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1.
Int J Mol Sci ; 22(23)2021 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-34884870

RESUMEN

The parasite species of genus Plasmodium causes Malaria, which remains a major global health problem due to parasite resistance to available Antimalarial drugs and increasing treatment costs. Consequently, computational prediction of new Antimalarial compounds with novel targets in the proteome of Plasmodium sp. is a very important goal for the pharmaceutical industry. We can expect that the success of the pre-clinical assay depends on the conditions of assay per se, the chemical structure of the drug, the structure of the target protein to be targeted, as well as on factors governing the expression of this protein in the proteome such as genes (Deoxyribonucleic acid, DNA) sequence and/or chromosomes structure. However, there are no reports of computational models that consider all these factors simultaneously. Some of the difficulties for this kind of analysis are the dispersion of data in different datasets, the high heterogeneity of data, etc. In this work, we analyzed three databases ChEMBL (Chemical database of the European Molecular Biology Laboratory), UniProt (Universal Protein Resource), and NCBI-GDV (National Center for Biotechnology Information-Genome Data Viewer) to achieve this goal. The ChEMBL dataset contains outcomes for 17,758 unique assays of potential Antimalarial compounds including numeric descriptors (variables) for the structure of compounds as well as a huge amount of information about the conditions of assays. The NCBI-GDV and UniProt datasets include the sequence of genes, proteins, and their functions. In addition, we also created two partitions (cassayj = caj and cdataj = cdj) of categorical variables from theChEMBL dataset. These partitions contain variables that encode information about experimental conditions of preclinical assays (caj) or about the nature and quality of data (cdj). These categorical variables include information about 22 parameters of biological activity (ca0), 28 target proteins (ca1), and 9 organisms of assay (ca2), etc. We also created another partition of (cprotj = cpj) including categorical variables with biological information about the target proteins, genes, and chromosomes. These variables cover32 genes (cp0), 10 chromosomes (cp1), gene orientation (cp2), and 31 protein functions (cp3). We used a Perturbation-Theory Machine Learning Information Fusion (IFPTML) algorithm to map all this information (from three databases) into and train a predictive model. Shannon's entropy measure Shk (numerical variables) was used to quantify the information about the structure of drugs, protein sequences, gene sequences, and chromosomes in the same information scale. Perturbation Theory Operators (PTOs) with the form of Moving Average (MA) operators have been used to quantify perturbations (deviations) in the structural variables with respect to their expected values for different subsets (partitions) of categorical variables. We obtained three IFPTML models using General Discriminant Analysis (GDA), Classification Tree with Univariate Splits (CTUS), and Classification Tree with Linear Combinations (CTLC). The IFPTML-CTLC presented the better performance with Sensitivity Sn(%) = 83.6/85.1, and Specificity Sp(%) = 89.8/89.7 for training/validation sets, respectively. This model could become a useful tool for the optimization of preclinical assays of new Antimalarial compounds vs. different proteins in the proteome of Plasmodium.


Asunto(s)
Antimaláricos/farmacología , Descubrimiento de Drogas/métodos , Aprendizaje Automático , Plasmodium falciparum/genética , Algoritmos , Antimaláricos/química , Bases de Datos Farmacéuticas , Evaluación Preclínica de Medicamentos , Genoma de Protozoos , Cadenas de Markov , Modelos Teóricos , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Reproducibilidad de los Resultados
2.
Mol Microbiol ; 110(4): 562-575, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30194883

RESUMEN

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.


