RESUMEN
Se realizó un estudio cualitativo, etnográfico para conocer y analizar la percepción de los pacientes extranjeros respecto de su vínculo con el sistema de salud en Argentina, considerando la cultura de origen y proceso migratorio mediante trece entrevistas semiestructuradas. Se incluyeron pacientes extranjeros mayores de 18 años que consultaron a un Centro de Atención Primaria de la Salud (CeMAP) de un agente del subsitema sanitario de la seguridad social argentina. Los mismos fueron seleccionados de manera intencional y por conveniencia. Las transcripciones se analizaron según la teoría fundamentada. En las entrevistas se destacó una relación médico-paciente más estrecha en comparación con la del país de origen, destacándose la calidez, compromiso y mayor comunicación con el binomio paciente-familia, siendo notables estas diferencias en la etapa del embarazo y en el ámbito de la salud sexual y reproductiva. Se han hallado diferencias sustanciales con el país de origen en temáticas como control prenatal y acompañamiento del parto, alimentación, medicinas tradicionales, accesibilidad al sistema sanitario, siendo influyente el tiempo de residencia en Argentina para lograr el empoderamiento en relación con el sistema de salud. Esto nos estimula a seguir trabajando en la cultura y proceso migratorio de los pacientes, explorar su cosmovisión, para propiciar un enfoque intercultural que permita adquirir herramientas para la atención de dicha población (AU)
A qualitative, ethnographic study was carried out to identify and analyze the perception and beliefs of foreign patients, regarding their experiences in Argentina Ìs healthcare system, taking into consideration their culture of origin and migratory process through semi-structured interviews. It're included foreign patients over 18 years of age who consulted at a Primary Health Care Center (CeMAP) of an agent of the Argentine social security health system. They were selected intentionally and for convenience. Thirteen semi-structured in-depth interviews were recorded and the transcripts were analyzed according to the Fundamental Theory. In the interviews, a closer patient-physician relationship stood out when compared with the country of origin, highlighting the warmth, commitment and greater communication with the patient and its family, these differences were more notable during prenatal, reproductive and sexual care. Substantial differences have been found with the country of origin in topics such as prenatal post-partum care, nutrition, traditional medicines, and accessibility to the health system. There was a positive impact of length of residency in Argentina on patient empowerment within the healthcare system. This is an estimate to continue working on the culture and the migration process of patients, to explore their worldview, to propose an intercultural approach that allows us to acquire tools for the care of this population (AU)
Asunto(s)
Humanos , Adulto , Relaciones Médico-Paciente , Atención Primaria de Salud , Migración Humana , Asistencia Sanitaria Culturalmente Competente/etnología , Accesibilidad a los Servicios de Salud , Paraguay , Perú , Seguridad Social , Bolivia , Chile , Colombia , República Dominicana , MéxicoRESUMEN
We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodiumberghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1alphaa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC(50) values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1alphaa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.
Asunto(s)
Antimaláricos/farmacología , Luciferasas/sangre , Malaria/parasitología , Parasitemia/diagnóstico , Plasmodium berghei/enzimología , Animales , Animales Modificados Genéticamente , Luciferasas/genética , Malaria/tratamiento farmacológico , Ratones , Plasmodium berghei/genéticaRESUMEN
This study used behavioral and electrophysiological techniques to examine age-related changes in the feeding behavior and chemosensory processing in the pond snail, Lymnaea stagnalis. Increasing age was associated with a 50% decrease in long-term food consumption. Analysis of short-term sucrose-evoked feeding bouts showed an age-related increase in the number of animals that failed to respond to the stimulus. Of the animals that did respond increasing age was associated with a decrease in the number of sucrose-evoked bites and a increase in the duration of the swallow phase. These changes were observed with both 0.01 and 0.05M sucrose stimuli but were not seen when 0.1M sucrose was used as the stimulus. Electrophysiological analysis of the chemosensory pathway in semi-intact lip-CNS preparations failed to demonstrate a significant change in the neuronal information entering the cerebral ganglia from the lips via the median lip nerve, but did demonstrate an age-related deficit in the neuronal output from the cerebral ganglia. This deficit was also dependent on the sucrose concentration and mirrored the concentration-dependent changes in feeding behavior. In summary, aging appeared to affect central but not peripheral processing of chemosensory information and suggests that this deficit contributes to the age-related changes in feeding behavior.
