RESUMEN
Glioblastoma multiforme (GBM) is a heterogeneous disease despite its seemingly uniform pathology. Deconvolution of The Cancer Genome Atlas's GBM gene expression data has unveiled the existence of distinct gene expression signature underlying discrete GBM subtypes. Recent conflicting findings proposed that microRNA (miRNA)-10b exclusively regulates glioma growth or invasion but not both. We showed that silencing of miRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subtype of GBM reduces its growth, invasion and angiogenesis while promoting apoptosis in vitro. In an orthotopic human glioma mouse model, inhibition of miRNA-10b diminishes the invasiveness, angiogenicity and growth of the mesenchymal subtype-like glioma cells in the brain and significantly prolonged survival of glioma-bearing mice. We demonstrated that the pleiotropic nature of miRNA-10b was due to its suppression of multiple tumor suppressors, including TP53, FOXO3, CYLD, PAX6, PTCH1, HOXD10 and NOTCH1. In particular, siRNA-mediated knockdown experiments identified TP53, PAX6, NOTCH1 and HOXD10 as invasion regulatory genes in our mesenchymal subtype-like glioma cells. By interrogating the REMBRANDT, we noted that dysregulation of many direct targets of miRNA-10b was associated with significantly poorer patient survival. Thus, our study uncovers a novel role for miRNA-10b in regulating angiogenesis and suggests that miRNA-10b may be a pleiotropic regulator of gliomagenesis.
Asunto(s)
Apoptosis , MicroARNs/metabolismo , Neovascularización Patológica , Animales , Baculoviridae/genética , Línea Celular Tumoral , Movimiento Celular , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , MicroARNs/antagonistas & inhibidores , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/antagonistas & inhibidores , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 µM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 µM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.
Asunto(s)
Proteínas de Artrópodos/farmacología , Hepatocitos/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Péptidos/farmacología , Venenos de Escorpión/química , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismoRESUMEN
PURPOSE: To study planning strategies that can be used in small animal radiation-induced lung toxicity experiments using 6 MV accelerator with high density MLC. METHODS: Three different types of plans were designed on CT images of a Sprague Dawley rat model to irradiate 50% of the total lung volume (lung divided into apex and base) with a prescription dose of 24 Gy to the partial lung. Two VMAT arc therapy plans were optimized to cover to the prescription dose, either the apex or base of the lung. Two AP- PA plans were designed to completely block either lung apex or base while irradiating the remaining 50% of the lung. Finally, two AP-PA plans were designed to cover, to the prescription dose, the apex or base of the lung. The plans were designed and optimized using the Eclipse AAA algorithm and recalculated using the MMCTP/EGS/Beam Monte Carlo system. RESULTS: When completely blocking the lung base, the apex will be underdosed by up to 30%; when completely covering the apex by the prescribed dose, the base will receive overdosing (V50%=73%). The VMAT plan leads to a more conformal dose distribution and spares unnecessary skin exposure when compared to AP-PA MV or kV delivery. Despite the small size of rat model, the 6 MV VMAT delivery is superior in terms of dose conformality and sparing of the heart and the non-irradiated 50% of the lung compared to the standard, simpler, AP-PA delivery. MC dosimetry in lung shows that the delivered dose is 10% higher than predicted by AAA because of the predominance of small fields in the delivery. CONCLUSIONS: Clinical state-of- the-art planning and delivery techniques can be scaled down accurately to rats. The use of these techniques is essential in small animal studies to render conclusions of radiation response investigations translatable to human studies.
RESUMEN
Stroke is one of the leading causes of death and disability worldwide. There are two major types of stroke: cerebral ischemia caused by obstruction of blood vessels in the brain and haemorrhagic stroke that is triggered by the disruption of blood vessels. Thrombolytic therapy involving recombinant tissue plasminogen activator (rtPA) has been shown to be beneficial only when used within 4.5 hours of onset of acute ischemic stroke. rtPA treatment beyond this time window has been found to be unsuitable and usually resulting in haemorrhagic transformation. Stroke is a multifactorial disease that forms a possible end state for majority of patients suffering from diabetes, atherosclerosis and hypertension which are known risk factors. Although the biochemistry of stroke and related diseases is quite well understood, the knowledge on the molecular mechanisms underlying these diseases is still at its infancy. microRNAs that form a unique class of endogenous riboregulators of gene function, offer tremendous potential in unraveling the mechanisms underlying stroke pathogenesis. microRNA expression also reflects the response of individuals to drugs and therapy. Several microRNAs and their target genes, known to be involved in endothelial dysfunction, dysregulation of neurovascular integrity, edema formation, pro-apoptosis, inflammation and extra-cellular matrix remodeling contribute to the critical processes in the pathogenesis of stroke. In this review, we will also be discussing the role of microRNAs as possible diagnostic and prognostic biomarkers as well as potential therapeutic targets in stroke pathogenesis.
