RESUMEN

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Asunto(s)

Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica/veterinaria , Microscopía/veterinaria , Parasitología/métodos , Animales , Fluorescencia , Interacciones Huésped-Parásitos , Imagenología Tridimensional , Inmunohistoquímica , Luz , Microscopía/métodos , Microscopía Confocal/métodos , Microscopía Confocal/veterinaria , Microscopía Electrónica/métodos , Microscopía Electrónica de Transmisión/métodos , Microscopía Electrónica de Transmisión/veterinaria , Microscopía Fluorescente/métodos , Microscopía Fluorescente/veterinaria , Parasitología/instrumentación

RESUMEN

Transgenic Leishmania infantum promastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested.

Asunto(s)

Antiprotozoarios/farmacología , Leishmania infantum/efectos de los fármacos , Proteínas Luminiscentes/biosíntesis , Alopurinol/farmacología , Animales , Biomarcadores , Cicloheximida/farmacología , Proteínas Fluorescentes Verdes , Leishmania infantum/crecimiento & desarrollo , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología

RESUMEN

Toxoplasma gondii is a common human pathogen causing serious, even fatal, disease in the developing fetus and in immunocompromised patients. Despite its ability to reproduce sexually and its broad geographic and host range, Toxoplasma has a clonal population structure comprised principally of three lines. We have analyzed 15 polymorphic loci in the archetypal type I, II, and III strains and found that polymorphism was limited to, at most, two rather than three allelic classes and no polymorphism was detected between alleles in strains of a given type. Multilocus analysis of 10 nonarchetypal isolates likewise clustered the vast majority of alleles into the same two distinct ancestries. These data strongly suggest that the currently predominant genotypes exist as a pandemic outbreak from a genetic mixing of two discrete ancestral lines. To determine if such mixing could lead to the extreme virulence observed for some strains, we examined the F(1) progeny of a cross between a type II and III strain, both of which are relatively avirulent in mice. Among the progeny were recombinants that were at least 3 logs more virulent than either parent. Thus, sexual recombination, by combining polymorphisms in two distinct and competing clonal lines, can be a powerful force driving the natural evolution of virulence in this highly successful pathogen.

Asunto(s)

Recombinación Genética , Toxoplasma/genética , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Toxoplasmosis/parasitología , Alelos , Animales , Secuencia de Bases , Cruzamientos Genéticos , Genes Protozoarios , Variación Genética , Genotipo , Humanos , Intrones , Dosificación Letal Mediana , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Toxoplasma/clasificación , Toxoplasma/aislamiento & purificación , Virulencia/genética

RESUMEN

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

Asunto(s)

Antígenos de Protozoos/fisiología , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Southern Blotting , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peso Molecular , Toxoplasma/patogenicidad

RESUMEN

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)-green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5' flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3' flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1-GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.

Asunto(s)

Giardia/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Regiones no Traducidas 3' , Animales , Transporte Biológico , Compartimento Celular , Pared Celular/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos

RESUMEN

Neospora caninum is an apicomplexan parasite which causes neosporosis, namely stillbirth and abortion in cattle, and neuromuscular disease in dogs. Although N. caninum is phylogenetically and biologically closely related to Toxoplasma gondii, it is antigenically clearly distinct. In analogy to T. gondii, three stages have been identified. These are: (i) asexually proliferating tachyzoites; (ii) tissue cysts harbouring slowly dividing bradyzoites; and (iii) oocysts containing sporozoites. The sexually produced stage of this parasite has only recently been identified, and has been shown to be shed with the faeces from dogs orally infected with N. caninum tissue cysts. Thus dogs are definitive hosts of N. caninum. Tachyzoites can be cultivated in vitro using similar techniques as previously described for T. gondii. Methods for generating tissue cysts containing N. caninum bradyzoites in mice, and purification of these cysts, have been developed. A number of studies have been undertaken to identify and characterise at the molecular level specific antigenic components of N. caninum in order to improve serological diagnosis and to enhance the current view on the many open questions concerning the cell biology of this parasite and its interactions with the host on the immunological and cellular level. The aim of this paper is to provide an overview on the approaches used for detection of antigens in N. caninum. The studies discussed here have had a great impact in the elucidation of the immunological and pathogenetic events during infection, as well as the development of potential new immunotherapeutic tools for future vaccination against N. caninum infection.

