica/prevención & control , Selenito de Sodio/farmacología , Adhesión Celular , División Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Metástasis de la Neoplasia/prevención & control , Ácido Selénico , Compuestos de Selenio/farmacología , Inhibidor Tisular de Metaloproteinasa-1/genética , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Intracoronary stent implantation resulted in the complete or near complete dilatation of high grade occlusions of the left anterior descending coronary arteries in the four patients in whom it was undertaken. Intracoronary stent implantation is a useful adjunct to Percutaneous Transluminal Angioplasty (PTCA) and is applicable in selected patients with symptomatic ischaemic heart disease in a developing country with limited health resources like Jamaica. This is so since financial data presented here document the significant savings this technique (when appropriately utilised) could realise compared to the use of balloon angioplasty alone.
Asunto(s)
Anciano , Humanos , Masculino , Persona de Mediana Edad , Stents , Enfermedad Coronaria , Angioplastia de Balón/métodos , Stents , Enfermedad Coronaria , Electrocardiografía , Jamaica , Angioplastia de Balón/economía , Ahorro de CostoRESUMEN
Intracoronary stent implantation resulted in the complete or near complete dilatation of high gread occlusions of the left anterior descending coronary arteries in the four patients in whom it was undertaken. Intracoronary stent implatation is a useful adjuct to Percutaneous Transluminal Angioplasty (PTCA) and is applicable in selected patients with symptomatic ischaemic heart disease in a developing country with limited health resources like Jamaica. This is so since financial data presented here document the significant savings this technique (when appropriately utilised) could realise compared to the use of baloon angioplasty alone. (AU)
Asunto(s)
Persona de Mediana Edad , Anciano , Informes de Casos , Humanos , Masculino , Stents/economía , Enfermedad Coronaria/terapia , Angioplastia de Balón/métodos , Enfermedad Coronaria/diagnóstico , Angioplastia de Balón/economía , Electrocardiografía , Ahorro de Costo , JamaicaRESUMEN
The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.
Asunto(s)
Chaperoninas/metabolismo , Citosol/metabolismo , Productos del Gen gag/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Virus del Mono Mason-Pfizer/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas Nucleares/metabolismo , Cápside/metabolismo , Chaperonina con TCP-1 , Productos del Gen gag/genética , Virus del Mono Mason-Pfizer/genética , Pliegue de Proteína , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas , Ensamble de Virus , Región del Complejo T del GenomaRESUMEN
The effect of bioflavonoids extracted from the bark of Pinus maritima, Pycnogenol (PYC), on gene expression of the proinflammatory cytokines interleukin-1beta (IL-1beta) and interleukin-2 (IL-2) were investigated in RAW 264.7 cells and Jurkat E6.1 cells, respectively. PYC exerted strong scavenging activities against reactive oxygen species (ROS) generated by H2O2 in RAW 264.7. In situ ELISA, immunoblot analysis, and competitive RT-PCR demonstrated that pretreatment of LPS-stimulated RAW 264.7 cells with PYC dose-dependently reduced both the production of IL-1beta and its mRNA levels. Furthermore, in the same cells, PYC blocked the activation of nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1), two major transcription factors centrally involved in IL-1beta gene expression. Concordantly, pretreatment of the cells with PYC abolished the LPS-induced IkappaB degradation. We also investigated the effect of PYC on IL-2 gene expression in phorbol 12-myristate 13acetate plus ionomycin (PMA/Io)-stimulated human T-cell line Jurkat E6.1. PYC inhibited the PMA/Io-induced IL-2 mRNA expression. However, as demonstrated in a reporter gene assay system, the mechanism of IL-2 gene transcriptional regulation by PYC was different from the regulation of IL-1beta. PYC inhibited both NF-AT and AP-1 chloramphenicol acetyltransferase (CAT) activities in transiently transfected Jurkat E6.1, but not NF-kappaB CAT activity. We also found that PYC can destabilize PMA/Io-induced IL-2 mRNA by posttranscriptional regulation. All these results suggest that bioflavonids can be useful therapeutic agents in treating many inflammatory, autoimmune, and cardiovascular diseases based on its diverse action mechanisms.
Asunto(s)
Antioxidantes/farmacología , Flavonoides/farmacología , Depuradores de Radicales Libres/farmacología , Proteínas I-kappa B , Interleucina-1/biosíntesis , Interleucina-2/biosíntesis , Pinus/química , Corteza de la Planta/química , Animales , Antioxidantes/aislamiento & purificación , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas de Unión al ADN/metabolismo , Flavonoides/aislamiento & purificación , Depuradores de Radicales Libres/aislamiento & purificación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Interleucina-1/genética , Interleucina-2/genética , Ionomicina/farmacología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Inhibidor NF-kappaB alfa , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Extractos Vegetales , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.