Asunto(s)
MicroARNs/metabolismo , Accidente Cerebrovascular/fisiopatología , Animales , Isquemia Encefálica/fisiopatología , Isquemia Encefálica/terapia , Hemorragia Cerebral/fisiopatología , Hemorragia Cerebral/terapia , Humanos , MicroARNs/uso terapéutico , Factores de Riesgo , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/terapiaRESUMEN
BACKGROUND: Over the past 10 years clinical trials aimed at finding suitable neuroprotectants against the debilitating effects of stroke have met with no success. Identifying novel neuroprotectants that can reverse the effects of stroke is becoming a challenge to both clinicians and scientists. OBJECTIVES: This review focused on the current status on the topic and highlights some of the neuroprotectants that are worth examining or re-examining further. METHODS: Recent findings on the subject have been included. CONCLUSION: Many neuroprotectants that have worked in preclinical evaluations have been found to be ineffective in clinical trials. Nevertheless, some of them are still considered to be worth re-examining. Non-coding small RNAs (riboregulators) as novel therapeutic leads have also been introduced in this review.
Asunto(s)
Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Ensayos Clínicos como Asunto , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Agonistas del GABA/farmacología , Agonistas del GABA/uso terapéutico , Humanos , Hipotermia Inducida , Activación del Canal Iónico , Canales Iónicos/agonistas , Canales Iónicos/antagonistas & inhibidores , Compuestos de Magnesio/farmacología , Compuestos de Magnesio/uso terapéutico , Antagonistas de Narcóticos , Fármacos Neuroprotectores/farmacología , Nootrópicos/farmacología , Nootrópicos/uso terapéutico , Accidente Cerebrovascular/fisiopatología , Accidente Cerebrovascular/terapiaRESUMEN
Lowering of low-density lipoprotein cholesterol is well achieved by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). Statins inhibit the conversion of HMG-CoA to mevalonate, a precursor for cholesterol and coenzyme Q10 (CoQ10). In HepG2 cells, simvastatin decreased mitochondrial CoQ10 levels, and at higher concentrations was associated with a moderately higher degree of cell death, increased DNA oxidative damage and a reduction in ATP synthesis. Supplementation of CoQ10, reduced cell death and DNA oxidative stress, and increased ATP synthesis. It is suggested that CoQ10 deficiency plays an important role in statin-induced hepatopathy, and that CoQ10 supplementation protects HepG2 cells from this complication.
Asunto(s)
Mitocondrias/efectos de los fármacos , Simvastatina/farmacología , Ubiquinona/análogos & derivados , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Coenzimas/metabolismo , Coenzimas/farmacología , Daño del ADN , Relación Dosis-Respuesta a Droga , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ubiquinona/metabolismo , Ubiquinona/farmacologíaRESUMEN
Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However, although bites can be deadly, snake venom is a natural biological resource that contains several components of potential therapeutic value. Venom has been used in the treatment of a variety of pathophysiological conditions in Ayurveda, homeopathy and folk medicine. With the advent of biotechnology, the efficacy of such treatments has been substantiated by purifying components of venom and delineating their therapeutic properties. This review will focus on certain snake venom components and their applications in health and disease.
