RESUMEN
The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Cromatina/química , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Empalme Alternativo , Camptotecina/química , Antígeno Carcinoembrionario/genética , Etopósido/química , Proteínas Ligadas a GPI/genética , Perfilación de la Expresión Génica , Células HEK293 , Proteína HMGB1/metabolismo , Histonas/metabolismo , Humanos , Familia de Multigenes , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , Regiones Promotoras Genéticas , Dominios Proteicos , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína AIRERESUMEN
Autoimmune regulator (Aire) has a unique expression pattern in thymic medullary epithelial cells (mTECs), in which it plays a critical role in the activation of tissue-specific antigens. The expression of Aire in mTECs is activated by receptor activator of nuclear factor κB (RANK) signaling; however, the molecular mechanism behind this activation is unknown. Here, we characterize a conserved noncoding sequence 1 (CNS1) containing two NF-κB binding sites upstream of the Aire coding region. We show that CNS1-deficient mice lack thymic expression of Aire and share several features of Aire-knockout mice, including downregulation of Aire-dependent genes, impaired terminal differentiation of the mTEC population, and reduced production of thymic Treg cells. In addition, we show that CNS1 is indispensable for RANK-induced Aire expression and that CNS1 is activated by NF-κB pathway complexes containing RelA. Together, our results indicate that CNS1 is a critical link between RANK signaling, NF-κB activation, and thymic expression of Aire.
Asunto(s)
FN-kappa B/fisiología , Timo/metabolismo , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Células Epiteliales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Activador del Factor Nuclear kappa-B/fisiología , Transducción de Señal , Timo/citología , Factores de Transcripción/genética , Proteína AIRERESUMEN
The gene WFS1 encodes a protein with unknown function although its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study, we aimed to find the functional networks influenced by the time-dependent silencing of WFS1 in HEK cells. We performed whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72, and 96 h after transfection with three different WFS1 siRNAs. To verify silencing we performed quantitative RT-PCR and Western blot analysis. Analysis was conducted in two ways. First we analyzed the overall effect of the siRNA treatment on the gene expression profile. As a next step we performed time-course analysis separately for different siRNAs and combined for all siRNAs. Quantitative RT-PCR and Western blot analysis confirmed clear silencing of the expression of WFS1 after 48 h. Significant (FDR value<10%) changes in the expression of 11 genes was identified with most of these genes being related to the mitochondrial dysfunction and apoptosis. Time-course analysis confirmed significant correlations between WFS1 silencing and changes in the expression profiles of several genes. The pathways that were influenced significantly by WFS1 silencing were related to mitochondrial damage and neurodegenerative diseases. Our findings suggest a role of WFS1 gene in cell survival and its involvement in degenerative diseases.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Neurodegenerativas/metabolismo , Animales , Western Blotting , Línea Celular , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Modelos Teóricos , Enfermedades Neurodegenerativas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Síndrome de Wolfram/metabolismoRESUMEN
The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b (miR-146a/b), both of which are known to suppress immune responses in a variety of conditions. Here, we studied how constitutive deficiency of miR-146b (Mir146b-/-) affects lipopolysaccharide (LPS)-induced neuroinflammation in mice. Our experiments demonstrated that miR-146b deficiency results in the attenuation of LPS-induced neuroinflammation, as it was evidenced by the reduction of sickness behavior, a decrease in the inflammatory status of microglia, and the loss of morphological signs of microglial activation in the hippocampus. Gene expression analysis revealed that LPS-induced upregulation of hippocampal pro-inflammatory cytokines is attenuated in Mir146b-/- mice, compared to wild-type (WT) mice. In addition, reduced expression of the NF-κB nuclear protein p65, reduced miR-146 family target TLR4 expression and relatively stronger upregulation of miR-146a was found in Mir146b-/- mice as compared to WT mice upon LPS challenge. Compensatory upregulation of miR-146a can explain the attenuation of the LPS-induced neuroinflammation. This was supported by experiments conducted with miR-146a/b deficient mice (Mir146a/b-/-), which demonstrated that additional deletion of the miR-146a led to the restoration of LPS-induced sickness behavior and proinflammatory cytokines. Our experiments also showed that the observed upregulation of miR-146a in Mir146b-/- mice is due to the overexpression of a miR-146a transcription inducer, interferon regulatory factor 7 (Irf7). Altogether, our results show the existence of crosstalk between miR-146a and mir-146b in the regulation of LPS-induced neuroinflammation.