Asunto(s)
Envejecimiento/fisiología , Células Quimiorreceptoras/fisiología , Conducta Alimentaria/fisiología , Lymnaea/fisiología , Procesamiento Proteico-Postraduccional , Animales , Células Quimiorreceptoras/metabolismo , Ingestión de Alimentos/fisiología , Red Nerviosa/fisiologíaRESUMEN
We recently, characterized a novel peptide of the egg-laying controlling caudodorsal cells (CDC) of Lymnaea stagnalis. Here, we show that the novel peptide has autoinhibitory actions and its expression is significantly up-regulated in reproductively senescent animals. Intracellular recordings show that when bath-applied to the central nervous system in vitro, the peptide reduces the depolarizing after potential (DAP) in CDCs without affecting the action potential-threshold and -amplitude and the resting membrane potential. Moreover, peptide application can terminate an ongoing after discharge in the CDCs or, when electrical stimulation fails to induce an after discharge, can terminate the long-lasting depolarization. Semiquantitative peptide profiling by mass spectrometry demonstrated correct processing and targeting of peptides in the CDC somata and axon terminals of reproductively senescent animals. Interestingly, the level of the autoinhibitory peptide was selectively increased in the CDCs of reproductively senescent animals. Our results indicate that a shift in balance between excitatory and inhibitory auto-transmitter peptides in the CDC system of old non-egg-laying animals, plays a role in after discharge failure in CDCs of reproductively senescent Lymnaea.
Asunto(s)
Envejecimiento/fisiología , Lymnaea/fisiología , Neuronas/efectos de los fármacos , Neuropéptidos/fisiología , Reproducción/fisiología , Potenciales de Acción/fisiología , Animales , Estimulación Eléctrica , Electrofisiología , Neuropéptidos/síntesis química , Neuropéptidos/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regulación hacia Arriba/fisiologíaAsunto(s)
Vacunas contra la Malaria , Malaria/inmunología , Plasmodium berghei/inmunología , Esporozoítos/inmunología , Animales , Apoptosis , Genes Protozoarios , Ingeniería Genética , Hepatocitos/parasitología , Hepatocitos/fisiología , Humanos , Esquemas de Inmunización , Inmunización Secundaria , Malaria/prevención & control , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Ratones , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Esporozoítos/genética , Esporozoítos/crecimiento & desarrollo , Vacunación , Vacunas Atenuadas/inmunologíaRESUMEN
During brain aging neuronal degradation occurs. In some neurons this may result in degeneration and cell death, still other neurons may survive and maintain their basic properties. The present study deals with survival of the egg-laying controlling neuroendocrine caudodorsal cells (CDCs) during reproductive senescence of the pond snail Lymnaea stagnalis. In senescent animals CDCs exhibited reduced branching patterns but still maintained their electrophysiological characteristics. In the isolated CNS the cells could still respond with an afterdischarge upon electrical stimulation. After an extended period of no egg laying of Lymnaea CDCs failed to exhibit an afterdischarge. In senescent CDCs that failed an afterdischarge, discharge activity could be restored by exposure to peptides released by CDCs from reproductive animals. Moreover, raising the intracellular cAMP level could induce discharge activity in CDCs with afterdischarge failure. Discharge activity also occurred during depolarization of senescent CDCs by exposure of the cells to saline with a high potassium concentration. These results indicate that in senescent CDCs the pacemaking mechanism of the afterdischarge is still intact but that the initial activation fails. Chemical (auto)transmission of CDCs in such animals was indeed reduced as indicated by the small amplitude of the depolarizing afterpotential (DAP) induced by electrical stimulation. Interestingly, CDCs of senescent animals contained a relative large amount of a particular small peptide. The artificially synthesized peptide appeared to suppress DAP induction in CDCs. Possibly, release of the peptide contributes to the prevention of afterdischarge induction in senescent CDCs. The results so far indicate that in senescent Lymnaea neurons electrophysiological functions persist even after long periods of inactivity and severe morphological reduction.
Asunto(s)
Envejecimiento/fisiología , Sistema Nervioso Central/anatomía & histología , Sistemas Neurosecretores/fisiología , Oviposición/fisiología , Animales , Sistema Nervioso Central/fisiología , AMP Cíclico/metabolismo , Electrofisiología , Femenino , Moluscos , Neuronas/metabolismo , Sistemas Neurosecretores/citología , Péptidos/química , Potasio/química , Potasio/metabolismo , Reproducción/fisiología , Factores de TiempoRESUMEN
Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.