Asunto(s)
Hemostasis , Neurotoxinas/química , Venenos de Serpiente/química , Animales , Sistema Cardiovascular/efectos de los fármacos , Desintegrinas/química , Desintegrinas/farmacología , Fibrinógeno/química , Fibrinógeno/farmacología , Lectinas Tipo C/química , Músculos/efectos de los fármacos , Fosfolipasas A/metabolismo , Fosfolipasas A/farmacología , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Estructura Terciaria de Proteína , Protrombina/química , Protrombina/farmacología , Transducción de Señal , Trombina/química , Trombina/farmacologíaRESUMEN
Various clinical manifestations leading to death have been documented in most cases of bites caused by venomous snakes. Cobra envenomation is an extremely variable process and known to cause profound neurological abnormalities. The complexity of cobra venom can induce multiple-organ failure, leading to death in case of severe envenomation. Intramuscular administration of Malayan spitting cobra (Naja sputatrix) crude venom at 1 microg/g dose caused death in mice in approximately 3 h. Analysis of gene expression profiles in the heart, brain, kidney, liver and lung revealed 203 genes whose expression was altered by at least 3-fold in response to venom treatment. Of these, 50% were differentially expressed in the heart and included genes involved in inflammation, apoptosis, ion transport and energy metabolism. Electrocardiogram recordings and serum troponin T measurements indicated declining cardiac function and myocardial damage. This not only sheds light on the cardiotoxicity of cobra venom but also reveals the molecular networks affected during envenomation.
Asunto(s)
Venenos Elapídicos/toxicidad , Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Miocardio/metabolismo , Animales , Venenos Elapídicos/administración & dosificación , Electrocardiografía/efectos de los fármacos , Perfilación de la Expresión Génica , Corazón/fisiopatología , Cardiopatías/inducido químicamente , Cardiopatías/fisiopatología , Inyecciones Intramusculares , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Mordeduras de Serpientes , Distribución Tisular , Troponina T/sangreRESUMEN
The NGF (nerve growth factor) from Naja sputatrix has been purified by gel filtration followed by reversed-phase HPLC. The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse NGF. Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF, sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein. Sputa NGF has been found to be non-toxic under both in vivo and in vitro conditions. The induction of neurite outgrowth by this NGF has been found to involve the high-affinity trkA-p75NTR complex of receptors. The pro-survival mechanism of p75NTR has been mediated by the activation of nuclear factor kappaB gene by a corresponding down-regulation of inhibitory kappaB gene. Real-time PCR and protein profiling (by surface-enhanced laser-desorption-ionization time-of-flight) have confirmed that sputa NGF up-regulates the expression of the endogenous NGF in PC12 cells. Preliminary microarray analysis has also shown that sputa NGF is capable of promoting additional beneficial effects such as the up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type calcium channel. Hence, sputa NGF forms a new and useful NGF.
Asunto(s)
Venenos Elapídicos/química , Factor de Crecimiento Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Elapidae , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/aislamiento & purificación , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Ratas , Receptor de Factor de Crecimiento Nervioso/biosíntesis , Receptor trkA/biosíntesis , Proteínas Recombinantes/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Fetal lung development is dependent upon secretion of liquid into the future airways which must be cleared at birth to establish air-breathing. Aquaporins (AQP) 1, 3, 4 and 5 are membranous water channel proteins that are present in the lung after birth in rodents, with little expression before birth. Our aim was to describe the changes in AQP1, 3, 4 and 5 expression and protein levels in the fetal lung of a long-gestation species (sheep) and in response to physiological factors known to alter fetal lung liquid dynamics. Both mRNA and high protein levels were detected for AQP1, 3, 4 and 5 by day 100 (term is ~150 days in ovine fetuses). A cortisol infusion (120-131 days) significantly (P < 0.05) increased AQP1 (0.9 +/- 0.2 (n = 4) vs.1.8 +/- 0.3 (n = 5)) and AQP5 (8.8 +/- 0.6 vs. 14.1 +/- 1.2) mRNA levels in fetal lung (measured by real-time PCR). Ten days of tracheal obstruction significantly (P < 0.05) decreased AQP5 mRNA levels (6.1 +/- 0.9 (n = 5) vs. 2.7 +/- 0.3 (n = 5)). Immunohistochemistry was used to show that protein levels changed in parallel with the mRNA changes. These findings suggest that AQPs could be involved in lung liquid production and reabsorption during fetal development in long-gestation species.