Asunto(s)

Antígenos de Protozoos/análisis , Coccidiosis/veterinaria , Neospora/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Bovinos , Coccidiosis/diagnóstico , Coccidiosis/inmunología , Coccidiosis/parasitología , Perros , Interacciones Huésped-Parásitos , Ratones , Datos de Secuencia Molecular , Neospora/aislamiento & purificación

RESUMEN

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.

Asunto(s)

Antígenos de Protozoos/análisis , Glicosilfosfatidilinositoles/análisis , Proteínas Protozoarias/análisis , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/fisiología , Antígenos de Superficie/análisis , Antígenos de Superficie/fisiología , Glicosilfosfatidilinositoles/fisiología , Humanos , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Conejos

RESUMEN

Toxoplasma gondii is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on approximately 7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18-28, 1998). Here, we report the results from analysis of approximately 2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in Toxoplasma.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Biblioteca de Genes , Humanos , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular

RESUMEN

As for any intracellular parasite, the surface of the Apicomplexan parasite Toxoplasma gondii must fulfil many functions including a role in attachment, signalling, invasion, transport and interaction with the immune response of the host. In this review, we describe the current state of knowledge on the molecules that are found on the surface of the different developmental stages of this parasite and speculate as to how at least some of these multiple functions are fulfilled. Special emphasis is given to the growing family of surface antigens that are related to the tachyzoite-specific surface antigen 1. We conclude that the surface (of tachyzoites, at least) is both more and less complex than previously thought: there are more proteins present but their sequences suggest that the majority may share a similar overall structure typified by surface antigen 1.

Asunto(s)

Antígenos de Protozoos/análisis , Antígenos de Superficie/análisis , Toxoplasma/química , Animales , Estadios del Ciclo de Vida , Toxoplasma/crecimiento & desarrollo

RESUMEN

To accelerate gene discovery and facilitate genetic mapping in the protozoan parasite Toxoplasma gondii, we have generated >7000 new ESTs from the 5' ends of randomly selected tachyzoite cDNAs. Comparison of the ESTs with the existing gene databases identified possible functions for more than 500 new T. gondii genes by virtue of sequence motifs shared with conserved protein families, including factors involved in transcription, translation, protein secretion, signal transduction, cytoskeleton organization, and metabolism. Despite this success in identifying new genes, more than 50% of the ESTs correspond to genes of unknown function, reflecting the divergent evolutionary status of this parasite. A newly recognized class of genes was identified based on its similarity to sequences known only from other members of the same phylum, therefore identifying sequences that are apparently restricted to the Apicomplexa. Such genes may underlie pathways common to this group of medically important parasites, therefore identifying potential targets for intervention.

Asunto(s)

Apicomplexa/genética , Expresión Génica , Genes Protozoarios , Familia de Multigenes , Toxoplasma/genética , Animales , Biología Computacional/métodos , Secuencia Conservada , ADN Complementario/análisis , Humanos , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Homología de Secuencia de Ácido Nucleico

RESUMEN

Previous investigations of the major surface antigen (SAG1) promoter of Toxoplasma gondii indicated an ability to function bi-directionally in transient transformation assays at least. This suggests there might be another tachyzoite-specific gene being divergently transcribed from the SAG1 promoter in its normal chromosomal location. To investigate this possibility we have characterized the region upstream of SAG1 and report here a co-directional transcription unit coding for a probable GPI-anchored surface protein with homology to SAG1 and SAG3. This antigen, which had not previously been identified in surface iodination experiments is given the acronym SRS1, for SAG1-related sequence 1. Genomic organization and sequence of a full-length cDNA of SRS1 are presented. Antisera against a recombinant SRS1 protein produced in Escherichia coli, recognize a specific band of 46 kDa in parasite lysates which corresponds to the largest of the GPI-anchored proteins by Western blot. The possible role of this previously unidentified surface antigen is discussed.