Asunto(s)
Lipopolisacáridos , MicroARNs , Ratones , Animales , Lipopolisacáridos/toxicidad , Inflamación/genética , MicroARNs/metabolismo , Regulación hacia Arriba , Citocinas/metabolismoRESUMEN
PURPOSE: Nanotechnology applied to cancer treatment is a growing area of research in nanomedicine with magnetic nanoparticle-mediated anti-cancer drug delivery systems offering least possible side effects. To that end, both structural and chemical properties of commercial cobalt metal nanoparticles were studied using label-free confocal Raman spectroscopy. MATERIALS AND METHODS: Crystal structure and morphology of cobalt nanoparticles were studied by XRD and TEM. Magnetic properties were studied with SQUID and PPMS. Confocal Raman microscopy has high spatial resolution and compositional sensitivity. It, therefore, serves as a label-free tool to trace nanoparticles within cells and investigate the interaction between coating-free cobalt metal nanoparticles and cancer cells. The toxicity of cobalt nanoparticles against human cells was assessed by MTT assay. RESULTS: Superparamagnetic Co metal nanoparticle uptake by MCF7 and HCT116 cancer cells and DPSC mesenchymal stem cells was investigated by confocal Raman microscopy. The Raman nanoparticle signature also allowed accurate detection of the nanoparticle within the cell without labelling. A rapid uptake of the cobalt nanoparticles followed by rapid apoptosis was observed. Their low cytotoxicity, assessed by means of MTT assay against human embryonic kidney (HEK) cells, makes them promising candidates for the development of targeted therapies. Moreover, under a laser irradiation of 20mW with a wavelength of 532nm, it is possible to bring about local heating leading to combustion of the cobalt metal nanoparticles within cells, whereupon opening new routes for cancer phototherapy. CONCLUSION: Label-free confocal Raman spectroscopy enables accurately localizing the Co metal nanoparticles in cellular environments. The interaction between the surfactant-free cobalt metal nanoparticles and cancer cells was investigated. The facile endocytosis in cancer cells shows that these nanoparticles have potential in engendering their apoptosis. This preliminary study demonstrates the feasibility and relevance of cobalt nanomaterials for applications in nanomedicine such as phototherapy, hyperthermia or stem cell delivery.
Asunto(s)
Cobalto/farmacocinética , Nanopartículas del Metal/química , Neoplasias/tratamiento farmacológico , Línea Celular Tumoral , Cobalto/química , Células HCT116 , Células HEK293 , Humanos , Fenómenos Magnéticos , Nanopartículas del Metal/administración & dosificación , Microscopía Confocal , Microscopía Electrónica de Transmisión , Neoplasias/patología , Espectrometría Raman/métodos , Difracción de Rayos XRESUMEN
Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.
Asunto(s)
Bilirrubina/farmacología , Plasmodium falciparum/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Antígenos de Protozoos/fisiología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Hemoproteínas/antagonistas & inhibidores , Hemoproteínas/biosíntesis , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Estrés Oxidativo/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismoRESUMEN
Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.