Asunto(s)
Proteínas de Dominio MADS/genética , Micelio/metabolismo , Phytophthora infestans/crecimiento & desarrollo , Phytophthora infestans/genética , Esporas Protozoarias/crecimiento & desarrollo , Esporas Protozoarias/genética , Regulación de la Expresión Génica , Silenciador del Gen , Genoma de Protozoos/genética , Phytophthora infestans/metabolismo , Enfermedades de las Plantas/parasitología , Solanum tuberosum/parasitología , Esporas Protozoarias/metabolismo , Factores de Transcripción/metabolismo
3.
Mol Plant Microbe Interact ; 31(12): 1227-1229, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29969057

RESUMEN

The Plasmodiophorida (Phytomyxea, Rhizaria) are a group of protists that infect plants. Of this group, Spongospora subterranea causes major problems for the potato industry by causing powdery scab and root galling of potatoes and as vector for the Potato mop-top virus (PMTV) (genus Pomovirus, family Virgaviridae). A single tuber isolate (SSUBK13) of this uncultivable protist was used to generate DNA for Illumina sequencing. The data were assembled to a draft genome of 28.08 Mb consisting of 2,340 contigs and an L50 of 280. A total of 10,778 genes were predicted and 93% of the BUSCO genes were detected. The presented genome assembly is only the second genome of a plasmodiophorid. The data will accelerate functional genomics to study poorly understood interaction of plasmodiophorids and their hosts.


Asunto(s)
Genoma de Protozoos/genética , Enfermedades de las Plantas/parasitología , Plasmodiophorida/genética , Solanum tuberosum/parasitología
4.
Genome Biol ; 17(1): 124, 2016 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-27286965

RESUMEN

BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.


Asunto(s)
Genoma de Protozoos , Enfermedades de las Plantas/parasitología , Solanum tuberosum/parasitología , Tylenchoidea/genética , Tylenchoidea/patogenicidad , Animales , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Transferencia de Gen Horizontal , Islas Genómicas , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Estadios del Ciclo de Vida , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Sitios de Empalme de ARN , Empalme del ARN , Transcriptoma , Tylenchoidea/crecimiento & desarrollo , Virulencia/genética
5.
Methods Mol Biol ; 1403: 603-22, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27076156

RESUMEN

Transfection technology is an important tool in the investigation of gene function and the modulation of gene expression, thereby contributing to the advancement of basic cellular research, drug discovery, and target validation. Creation of the mutant cells through gene disruption and exogenous protein expression with noticeable phenotype like reporter genes are among other key applications. In this chapter, protocols for generating recombinant Leishmania expressing EGFP or EGFP-Luciferase and their applications are given in detail.


Asunto(s)
Genoma de Protozoos/genética , Leishmania/genética , Leishmania/inmunología , Vacunas Antiprotozoos/inmunología , Transfección/métodos , Animales , Clonación Molecular , Evaluación Preclínica de Medicamentos , Genotipo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Imagen Óptica
6.
Artículo en Inglés | MEDLINE | ID: mdl-24438302

RESUMEN

Spongospora subterranea is a soil-borne obligate parasite responsible for potato powdery scab disease. S. subterranea is a member of the order Plasmodiophorida, a protist taxa that is related to Cercozoa and Foraminifera but the fine details of these relationships remain unresolved. Currently there is only one available complete mtDNA sequence of a cercozoan, Bigelowiella natans. In this work, the mitochondrial sequence of a S. subterranea isolate infecting an Andean variety of S. tuberosum ssp. andigena (Diacol-Capiro) is presented. The mtDNA codes for 16 proteins of the respiratory chain, 11 ribosomal proteins, 3 ribosomal RNAs, 24 tRNAs, a RNA processing RNaseP, a RNA-directed polymerase, and two proteins of unknown function. This is the first report of a mtDNA genome sequence from a plasmodiophorid and will be useful in clarifying the phylogenetic relationship of this group to other members in the supergroup Rhizaria once more mtDNA sequences are available.