Asunto(s)
Anopheles/parasitología , Malaria/transmisión , Plasmodium/fisiología , Aedes/parasitología , Animales , Vectores de Enfermedades , Femenino , Interacciones Huésped-Parásitos , Humanos , Malaria/parasitología , Oocitos , Plasmodium berghei/fisiología , Plasmodium gallinaceum/fisiología , Glándulas Salivales/parasitología , Especificidad de la EspecieRESUMEN
Recovery after crush of neuroendocrine caudodorsal cells (CDCs) in the aging brain of the mollusc Lymnaea stagnalis, was determined as a measure of neuronal plasticity. Neuronal plasticity was determined in differently aged animals containing intact (young: 170 days) or morphologically and physiologically degraded (middle-aged: 305 days and old: 443 days) CDCs. Branching patterns and electrical and chemical connectivity and afterdischarge activity of CDCs were studied. Immediately after crush, electrical coupling and chemical transmission were absent. In all age groups partial recovery occurred within about 20 days. CDCs in old animals exhibited restricted recovery of electrical coupling and enhanced recovery of chemical transmission. In young and middle-aged animals normal afterdischarges occurred from day 8 on. In old animals abnormal afterdischarges occurred starting at day 0, becoming normal by day 12 after crush. Recovery of CDC branching was partial in all age groups. It is concluded that in the aging brain recovery of CDCs after injury does occur but is differentially restricted. Our results suggest that senescent degraded neurons still possess a considerable degree of plasticity.
Asunto(s)
Envejecimiento/patología , Lesiones Encefálicas/patología , Lymnaea/fisiología , Neuronas/patología , Sistemas Neurosecretores/patología , Animales , Electrofisiología , Colorantes Fluorescentes , Isoquinolinas , Potenciales de la Membrana/fisiología , Compresión Nerviosa , Plasticidad Neuronal/fisiologíaRESUMEN
Comparative genomics allows inferences to be drawn about the coding potential of related genomes, and the evolutionary forces that have influenced genome organisation. Early comparisons have indicated that there is significant synteny (conserved physical association of genes) between the human parasite Plasmodium falciparum and the malaria parasites of rodents, such as Plasmodium berghei. The various Plasmodium genome initiatives have now provided the opportunity to perform comparative genomics within different species of malaria parasites in more detail, allowing the discovery of orthologues and paralogues of less well conserved genes and addressing questions of conservation, evolution and structure of multi-gene families. A remarkable level of conservation is being revealed, illustrated here by a comparison of members of one of the first conserved gene families to emerge from the sequencing initiatives, the P48/45 gene family. We have identified two additional members in this family, Pf36p and Pfs38, and shown that all members are conserved in P. falciparum and P. berghei, opening the way for functional analyses in the latter more accessible rodent malaria model. In addition, it has been shown that direct comparison of a 13.6 kb contig of a chromosome of P. berghei and the orthologous region in P. falciparum reveals an unexpected high level of conservation of gene organisation and complexity. The results of this comparison highlight the value of a comparative approach to elucidate the gene content of complex loci and improve its annotation
Asunto(s)
Genes Protozoarios , Genómica , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Genoma de Protozoos , Humanos , Malaria/parasitología , Familia de Multigenes , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.
Asunto(s)
Antígenos de Protozoos/fisiología , Antígenos de Superficie/fisiología , Plasmodium berghei/crecimiento & desarrollo , Proteínas Protozoarias , Animales , Anopheles/parasitología , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Sistema Digestivo/parasitología , Epitelio , Plasmodium berghei/genéticaRESUMEN
A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.
Asunto(s)
Secuencia Conservada/genética , Exones/genética , Orden Génico/genética , Genes Protozoarios/genética , Intrones/genética , Plasmodium berghei/genética , Plasmodium falciparum/genética , Empalme Alternativo/genética , Animales , Southern Blotting , Cromosomas/genética , Clonación Molecular , Biología Computacional , Mapeo Contig , Bases de Datos como Asunto , Genes Sobrepuestos/genética , Células Germinativas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Plasmodium berghei/citología , Plasmodium falciparum/citología , ARN Protozoario/análisis , ARN Protozoario/genética , Análisis de Secuencia de ADN , Transcripción Genética/genéticaRESUMEN
Excitability changes during reproductive senescence were investigated in the neurosecretory caudodorsal cells (CDCs) that control egg laying in the mollusc Lymnaea stagnalis. CDCs in the isolated central nervous system (CNS) were exposed to different discharge inducing treatments. Senescent CDCs (of animals 8 weeks after laying their last egg mass) and inhibited (I-) state CDCs (of egg-laying (EL) animals) were used. We showed that senescent and I-state CDCs closely resemble each other electrophysiologically. Electrical stimulation did not induce an afterdischarge in either type of CDC but exposure to release products of CDCs from EL animals or to saline with high potassium concentration did induce discharge activity. Also, 8-chlorophenylthio (8-CPT)-cAMP (10(-5) M) induced discharge activity. Exposure to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (10(-3) M) or to the adenylate cyclase activator Forskolin (10(-4) M), restored afterdischarge induction by electrical stimulation. Application of IBMX (10(-3) M) and Forskolin (10(-4) M) together induced discharges in the absence of electrical stimulation. Our results suggest that in senescent CDCs changes in the intracellular cAMP pathway may underlie afterdischarge failure.