Asunto(s)
Colina Quinasa/química , Plasmodium falciparum/enzimología , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Eritrocitos/metabolismo , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilcolina/química , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Hepatic dysfunction is a common clinical complication in malaria, although its pathogenesis remains largely unknown. Using a variety of in vivo and ex vivo approaches, we have shown for the first time that malarial infection induces hepatic apoptosis through augmentation of oxidative stress. Apoptosis in hepatocyte has been confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin-nick-end labeling assay (TUNEL) and caspase-3 activation. Gene expression analysis using RT-PCR indicates the significant down-regulation of Bcl-2 and up-regulation of Bax expression in liver of malaria infected mice suggesting the involvement of mitochondrial pathway of apoptosis. The levels of Fas expression and caspase-8 activity in infected liver were same as that of uninfected control mice indicating death receptor (Fas) pathway did not contribute to liver apoptosis during malarial infection. Moreover, evidence has been presented by confocal microscopy to show the translocation of Bax from cytosol to mitochondria in apoptotic hepatocyte, resulting in opening of permeability transition pores, which in turn decreases mitochondrial membrane potential and induces cytochrome c release into cytosol. Malarial infection induces the generation of hydroxyl radical (*OH) in liver, which may be responsible for the induction of oxidative stress and apoptosis as administration of *OH specific antioxidant as well as spin trap, alpha-phenyl-tert-butyl-nitrone in malaria-infected mice significantly inhibits the development of oxidative stress as well as induction of apoptosis. Thus, results suggest the implication of oxidative stress induced-mitochondrial pathway of apoptosis in the pathophysiology of hepatic dysfunction in malaria.
Asunto(s)
Apoptosis , Hígado/patología , Malaria/metabolismo , Malaria/patología , Mitocondrias Hepáticas/metabolismo , Estrés Oxidativo , Animales , Hepatocitos/patología , Radical Hidroxilo/metabolismo , Hígado/metabolismo , Ratones , Plasmodium yoeliiRESUMEN
Digestion of hemoglobin in the food vacuole of the malaria parasite produces very high quantities of redox active toxic free heme. Hemozoin (beta-hematin) formation is a unique process adopted by Plasmodium sp. to detoxify free heme. Hemozoin formation is a validated target for most of the well-known existing antimalarial drugs and considered to be a suitable target to develop new antimalarials. Here we discuss the possible mechanisms of free heme detoxification in the malaria parasite and the mechanistic details of compounds, which offer antimalarial activity by inhibiting hemozoin formation. The chemical nature of new antimalarial compounds showing antimalarial activity through the inhibition of hemozoin formation has also been incorporated, which may help to design future antimalarials with therapeutic potential against multi-drug resistant malaria.
Asunto(s)
Antimaláricos , Hemoproteínas/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/química , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/microbiología , Estructura Molecular , Plasmodium falciparum/metabolismo , Relación Estructura-ActividadRESUMEN
Activation of the mitochondrial apoptosis pathway by oxidative stress has been implicated in hepatocyte apoptosis during malaria. Because mitochondria are the source and target of reactive oxygen species (ROS), we have investigated whether hepatocyte apoptosis is linked to mitochondrial pathology and mitochondrial ROS generation during malaria. Malarial infection induces mitochondrial pathology by inhibiting mitochondrial respiration, dehydrogenases, and transmembrane potential and damaging the ultrastructure as evident from transmission electron microscopic studies. Mitochondrial GSH depletion and formation of protein carbonyl indicate that mitochondrial pathology is associated with mitochondrial oxidative stress. Fluorescence imaging of hepatocytes documents intramitochondrial superoxide anion (O(2)(-)) generation during malaria. O(2)(-) inactivates mitochondrial aconitase to release iron from iron-sulfur clusters, which forms the hydroxyl radical ((.)OH) interacting with H(2)O(2) produced concurrently. Malarial infection inactivates mitochondrial aconitase, and carbonylation of aconitase is evident from Western immunoblotting. The release of iron has been documented by fluorescence imaging of hepatocytes using Phen Green SK, and mitochondrial (.)OH generation has been confirmed. During malaria, the depletion of cardiolipin and formation of the mitochondrial permeability transition pore favor cytochrome c release to activate caspase-9. Interestingly, mitochondrial (.)OH generation correlates with the activation of both caspase-9 and caspase-3 with the progress of malarial infection, indicating the critical role of (.)OH.