Asunto(s)
Adenoviridae/genética , Mutación , Proteínas Nucleares , Proteínas/genética , Replicación Viral , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales Cultivadas/virología , Proteína p14ARF Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Currently, bioflavonoids have been known to have strong antioxidant capacities, and a variety of efforts have been made to identify the utilities of bioflavonoids in treating various diseases based on their antioxidant capacities. The effects of bioflavonoids extracted from the bark of Pinus maritima Pycnogenol (PYC) on free radical formation, activation of redox sensitive transcription factors, as well as interleukin-1 beta (IL-1 beta) production were investigated in murine macrophage cell lines. PYC exerted strong scavenging activities against reactive oxygen species generated either by H(2)O(2) or PMA in RAW 264.7 and IC-21 cells, respectively. In situ ELISA, immunoblot analysis, and competitive RT-PCR demonstrated that PYC pretreatment of LPS-stimulated RAW 264.7 cells dose-dependently reduced both the production of IL-1 beta and its mRNA levels. Furthermore, in the same cells, PYC blocked the activation of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1), two major transcription factors centrally involved in IL-1 beta gene expression. When RAW 264.7 cells were stimulated with LPS, the inhibitor protein I kappa B largely disappeared from cytosolic fractions. However, pretreatment of the cells with PYC abolished the LPS-induced I kappa B degradation. These results suggest that PYC can inhibit the expression of the proinflammatory cytokine IL-1 by regulating redox-sensitive transcription factors. This study may support the possibility that bioflavonoids including PYC can be used as antiinflammatory and immunosuppressive drugs based on their radical scavenging activities.
Asunto(s)
Flavonoides/farmacología , Interleucina-1/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Animales , Línea Celular , ADN/metabolismo , Depuradores de Radicales Libres/farmacología , Interleucina-1/farmacología , Mediciones Luminiscentes , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , ARN Mensajero/análisis , Factor de Transcripción AP-1/metabolismoRESUMEN
Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, functions as an anticarcinogen and antioxidant. In the present study, we investigated the effect of CHL on nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Treatment with CHL inhibited nitric oxide production in the LPS-stimulated RAW 264. 7 cells in a dose-related manner. Competitive RT-PCR analysis, using a DNA competitor as an internal standard, demonstrated that the treatment with 1, 10, and 50 microM CHL decreased LPS-induced iNOS mRNA expression in a concentration-dependent manner. Since the expression of the iNOS gene is mainly regulated by NF-kappaB, we then examined the effects of CHL on the NF-kappaB DNA binding activity, using an electrophoretic mobility shift assay. CHL down-regulated the NF-kappaB DNA binding on its cognate recognition site at the concentrations just noted. Employing a transfection and reporter gene expression system with p(NF-kappaB)(3)-chloramphenicol acetyl transferase (CAT), the treatment of CHL produced a dose-dependent inhibition of CAT activity in RAW 264.7 cells. Furthermore, CHL partially restored LPS-decreased IkappaBalpha, an inhibitory protein against NF-kappaB activation, in the cytosolic extract from the LPS-treated cells determined by immunoblot analysis. CHL also protected the hydroxyl radical-induced cytotoxicity in RAW 264.7 cells, indicating its antioxidant effect. These results suggest that CHL suppresses the nitric oxide production and iNOS mRNA expression mediated by the inhibition of NF-kappaB activation, and its action mechanism may be based on its antioxidant effect.
Asunto(s)
Antimutagênicos/farmacología , Clorofilidas/farmacología , Inhibidores Enzimáticos/farmacología , Macrófagos/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , ADN/metabolismo , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis , Genes Reporteros , Lipopolisacáridos/farmacología , Activación de Linfocitos , Macrófagos/enzimología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salmonella typhimurium , TransfecciónRESUMEN
Based on the assumption that foreign DNA sequences may have increased chance of integration into the host genome if they are flanked by high copy-numbered genomic sequences such as SINEs (short interspersed elements), we investigated the integration frequency of Lac Z reporter gene flanked by a fused B1/B2 in an in vivo system using pronuclear microinjection technique in the mouse. The SINE-flanked DNA showed a 4-fold increased integration frequency of the reporter gene than the control DNA (63% vs. 16%). Moreover, the level of beta-galactosidase expression, estimated from the X-Gal staining intensity in transgenic embryos, was greatly higher in SINE-carrying DNA. These results suggest that the SINE sequences can serve a very useful tool in improving the efficiency of current transgenic animal technology.