Asunto(s)
Envejecimiento/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/efectos de los fármacos , AMP Cíclico/farmacología , Lymnaea/efectos de los fármacos , Lymnaea/fisiología , Reproducción/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Sistema Nervioso Central/fisiología , Colforsina/farmacología , AMP Cíclico/análogos & derivados , Estimulación Eléctrica , Electrofisiología , Lymnaea/citología , Oviposición/efectos de los fármacos , Óvulo/efectos de los fármacos , Óvulo/fisiología , Potasio/farmacología , Transducción de Señal/efectos de los fármacos , Tionucleótidos/farmacologíaRESUMEN
Unlike most eukaryotes, many apicomplexan parasites contain only a few unlinked copies of ribosomal RNA (rRNA) genes. Based on stage-specific expression of these genes and structural differences among the rRNA molecules it has been suggested that Plasmodium spp. produce functionally different ribosomes in different developmental stages. This hypothesis was investigated through comparison of the structure of the large subunit rRNA molecules of the rodent malaria parasite, Plasmodium berghei, and by disruption of both of the rRNA gene units that are transcribed exclusively during development of this parasite in the mosquito (S-type rRNA gene units). In contrast to the human parasite, Plasmodium falciparum, we did not find evidence of structural differences in core regions of the distinct large subunit rRNAs which are known to be associated with catalytic activity including the GTPase site that varies in P. falciparum. Knockout P. berghei parasites lacking either of the S-type gene units were able to complete development in both the vertebrate and mosquito hosts. These results formally exclude the hypothesis that two functionally different ribosome types distinct from the predominantly blood stage-expressed A-type ribosomes, are required for development of all Plasmodium species in the mosquito. The maintenance of two functionally equivalent rRNA genes might now be explained as a gene dosage phenomenon.
Asunto(s)
Plasmodium berghei/fisiología , Ribosomas/fisiología , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fenotipo , Plasmodium berghei/genética , ARN Ribosómico/genética , Ribosomas/genéticaRESUMEN
The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle. To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2). The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes. We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies. Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S. pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect. However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature. Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery.
Asunto(s)
Ciclo Celular/fisiología , Plasmodium vivax/genética , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , Schizosaccharomyces/citología , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Quinasas CDC2-CDC28 , Clonación Molecular , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/fisiología , ADN Complementario/química , Regulación de la Expresión Génica , Biblioteca de Genes , Prueba de Complementación Genética , Humanos , Malaria Vivax/parasitología , Datos de Secuencia Molecular , Plasmodium vivax/citología , Proteínas Quinasas/química , Proteínas Protozoarias/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Fertilization and zygote development are obligate features of the malaria parasite life cycle and occur during parasite transmission to mosquitoes. The surface protein PFS48/45 is expressed by male and female gametes of Plasmodium falciparum and PFS48/45 antibodies prevent zygote development and transmission. Here, gene disruption was used to show that Pfs48/45 and the ortholog Pbs48/45 from a rodent malaria parasite P. berghei play a conserved and important role in fertilization. p48/45- parasites had a reduced capacity to produce oocysts in mosquitoes due to greatly reduced zygote formation. Unexpectedly, only male gamete fertility of p48/45- parasites was affected, failing to penetrate otherwise fertile female gametes. P48/45 is shown to be a surface protein of malaria parasites with a demonstrable role in fertilization.