Asunto(s)
Acuaporinas/biosíntesis , Acuaporinas/genética , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Pulmón/metabolismo , Envejecimiento/genética , Envejecimiento/fisiología , Algoritmos , Secuencia de Aminoácidos , Animales , Antiinflamatorios/farmacología , Western Blotting , Clonación Molecular , ADN Complementario/biosíntesis , Femenino , Hidrocortisona/farmacología , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/genética , Embarazo , ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos , Tráquea/fisiologíaRESUMEN
An Australian common brown snake, Pseudonaja textilis, is known to contain highly lethal neurotoxins. Among them, a long-chain alpha-neurotoxin, pseudonajatoxin b, has been identified. In this report, while presenting evidence for the presence of at least four such long-chain alpha-neurotoxins in the venom of P. textilis, we describe the characteristics of both the mRNA and the gene responsible for the synthesis of these neurotoxins. A precursor toxin synthesized from the gene has been identified as being capable of producing the isoforms possibly by post-translational modifications at its C-terminal end. Recombinant toxins corresponding to the precursor and its product have been found to possess similar binding affinities for muscular acetylcholine receptors (IC(50)=3x10(-8) M) and a lethality, LD(50), of 0.15 microg/g in mice.
Asunto(s)
Venenos Elapídicos/química , Venenos Elapídicos/genética , Elapidae/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Australia , Secuencia de Bases , Clonación Molecular , Venenos Elapídicos/toxicidad , Genes , Hígado/metabolismo , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/toxicidad , Filogenia , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Precursores de Proteínas/química , Precursores de Proteínas/toxicidad , Proteínas Recombinantes/química , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We have developed a new method for quantification of promoter activity in cell lines transfected with recombinant plasmids containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) by real-time PCR. As the efficiency of transfection has a direct influence on the total mRNA produced, we have used the neomycin-resistance gene present within the same vector DNA to normalize the measurement of mRNA levels. Three promoters from genes encoding toxins (pre-synaptic neurotoxin phospholipase A(2), post-synaptic alpha neurotoxin and cardiotoxin), believed to have evolved from the same ancestor but exhibiting different promoter activities, have been employed in this study to demonstrate the feasibility and accuracy of the method in CAT gene reporter analysis.
Asunto(s)
Regulación de la Expresión Génica , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/genética , Toxinas Biológicas/genética , Transcripción Genética/genética , Animales , Células CHO , Cloranfenicol O-Acetiltransferasa/genética , Proteínas Cardiotóxicas de Elápidos/genética , Cricetinae , Resistencia a Medicamentos/genética , Genes Reporteros/genética , Vectores Genéticos/genética , Cinética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Neurotoxinas/genética , Fosfolipasas A/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Sensibilidad y Especificidad , Moldes Genéticos , Factores de Tiempo , TransfecciónRESUMEN
We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.
Asunto(s)
Calcio/metabolismo , Péptidos/farmacología , Bloqueadores de los Canales de Potasio , Venenos de Escorpión/genética , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cromatografía en Agarosa , Clonación Molecular , Cricetinae , Cisteína/química , ADN Complementario/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Unión Proteica , Conformación Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venenos de Escorpión/química , Escorpiones , Homología de Secuencia de AminoácidoRESUMEN
This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.
Asunto(s)
Proteínas Cardiotóxicas de Elápidos/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Células CHO , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Proteínas Cardiotóxicas de Elápidos/química , Proteínas Cardiotóxicas de Elápidos/metabolismo , Cricetinae , ADN Complementario , Regulación de la Expresión Génica , Datos de Secuencia Molecular , ARN Mensajero/genética , Eliminación de Secuencia , TATA Box , Factores de Transcripción/metabolismoAsunto(s)
Blastocystis/genética , Factor 1 de Elongación Peptídica/genética , Polimorfismo Genético/genética , Animales , Blastocystis/clasificación , Infecciones por Blastocystis/parasitología , Variación Genética/genética , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , RatasRESUMEN
The elongation factor-1 alpha (EF-1alpha) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEalpha) that codes for a major part of the coding region of the EF-lalpha protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5' and 3' ends of the EF-1alpha gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5' end of B. hominis C EF-1alpha were identified. A comparative study on the deduced amino acid sequences of BEalpha of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the species. A phylogenetic reconstruction analysis with other eukaryotic EF-1alpha sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.