Asunto(s)
Genoma Mitocondrial , Genoma de Protozoos , Plasmodiophorida/genética , Solanum tuberosum/parasitología , Composición de Base/genética , Emparejamiento Base/genética , Secuencia de Bases , ADN Circular/genética , ADN Mitocondrial/genética , Sistemas de Lectura Abierta/genética , Enfermedades de las Plantas/parasitología , ARN de Transferencia/genética
7.
Sci Rep ; 5: 8771, 2015 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-25740547

RESUMEN

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host-pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento , Kinetoplastida/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria/métodos , Bibliotecas de Moléculas Pequeñas , Animales , Antiprotozoarios/farmacología , Línea Celular , Genoma de Protozoos , Humanos , Kinetoplastida/clasificación , Kinetoplastida/genética , Ratones , Filogenia
8.
J Biotechnol ; 202: 135-45, 2015 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-25527385

RESUMEN

Euglenoid flagellates are mainly fresh water protists growing in highly diverse environments making them well-suited for a multiplicity of biotechnology applications. Phototrophic euglenids possesses complex chloroplasts of green algal origin bounded by three membranes. Euglena nuclear and plastid genome organization, gene structure and gene expression are distinctly different from other organisms. Our observations on the model organism Euglena gracilis indicate that transcription of both the plastid and nuclear genome is insensitive to environmental changes and that gene expression is regulated mainly at the post-transcriptional level. Euglena plastids have been proposed as a site for the production of proteins and value added metabolites of biotechnological interest. Euglena has been shown to be a suitable protist species to be used for production of several compounds that are used in the production of cosmeceuticals and nutraceuticals, such as α-tocopherol, wax esters, polyunsaturated fatty acids, biotin and tyrosine. The storage polysaccharide, paramylon, has immunostimulatory properties and has shown a promise for biomaterials production. Euglena biomass can be used as a nutritional supplement in aquaculture and in animal feed. Diverse applications of Euglena in environmental biotechnology include ecotoxicological risk assessment, heavy metal bioremediation, bioremediation of industrial wastewater and contaminated water.


Asunto(s)
Euglena/crecimiento & desarrollo , Euglena/metabolismo , Genoma de Protozoos , Biodegradación Ambiental , Biotecnología , Núcleo Celular/genética , Cloroplastos/genética , Cosmecéuticos/metabolismo , Suplementos Dietéticos , Euglena/genética , Modelos Biológicos , Filogenia
9.
Chembiochem ; 15(13): 1920-30, 2014 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-25111632

RESUMEN

Malaria, an infectious disease caused by eukaryotic parasites of the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1358 small-molecule kinase inhibitors to evaluate the role of both the human and the malaria kinomes in Plasmodium infection of liver cells, the parasites' obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood-stage malaria. Most of the screening hits inhibited both liver- and blood-stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests that several kinases are essential to malaria parasites, including cyclin-dependent kinases (CDKs), glycogen synthase kinases, and phosphoinositide-3-kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual-stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.


Asunto(s)
Genoma de Protozoos/genética , Malaria/genética , Plasmodium/genética , Proteínas Quinasas/genética , Animales , Antimaláricos/química , Biología Computacional , Evaluación Preclínica de Medicamentos , Humanos , Hígado/parasitología , Malaria/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Plasmodium/enzimología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas
10.
Transfusion ; 54(10): 2419-24, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24801273

RESUMEN

BACKGROUND: At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy. STUDY DESIGN AND METHODS: Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. RESULTS: Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 10(3) -8.5 × 10(4) copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. CONCLUSIONS: Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.


Asunto(s)
Donantes de Sangre , Emigrantes e Inmigrantes , Genoma de Protozoos , Malaria/parasitología , Plasmodium/genética , Adulto , Anciano , Anticuerpos Antiprotozoarios/sangre , Donantes de Sangre/estadística & datos numéricos , Transfusión de Sangre Autóloga/estadística & datos numéricos , Emigrantes e Inmigrantes/estadística & datos numéricos , Femenino , Variación Genética , Humanos , Italia/epidemiología , Malaria/sangre , Malaria/genética , Malaria/transmisión , Masculino , Persona de Mediana Edad , Plasmodium/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Análisis de Secuencia de ADN , Adulto Joven
11.
Exp Parasitol ; 135(3): 546-50, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24055215

RESUMEN

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.