Asunto(s)
Malaria/fisiopatología , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Anticuerpos , Culicidae , Femenino , Fertilidad/fisiología , Gametogénesis/fisiología , Genoma de Protozoos , Malaria/inmunología , Malaria/prevención & control , Vacunas contra la Malaria , Masculino , Datos de Secuencia Molecular , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , CigotoRESUMEN
The pond snail Lymnaea stagnalis has a maximum life span of about 22 months. At the age of about 250 days animals start to decrease egg laying activity and at about 500 days most animals ceased egg laying activity. At the age of cessation of egg laying the neurosecretory caudodorsal cells (CDCs) which control egg laying in Lymnaea exhibit reduced branching patterns. At this stage the cells still exhibit their physiological properties. CDCs still contain biologically active peptides and in the isolated CNS they still exhibit an afterdischarge upon electrical stimulation. Probably in the intact animal cessation of egg laying occurs because the CDCs are not activated anymore by natural egg laying inducing stimuli. In very old animals CDCs exhibit signs of degeneration indicating that cell death occur. After an extended period of no egg laying of Lymnaea physiological changes occur in the CDCs. CDCs from animals after an extended period of no egg laying failed to exhibit an afterdischarge. In such CDCs chemical and electrical coupling among the CDCs are reduced. Morphologically reduced CDCs predominantly fail to exhibit an afterdischarge. However, there are minimally branched CDCs that still could give an afterdischarge. Probably morphological reduction is not the only factor that defines afterdischarge failure. At present we suggest the following sequence of changes. 1. Morphological reduction of CDC branching patterns. 2. Cessation of egg laying. 3. Physiological changes in the CDCs resulting in afterdischarge failure. 4. Further morphological and physiological deterioration of CDCs.
Asunto(s)
Lymnaea/fisiología , Sistemas Neurosecretores/fisiología , Envejecimiento/fisiología , Animales , Femenino , Lymnaea/citología , Masculino , Sistemas Neurosecretores/citología , Oviposición/fisiología , Reproducción/fisiologíaRESUMEN
Plasmodium parasites are haploid unicellular organisms that cause malaria. In the last decade, transfection systems have been developed for both human and animal model species of Plasmodium, providing a broad range of genetic tools for the study of malaria parasite biology. Transient transfection has been used to provide insight into the regulation of gene expression by Plasmodium spp. The development of stable transfection technologies has provided the opportunity to express transgenes in Plasmodium spp., as well as elucidate the function of proteins by disrupting, modifying, or replacing the genes encoding them. These genetic tools represent an important breakthrough for malaria research and will significantly contribute to our understanding of the biology of the parasite. However, further developments in this technology are still required, especially because the full genome sequence of the major human malaria parasite Plasmodium falciparum will shortly be available. Ultimately, the biological information obtained through genetic manipulation of Plasmodium spp. will facilitate a more rational approach to vaccine and drug design.
Asunto(s)
Malaria/parasitología , Plasmodium/genética , Transfección/métodos , Animales , Resistencia a Medicamentos/genética , Humanos , MutagénesisRESUMEN
We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.
Asunto(s)
Marcación de Gen , Genes Protozoarios/genética , Genoma de Protozoos , Mutagénesis Sitio-Dirigida , Plasmodium berghei/genética , Eliminación de Secuencia/genética , Animales , Vectores Genéticos/síntesis química , Plasmodium berghei/enzimología , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/genética , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/genética , Transcripción Genética , TransfecciónRESUMEN
The current knowledge on genomes of non-falciparum malaria species and the potential of model malaria parasites for functional analyses are reviewed and compared with those of the most pathogenic human parasite, Plasmodium falciparum. There are remarkable similarities in overall genome composition among the different species at the level of chromosome organisation and chromosome number, conserved order of individual genes, and even conserved functions of specific gene domains and regulatory control elements. With the initiative taken to sequence the genome of P. falciparum, a wealth of information is already becoming available to the scientific community. In order to exploit the biological information content of a complete genome sequence, simple storage of the bulk of sequence data will be inadequate. The requirement for functional analyses to determine the biological role of the open reading frames is commonly accepted and knowledge of the genomes of the animal model malaria species will facilitate these analyses. Detailed comparative genome information and sequencing of additional Plasmodium genomes will provide a deeper insight into the evolutionary history of the species, the biology of the parasite, and its interactions with the mammalian host and mosquito vector. Therefore, an extended and integrated approach will enhance our knowledge of malaria and will ultimately lead to a more rational approach that identifies and evaluates new targets for anti-malarial drug and vaccine development.
Asunto(s)
Genoma de Protozoos , Malaria Falciparum/genética , Plasmodium falciparum/genética , Animales , Mapeo Cromosómico/veterinaria , Cromosomas , Clonación Molecular , Humanos , Familia de Multigenes , Regiones Promotoras GenéticasRESUMEN
Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.