Asunto(s)
Desarrollo Embrionario/fisiología , Genes Reporteros , Elementos de Nucleótido Esparcido Corto , beta-Galactosidasa/genética , Animales , ADN Superhelicoidal , Femenino , Expresión Génica , Ratones , Microinyecciones , Embarazo , TransgenesRESUMEN
We investigated whether mouse short interspersed elements (SINEs) could influence the recombination frequency of foreign DNA. Vectors harboring a reporter gene in combinations of SINEs B1 and/or B2 or a portion of long interspersed element-1 were prepared and tested in vitro by a colony assay using HC11 murine mammary epithelial cells and in vivo by microinjection into fertilized mouse eggs. In transfected HC11 cells, the number of colonies surviving G418 selection increased by 3.5-fold compared with control when the reporter was flanked by fused B1-B2 sequences. Similar results were obtained from microinjection study; in fetuses 11.5 days post coitum, transgene positives in control and SINE-flanked vectors were 16 and 53%, respectively. Individual B1- and B2-harboring vectors showed equivalent activities with each other, as determined by the colony assay (2.8-fold versus 3.2-fold compared with control). We determined the contribution of homologous recombination to the SINE-mediated increase in integration frequency through a polymerase chain reaction-based strategy; in more than half of embryos transgenes underwent homologous recombinations involving B1 sequences. These results demonstrate that the SINE sequences can increase the integration rate of foreign DNA and that such an increase is most likely due to the enhancement of homologous recombination.
Asunto(s)
ADN/genética , Recombinación Genética , Transfección , Animales , Secuencia de Bases , Vectores Genéticos , Ratones , Datos de Secuencia MolecularRESUMEN
Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been used for the treatment of several abnormal human conditions without apparent toxicity. Recent studies have revealed that CHL has the excellent chemopreventive potential. In the present investigation, we have found the inhibitory activities of CHL against 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in Salmonella typhimurium TA100 and also on DMBA-initiated and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-promoted mouse skin tumor formation. The incidence and the multiplicity of skin tumors were not significantly decreased in mice by a single topical application of CHL prior to the DMBA treatment, but there was a marked suppression of papillomagenesis in mice treated with CHL during the promotional stage. Furthermore, the formation of DMBA-induced papillomagenesis was reduced in all mice that had received CHL for 6 weeks following treatment with TPA for 6, 18 and 24 weeks. These results indicate that CHL can inhibit both tumor promotion and the progression of papillomagenesis in the two-stage mouse skin carcinogenesis induced by DMBA and TPA.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/antagonistas & inhibidores , Anticarcinógenos/uso terapéutico , Antimutagênicos/uso terapéutico , Clorofilidas/uso terapéutico , Neoplasias Cutáneas/prevención & control , Animales , Quimioprevención , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Sprague-Dawley , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
Effects of selenite and selenodiglutathione, an initial metabolite of selenite, on the induction of apoptosis and cytotoxicity were investigated in human promyelocytic leukemia HL-60 cells. Treatment of selenite or selenodiglutathione resulted in concentration-dependent cytotoxicity, measured by lactate dehydrogenase leakage assay, and by tetrazolium salt reduction assay. Selenodiglutathione has been shown to exert more cytotoxic effect than selenite in both assay systems. Time-course study of cellular selenium uptake suggests that the higher cytotoxicity of selenodiglutathione be largely due to faster and greater selenium uptake rate. Treatment with selenite or selenodiglutathione also induced apoptosis in a dose-dependent manner, as detected by enzyme-linked immunosorbent assay and by DNA fragmentation assay. The dose-response data of apoptosis induced by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating a relationship between the induction of apoptosis and cytotoxicity. Zn, which is a well-known inhibitor of apoptosis, dose-dependently blocked not only the induction of apoptosis, but also the membrane damage induced by selenium, corroborating this hypothesis. It was noted that the inhibition of apoptosis by Zn exerted little protective effect on cytotoxicity at higher concentrations of selenium, compared with a perfect protective effect at low concentration of selenium. These results suggest that cytotoxicity induced by selenium may be partially correlated with apoptosis.
Asunto(s)
Apoptosis , Glutatión/análogos & derivados , Compuestos de Organoselenio/farmacología , Selenito de Sodio/farmacología , Relación Dosis-Respuesta a Droga , Glutatión/antagonistas & inhibidores , Glutatión/farmacología , Células HL-60 , Humanos , Compuestos de Organoselenio/antagonistas & inhibidores , Selenito de Sodio/antagonistas & inhibidores , Factores de Tiempo , Zinc/farmacologíaRESUMEN
We have analyzed the regulatory roles of the first intron (intron-1) of the bovine beta-casein gene in the bovine beta-casein/CAT expression system using a mouse mammary epithelial cell line, HC11. After a combined treatment of HC11 cells with insulin, dexamethasone and prolactin, the induced expression of p beta c1.8/+ICAT vector including 2 kb intron-1 and 1.8 kb promoter was greatly increased to 23.5 folds, while that of p beta ca.8CAT basic vector with 1.8 kb promoter only, was 6.5. A classical enhancer activity was shown in the 2 kb intron fragment from the experiment in which the orientation and the position of the intron-1 on the vectors were changed. The enhancer activity was largely dependent on the lactogenic hormones, especially prolactin. A stepwise reduction of the inducibility in the 5' to 3' deletion analysis of the intron-1 indicates the existence of several functional elements in the region. In particular, an internal fragment (+1071 to +1490) was important for the prolactin-dependent enhancing activity of the intron-1. These results suggest that several elements in the intron-1 of the bovine beta-casein gene cooperatively interact not only with each other but also with its promoter for hormonal induction.