Asunto(s)
Apoptosis/fisiología , Hepatocitos/fisiología , Malaria/fisiopatología , Mitocondrias Hepáticas/fisiología , Estrés Oxidativo/fisiología , Plasmodium yoelii/fisiología , Aconitato Hidratasa/metabolismo , Animales , Cardiolipinas/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Hepatocitos/ultraestructura , Hierro/análisis , Hígado/ultraestructura , Malaria/patología , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/fisiología , Consumo de Oxígeno/fisiología , Parasitemia , Plasmodium yoelii/crecimiento & desarrollo , Superóxido Dismutasa/metabolismo , Superóxidos/análisisRESUMEN
A series of [(aryl)arylsufanylmethyl]pyridines (AASMP) have been synthesized. These compounds inhibited hemozoin formation, formed complexes (K(D) = 12 to 20 muM) with free heme (ferriprotoporphyrin IX) at a pH close to the pH of the parasite food vacuole, and exhibited antimalarial activity in vitro. The inhibition of hemozoin formation may develop oxidative stress in Plasmodium falciparum due to the accumulation of free heme. Interestingly, AASMP developed oxidative stress in the parasite, as evident from the decreased level of glutathione and increased formation of lipid peroxide, H(2)O(2), and hydroxyl radical (.OH) in P. falciparum. AASMP also caused mitochondrial dysfunction by decreasing mitochondrial potential (DeltaPsim) in malaria parasite, as measured by both flow cytometry and fluorescence microscopy. Furthermore, the generation of .OH may be mainly responsible for the antimalarial effect of AASMP since .OH scavengers such as mannitol, as well as spin trap alpha-phenyl-n-tertbutylnitrone, significantly protected P. falciparum from AASMP-mediated growth inhibition. Cytotoxicity testing of the active compounds showed selective activity against malaria parasite with selectivity indices greater than 100. AASMP also exhibited profound antimalarial activity in vivo against chloroquine resistant P. yoelii. Thus, AASMP represents a novel class of antimalarial.
Asunto(s)
Antimaláricos/farmacología , Malaria/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Plasmodium yoelii/efectos de los fármacos , Piridinas/química , Piridinas/farmacología , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/uso terapéutico , Benzoína/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Hemina/metabolismo , Humanos , Malaria/parasitología , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo , Pruebas de Sensibilidad Parasitaria , Piridinas/síntesis química , Piridinas/uso terapéuticoRESUMEN
We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and Bcl(xL) to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.
Asunto(s)
2-Piridinilmetilsulfinilbencimidazoles/farmacología , Antiinflamatorios no Esteroideos/farmacología , Citoprotección/efectos de los fármacos , Mucosa Gástrica/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Gastropatías/patología , Receptor fas/metabolismo , Animales , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Mucosa Gástrica/lesiones , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Indometacina/farmacología , Lansoprazol , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Gastropatías/inducido químicamente , Gastropatías/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
We showed earlier that malarial infection significantly induces liver apoptosis mediated by oxidative stress mechanisms. Thus, a nontoxic antioxidant-antiapoptotic molecule may be beneficial for hepatoprotection. Melatonin remarkably prevents hepatocyte apoptosis in mice induced during malaria as indicated by caspase 3 and TUNEL assays as well as transmission electron microscopy (TEM) of the liver tissue. The mitochondrial apoptotic pathway, which plays a critical role in liver cell death during malarial infection, was almost completely suppressed by melatonin as it corrects both the overexpression of Bax and down-regulation of bcl-2 as revealed by semiquantitative RT-PCR. Fluorometric studies using JC-1 documented that melatonin also restores mitochondrial transmembrane potential (DeltaPsim) in malaria-infected mice liver. The antiapoptotic effect of melatonin is associated with its antioxidant role because melatonin protects liver from oxidative stress induced during malaria by scavenging the hydroxyl radicals, preventing the depletion of reduced glutathione, inhibiting lipid peroxidation and protein carbonyl formation. The effective antioxidant dose of melatonin to protect liver from oxidative stress during malaria is 20 times lower than that of known antioxidants, vitamin C and vitamin E. Apoptosis of hepatocytes during malarial infection is well correlated with dysfunction of the liver while melatonin offers hepatoprotective effects as indicated by different liver function tests. Thus, melatonin may well be effective in combating oxidative stress-induced apoptosis and liver damage during malaria infection.