Asunto(s)
Vacunas contra la Malaria/normas , Malaria/prevención & control , Plasmodium berghei/inmunología , Vacunas de ADN/normas , Animales , Biolística , Mapeo Cromosómico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Genoma de Protozoos/genética , Genoma de Protozoos/inmunología , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Plásmidos/genética , Plásmidos/inmunología , Plasmodium berghei/genética , Ratas , Ratas Wistar , Organismos Libres de Patógenos Específicos
12.
Curr Biol ; 23(16): 1541-7, 2013 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-23891116

RESUMEN

Mikrocytos mackini is an intracellular protistan parasite of oysters whose position in the phylogenetic tree of eukaryotes has been a mystery for many years [1,2]. M. mackini is difficult to isolate, has not been cultured, and has no defining morphological feature. Furthermore, its only phylogenetic marker that has been successfully sequenced to date (the small subunit ribosomal RNA) is highly divergent and has failed to resolve its evolutionary position [2]. M. mackini is also one of the few eukaryotes that lacks mitochondria [1], making both its phylogenetic position and comparative analysis of mitochondrial function particularly important. Here, we have obtained transcriptomic data for M. mackini from enriched isolates and constructed a 119-gene phylogenomic data set. M. mackini proved to be among the fastest-evolving eukaryote lineages known to date, but, nevertheless, our analysis robustly placed it within Rhizaria. Searching the transcriptome for genetic evidence of a mitochondrion-related organelle (MRO) revealed only four mitochondrion-derived genes: IscS, IscU, mtHsp70, and FdxR. Interestingly, all four genes are involved in iron-sulfur cluster formation, a biochemical pathway common to other highly reduced "mitosomes" in unrelated MRO-containing lineages [7]. This is the first evidence of MRO in Rhizaria, and it suggests the parallel evolution of mitochondria to mitosomes in this supergroup.


Asunto(s)
ADN Protozoario/genética , Genoma de Protozoos , Filogenia , Rhizaria/genética , Animales , ADN Complementario/genética , ADN Complementario/metabolismo , ADN Protozoario/metabolismo , Evolución Molecular , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Ostreidae/parasitología , Rhizaria/clasificación , Rhizaria/metabolismo , Rhizaria/fisiología , Análisis de Secuencia de ADN
13.
Mol Cell Proteomics ; 12(2): 302-11, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23152538

RESUMEN

Arginine (arg) methylation is a widespread posttranslational modification of proteins that impacts numerous cellular processes such as chromatin remodeling, RNA processing, DNA repair, and cell signaling. Known arg methylproteins arise mostly from yeast and mammals, and are almost exclusively nuclear and cytoplasmic. Trypanosoma brucei is an early branching eukaryote whose genome encodes five putative protein arg methyltransferases, and thus likely contains a plethora of arg methylproteins. Additionally, trypanosomes and related organisms possess a unique mitochondrion that undergoes dramatic developmental regulation and uses novel RNA editing and mitochondrial DNA replication mechanisms. Here, we performed a global mass spectrometric analysis of the T. brucei mitochondrion to identify new arg methylproteins in this medically relevant parasite. Enabling factors of this work are use of a combination digestion with two orthogonal enzymes, an efficient offline two dimensional chromatography separation, and high-resolution mass spectrometry analysis with two complementary activations. This approach led to the comprehensive, sensitive and confident identification and localization of methylarg at a proteome level. We identified 167 arg methylproteins with wide-ranging functions including metabolism, transport, chaperoning, RNA processing, translation, and DNA replication. Our data suggest that arg methylproteins in trypanosome mitochondria possess both trypanosome-specific and evolutionarily conserved modifications, depending on the protein targeted. This study is the first comprehensive analysis of mitochondrial arg methylation in any organism, and represents a significant advance in our knowledge of the range of arg methylproteins and their sites of modification. Moreover, these studies establish T. brucei as a model organism for the study of posttranslational modifications.