Asunto(s)
Blastocystis hominis/genética , Factor 1 de Elongación Peptídica/genética , Filogenia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Infecciones por Blastocystis , Blastocystis hominis/química , Blastocystis hominis/clasificación , Paseo de Cromosoma , Clonación Molecular , Cartilla de ADN/química , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics. A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein. PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure. PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance. The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics. Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa. PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin. It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice. Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antitoxinas/química , Proteínas Sanguíneas/química , Boidae , Inhibidores Enzimáticos/química , Glicoproteínas/química , Fosfolipasas A/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Venenos de Víboras/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios no Esteroideos/farmacología , Antitoxinas/genética , Antitoxinas/aislamiento & purificación , Antitoxinas/fisiología , Secuencia de Bases , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/fisiología , Catálisis , Clonación Molecular , ADN Complementario/aislamiento & purificación , Edema/prevención & control , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Glicoproteínas/fisiología , Ratones , Datos de Secuencia Molecular , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Proteínas/antagonistas & inhibidores , Proteínas/toxicidad , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Venenos de Víboras/toxicidadRESUMEN
Phospholipases A(2) (PLA(2)s) catalyzing the hydrolysis of phospholipids form a family of proteins with diverse physiological and pharmacological properties. While there have been several reports on the cloning of PLA(2) cDNAs, very few studies have been carried out on the PLA(2) genes and, most importantly, no information has been available on the gene structure and function of group I venom PLA(2). This study, on the PLA(2) gene from a spitting cobra, besides being the very first report on any venom group I PLA(2) gene, constitutes the missing link in the biology and evolution of phospholipases. The 4-kb gene consists of four exons and three introns and resembles the human pancreatic PLA(2) gene. However, the size of intron 3 in particular is much smaller than that in the pancreatic gene. Interestingly, the information for the toxic and most of the pharmacological properties of the venom PLA(2) can be attributed to the end of exon 3 and the whole of exon 4 of the gene. This functional delineation fits in well with the theory of adaptive evolution exhibited by the venom PLA(2)s. We also show that the mammalian pancreatic and elapid PLA(2)s have similar paths of evolution (probably following gene duplication) from a common ancestral gene. Venom group II phospholipases, although evolved from the same ancestor, diverged early in evolution from the group I PLA(2) genes. Intriguingly, CAT reporter gene assays and DNase 1 footprinting studies on the promoter and its deletion constructs using CHO and HepG2 cell lines identified the possible involvement of cis elements such as Sp1, AP2, gamma-IRE, and (TG)(12) repeats in the expression of the gene in a tissue-specific manner.
Asunto(s)
Venenos Elapídicos/enzimología , Fosfolipasas A/genética , Filogenia , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN , Cartilla de ADN , Elapidae , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The structure and organization of five genes responsible for the synthesis of six isoforms of short-chain alpha-neurotoxins in Pseudonaja textilis venom are reported in this paper. This also forms the first report which describes the synthesis of two neurotoxin mRNA variants from one of these genes (Pt-sntx1) as a result of alternative splicing. Each gene consists of three exons which are separated by two introns and each has a functional promoter. The promoter activity was confirmed by both CAT assay and Real-Time PCR. A transcription initiation site, two putative TATA boxes, one CCAAT box and the transcription factor binding consensus sites for AP-1, GATA-2, c/EBPb were identified in the 5' non-coding region of each gene. Phylogenetic analysis showed that these five genes from P. textilis constituted a distinct group which has evolved by gene duplication followed by accelerated evolution from an ancestral gene.
Asunto(s)
Venenos Elapídicos , Neurotoxinas/genética , Venenos de Serpiente/genética , Serpientes/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , Evolución Molecular , Genes Reporteros , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , Proteínas de Reptiles , Alineación de Secuencia , Análisis de Secuencia de ADN , Venenos de Serpiente/química , Venenos de Serpiente/metabolismoRESUMEN
Four phospholipase A(2) (PLA(2)) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA(2) activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC(50) of 9.0 nM, whereas Superbin d was found to be least effective with an IC(50) of 3.0 microM. Superbins b and c were moderately effective with IC(50) values of 0.05 and 0.5 microM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA(2) isoforms in A. superbus venom. It has also provided complete information on the precursor PLA(2). The precursor PLA(2) contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA(2) (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA(2) enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA(2) enzymes containing an extra pancreatic loop also have been identified among the isoforms.