Asunto(s)
Apoptosis/efectos de los fármacos , Radicales Libres/metabolismo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Malaria/prevención & control , Melatonina/farmacología , Animales , Antioxidantes/farmacología , Caspasa 3/metabolismo , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Hepatocitos/patología , Hepatocitos/ultraestructura , Radical Hidroxilo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/parasitología , Hígado/patología , Pruebas de Función Hepática , Malaria/metabolismo , Malaria/patología , Ratones , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Plasmodium yoelii/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína X Asociada a bcl-2/genéticaRESUMEN
Choline kinase is the first enzyme in the Kennedy pathway (CDP-choline pathway) for the biosynthesis of the most essential phospholipid, phosphatidylcholine, in Plasmodium falciparum. In addition, choline kinase also plays a pivotal role in trapping essential polar head group choline inside the malaria parasite. Recently, Plasmodium falciparum choline kinase (PfCK) has been cloned, overexpressed, and purified. However, the function of this enzyme in parasite growth and survival has not been evaluated owing to the lack of a suitable inhibitor. Purified recombinant PfCK enabled us to identify an inhibitor of PfCK, hexadecyltrimethylammonium bromide (HDTAB), which has a very close structural resemblance to hexadecylphosphocholine (miltefosin), the well-known antiproliferative and antileishmanial drug. HDTAB inhibited PfCK in a dose-dependent manner and offered very potent antimalarial activity in vitro against Plasmodium falciparum. Moreover, HDTAB exhibited profound antimalarial activity in vivo against the rodent malaria parasite Plasmodium yoelii (N-67 strain). Interestingly, parasites at the trophozoite and schizont stages were found to be particularly sensitive to HDTAB. The stage-specific antimalarial effect of HDTAB correlated well with the expression pattern of PfCK in P. falciparum, which was observed by reverse transcription-PCR and immunofluorescence microscopy. Furthermore, the antimalarial activity of HDTAB paralleled the decrease in phosphatidylcholine content, which was found to correlate with the decreased phosphocholine generation. These results suggest that inhibition of choline kinase by HDTAB leads to decreased phosphocholine, which in turn causes a decrease in phosphatidylcholine biosynthesis, resulting in death of the parasite.
Asunto(s)
Compuestos de Cetrimonio/farmacología , Colina Quinasa/antagonistas & inhibidores , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Animales , Antiprotozoarios/farmacología , Cetrimonio , Compuestos de Cetrimonio/química , Colina Quinasa/genética , Relación Dosis-Respuesta a Droga , Malaria Falciparum/parasitología , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genéticaRESUMEN
A growing body of evidence has ascertained that apoptosis is not only restricted to metazoans but also exists in unicellular parasites. In Plasmodium falciparum, the presence of a putative gene having sequence homology with apoptosis related protein (PfARP) (Gene ID PFI0450c) has raised enormous interest to unravel the function of this unique protein in cell death of malaria parasite. To characterize this protein, the PfARP gene has been amplified from the P. falciparum transcriptome by RT-PCR and the amplified gene has been successfully cloned, over-expressed and purified to homogeneity. The purified PfARP exhibits minimum subunit MW of approximately 24kDa as evident from SDS-PAGE. CD analysis reveals that the alpha and beta content of the recombinant PfARP are 61% and 15%, respectively. Semiquantitative RT-PCR analysis indicates the expression of PfARP at both metabolically less active ring and highly active trophozoite stages of malaria parasite. Immunofluorescence microscopy further supports that PfARP expresses stage specifically with the highest expression at trophozite stage and very little in the schizont stage. PfARP is a cytosolic protein as evident from immunofluorescence microscopy. The role of this protein in P. falciparum cell death and stage progression is not yet known. The identification, purification and characterization would certainly be a step to initiate work on this protein to evaluate its role in P. falciparum growth, multiplication and stage progression.