Asunto(s)
Genoma de Protozoos , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Arginina/metabolismo , Cromatografía Líquida de Alta Presión , Replicación del ADN , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometría de Masas , Metilación , Mitocondrias/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Transporte de Proteínas , Proteína-Arginina N-Metiltransferasas/genética , Proteolisis , Proteoma/genética , Proteínas Protozoarias/genética , Edición de ARN , Trypanosoma brucei brucei/genética
14.
J Mol Evol ; 73(3-4): 70-3, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22037698

RESUMEN

In a process similar to exon splicing, ciliates use DNA splicing to produce a new somatic macronuclear genome from their germline micronuclear genome after sexual reproduction. This extra layer of DNA rearrangement permits novel mechanisms to create genetic complexity during both evolution and development. Here we describe a chimeric macronuclear chromosome in Oxytricha trifallax constructed from two smaller macronuclear chromosomes. To determine how the chimera was generated, we cloned and sequenced the corresponding germline loci. The chimera derives from a novel locus in the micronucleus that arose by partial duplication of the loci for the two smaller chromosomes. This suggests that an exon shuffling-like process, which we call MDS shuffling, enables ciliates to generate novel genetic material and gene products using different combinations of genomic DNA segments.


Asunto(s)
Quimera/genética , Duplicación Cromosómica , Oxytricha/genética , Alelos , Secuencia de Bases , Variaciones en el Número de Copia de ADN , Genes Protozoarios , Genoma de Protozoos , Micronúcleo Germinal/genética , Datos de Secuencia Molecular , Cebollas , Recombinación Genética
15.
PLoS One ; 3(6): e2484, 2008 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-18575593

RESUMEN

Mutations in PfCRT (Plasmodium falciparum chloroquine-resistant transporter), particularly the substitution at amino acid position 76, confer chloroquine (CQ) resistance in P. falciparum. Point mutations in the homolog of the mammalian multidrug resistance gene (pfmdr1) can also modulate the levels of CQ response. Moreover, parasites with the same pfcrt and pfmdr1 alleles exhibit a wide range of drug sensitivity, suggesting that additional genes contribute to levels of CQ resistance (CQR). Reemergence of CQ sensitive parasites after cessation of CQ use indicates that changes in PfCRT are deleterious to the parasite. Some CQR parasites, however, persist in the field and grow well in culture, which may reflect adaptive changes in the parasite genome to compensate for the mutations in PfCRT. Using three isogenic clones that have different drug resistance profiles corresponding to unique mutations in the pfcrt gene (106/1(K76), 106/1(76I), and 106/(76I-352K)), we investigated changes in gene expression in these parasites grown with and without CQ. We also conducted hybridizations of genomic DNA to identify copy number (CN) changes in parasite genes. RNA transcript levels from 45 genes were significantly altered in one or both mutants relative to the parent line, 106/1(K76). Most of the up-regulated genes are involved in invasion, cell growth and development, signal transduction, and transport activities. Of particular interest are genes encoding proteins involved in transport and/or regulation of cytoplasmic or compartmental pH such as the V-type H(+) pumping pyrophosphatase 2 (PfVP2), Ca(2+)/H(+) antiporter VCX1, a putative drug transporter and CN changes in pfmdr1. These changes may represent adaptations to altered functionality of PfCRT, a predicted member of drug/metabolite transporter superfamily found on the parasite food vacuole (FV) membrane. Further investigation of these genes may shed light on how the parasite compensates for functional changes accompanying drug resistance mutations in a gene coding for a membrane/drug transporter.


Asunto(s)
Genoma de Protozoos , Proteínas de Transporte de Membrana/genética , Mutación , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Animales , Antimaláricos/farmacología , Cloroquina/farmacología , Evaluación Preclínica de Medicamentos , Eliminación de Gen , Perfilación de la Expresión Génica , Concentración de Iones de Hidrógeno , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba
16.
Trends Parasitol ; 24(4): 184-9, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18329342

RESUMEN

Tyzzer discovered the genus Cryptosporidium a century ago, and for almost 70 years cryptosporidiosis was regarded as an infrequent and insignificant infection that occurred in the intestines of vertebrates and caused little or no disease. Its association with gastrointestinal illness in humans and animals was recognized only in the early 1980s. Over the next 25 years, information was generated on the disease's epidemiology, biology, cultivation, taxonomy and development of molecular tools. Milestones include: (i) recognition in 1980 of cryptosporidiosis as an acute enteric disease; (ii) its emergence as a chronic opportunistic infection that complicates AIDS; (iii) acknowledgement of impact on the water industry once it was shown to be waterborne; and (iv) study of Cryptosporidium genomics.


Asunto(s)
Antiprotozoarios/uso terapéutico , Criptosporidiosis , Cryptosporidium/patogenicidad , Genoma de Protozoos , Agua/parasitología , Animales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/epidemiología , Criptosporidiosis/inmunología , Criptosporidiosis/parasitología , Cryptosporidium/genética , Cryptosporidium/crecimiento & desarrollo , Cryptosporidium/inmunología , Diarrea/parasitología , Humanos , Huésped Inmunocomprometido , Estudios Retrospectivos
17.
Exp Parasitol ; 117(1): 87-92, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17475254

RESUMEN

A genomic approach was taken to study the effect of chloroquine (CQ) on Plasmodium falciparum cultures in multiple cell states, following short and long exposures to drug at varying concentrations. Six hundred genes from numerous functional groups were responsive to CQ amongst all cell states assayed in a micro-array analysis; however, the amplitude of fold-change was low in the majority of cases. Moreover, alterations in specific, functionally related cascades could not be discerned, leading us to believe there is no single signature response to CQ at the transcript level in P. falciparum. Instead, cell cycle changes appear to have a more pronounced effect on gene expression; only a fraction of the drug responsive loci (approximately 5%) were shared between two separate starting cultures that varied in staging profile in the current study, as well as a previous published analysis using SAGE technology [Gunasekera, A.M., Patankar, S., Schug, J., Eisen, G.,Wirth, D.F., 2003. Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum. Molecular Microbiology 50, 1229-1239]. These findings are important to report, given the striking contrast to similar studies in other model eukaryotic organisms.


Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Genoma de Protozoos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , ARN Protozoario/efectos de los fármacos , Animales , Northern Blotting , Regulación de la Expresión Génica/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Protozoario/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos
18.
Trop Med Int Health ; 12(2): 251-9, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17300633

RESUMEN

OBJECTIVES: To compare the efficacy and tolerability of dihydroartemisinin-piperaquine (DHA-PQP) with that of a 3-day regimen of mefloquine and artesunate (MAS3) for the treatment of uncomplicated falciparum malaria in Cambodia. METHOD: Randomized open-label non-inferiority study over 64 days. RESULTS: Four hundred and sixty-four patients were included in the study. The polymerase chain reaction genotyping-adjusted cure rates on day 63 were 97.5% (95% confidence interval, CI, 93.8-99.3) for DHA-PQP and 97.5% (95% CI, 93.8-99.3) for MAS3, P = 1. There were no serious adverse events, but significantly more episodes of vomiting (P = 0.03), dizziness (P = 0.002), palpitations (P = 0.04), and sleep disorders (P = 0.03) reported in the MAS3 treatment group, consistent with the side-effect profile of mefloquine. CONCLUSIONS: DHA-PQP was as efficacious as MAS3, but much better tolerated, making it more appropriate for use in a routine programme setting. This highly efficacious, safe and more affordable fixed-dose combination could become the treatment of choice for Plasmodium falciparum malaria in Cambodia.


Asunto(s)
Antimaláricos/uso terapéutico , Artemisininas/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Quinolinas/uso terapéutico , Sesquiterpenos/uso terapéutico , Adolescente , Adulto , Anciano , Anemia/complicaciones , Anemia/epidemiología , Antimaláricos/efectos adversos , Artemisininas/efectos adversos , Artesunato , Cambodia/epidemiología , Niño , Preescolar , Quimioterapia Combinada , Femenino , Genoma de Protozoos , Humanos , Lactante , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Malaria Vivax/tratamiento farmacológico , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Masculino , Mefloquina/uso terapéutico , Persona de Mediana Edad , Quinolinas/efectos adversos , Recurrencia , Sesquiterpenos/efectos adversos , Resultado del Tratamiento
19.
Proc Natl Acad Sci U S A ; 103(39): 14548-53, 2006 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-16983082

RESUMEN

With >1 million deaths annually, mostly among children in sub-Saharan Africa, malaria poses one of the most critical challenges in medicine today. Although introduction of the artemisinin class of antimalarial drugs has offered a temporary solution to the problem of drug resistance, new antimalarial drugs are needed to ensure effective control of the disease in the future. Herein, we have investigated members of the methionine aminopeptidase family as potential antimalarial targets. The Plasmodium falciparum methionine aminopeptidase 1b (PfMetAP1b), one of four MetAP proteins encoded in the P. falciparum genome, was cloned, overexpressed, purified, and used to screen a 175,000-compound library for inhibitors. A family of structurally related inhibitors containing a 2-(2-pyridinyl)-pyrimidine core was identified. Structure/activity studies led to the identification of a potent PfMetAP1b inhibitor, XC11, with an IC(50) of 112 nM. XC11 was highly selective for PfMetAP1b and did not exhibit significant cytotoxicity against primary human fibroblasts. Most importantly, XC11 inhibited the proliferation of P. falciparum strains 3D7 [chloroquine (CQ)-sensitive] and Dd2 (multidrug-resistant) in vitro and is active in mouse malaria models for both CQ-sensitive and CQ-resistant strains. These results suggest that PfMetAP1b is a promising target and XC11 is an important lead compound for the development of novel antimalarial drugs.


Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Antimaláricos/metabolismo , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/química , Animales , Antimaláricos/química , Antimaláricos/farmacología , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/parasitología , Genoma de Protozoos/genética , Humanos , Metionil Aminopeptidasas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Parásitos/efectos de los fármacos , Parásitos/crecimiento & desarrollo , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos
20.
Nucleic Acids Res ; 34(14): 4012-24, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16914442

RESUMEN

Proteins containing the 21st amino acid selenocysteine (Sec) are present in the three domains of life. However, within lower eukaryotes, particularly parasitic protists, the dependence on the trace element selenium is variable as many organisms lost the ability to utilize Sec. Herein, we analyzed the genomes of Trypanosoma and Leishmania for the presence of genes coding for Sec-containing proteins. The selenoproteomes of these flagellated protozoa have three selenoproteins, including distant homologs of mammalian SelK and SelT, and a novel multidomain selenoprotein designated SelTryp. In SelK and SelTryp, Sec is near the C-terminus, and in all three selenoproteins, it is within predicted redox motifs. SelTryp has neither Sec- nor cysteine-containing homologs in the human host and appears to be a Kinetoplastida-specific protein. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with 75Se. In addition, genes coding for components of the Sec insertion machinery were identified in the Kinetoplastida genomes. Finally, we found that Trypanosoma brucei brucei cells were highly sensitive to auranofin, a compound that specifically targets selenoproteins. Overall, these data establish that Trypanosoma, Leishmania and likely other Kinetoplastida utilize and depend on the trace element selenium, and this dependence is due to occurrence of selenium in at least three selenoproteins.


Asunto(s)
Genoma de Protozoos , Leishmania/genética , Proteínas Protozoarias/genética , Selenio/metabolismo , Selenoproteínas/genética , Trypanosoma/genética , Secuencia de Aminoácidos , Animales , Auranofina/farmacología , Secuencia de Bases , Biología Computacional , Genómica , Leishmania/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteoma/genética , Proteínas Protozoarias/química , ARN Protozoario/química , ARN de Transferencia/clasificación , Selenoproteínas/química , Alineación de Secuencia , Trypanosoma/efectos de los fármacos , Trypanosoma/metabolismo
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