Asunto(s)

Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Proteínas Protozoarias/genética , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos/análisis , Antígenos de Protozoos/química , Antígenos de Superficie/análisis , Antígenos de Superficie/química , Secuencia de Bases , ADN Complementario/genética , ADN Protozoario/genética , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Protozoario/genética , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Toxoplasma/genética , Transcripción Genética/genética

RESUMEN

Toxoplasma gondii has recently come under intense study as a model for intracellular parasitism because it has a number of properties that facilitate experimental manipulation. Attention is now being turned towards understanding the developmental biology of this complex parasite. The differentiation between the two asexual stages, the rapidly growing tachyzoites and the more slowly dividing, encysted bradyzoites, is of particular interest. Progression from the former to the latter is influenced by the host's immune response. This paper describes current progress on a number of research fronts, all aimed at understanding the triggers that push the tachyzoite-bradyzoite equilibrium in one or other direction and the changes that occur in gene expression (and ultimately metabolism and function). Chief among the techniques used for these studies are genetics and molecular genetics. Recent progress in these areas is described.

Asunto(s)

Mapeo Cromosómico , Hipoxantina Fosforribosiltransferasa/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/genética , Acetilglucosamina/metabolismo , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Quitina/metabolismo , Quistes , Genes Protozoarios , Prueba de Complementación Genética , Humanos , Hipoxantina Fosforribosiltransferasa/efectos de los fármacos , Nucleósido-Trifosfatasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selección Genética , Lugares Marcados de Secuencia , Toxoplasma/efectos de los fármacos , Xantinas/farmacología

RESUMEN

BACKGROUND/AIMS: The commonly used procedure to treat gallstone ileus is enterolithotomy in combination with late cholecystectomy for persistent symptoms. It is related to a high mortality rate up to 20%. To date it has been impossible to reduce the mortality associated with this accepted surgical technique. PATIENTS AND METHODS: A seven-year retrospective study with a follow up over a period of four years and five months. Sixteen patients with gallstone ileus were entered into the study. Twelve patients were female and fourteen were more than 70 years of age. Fourteen patients were treated by enterolithotomy as well as additional cholecystectomy and resection of the fistula in a one-stage repair. RESULTS: Mortality during hospitalization amounted to one (6%) and postoperative complications to five patients (31%). Follow-up over a period of four years and five months showed one patient had died 3 years postoperatively of an advanced renal tumor. All other patients were alive and without symptoms. CONCLUSION: Our results prove that the concomitant one-stage repair for gallstone ileus significantly reduces the overall mortality rate compared to the single enterolithotomy alone and later cholecystectomy based on patient symptoms.

Asunto(s)

Colecistectomía , Colelitiasis/complicaciones , Obstrucción Intestinal/etiología , Obstrucción Intestinal/cirugía , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Estudios Retrospectivos , Procedimientos Quirúrgicos Operativos/métodos

RESUMEN

Genetic analysis of the protozoan parasite Toxoplasma gondii has undergone a rapid expansion in recent years. This is due to effort in a number of laboratories that have worked on the development of molecular genetic techniques. It is also due, however, to the natural biology of this system (including a well-described sexual cycle) that makes possible genetic mapping of the F1 progeny from a cross. In this article, we present a detailed methodology for rapidly mapping natural polymorphisms between the ME49 and CEP strains for which extensive restriction fragment length polymorphism analysis has already been performed. The example we present shows that the failure to detect expression of bradyzoite-specific surface antigens in the CEP strain under conditions that promote differentiation in vitro is not a result of a general failure to express such genes; instead, it is apparently due to antigenic polymorphism in the gene products concerned. This conclusion was reached rapidly and definitively by genetic mapping, whereas molecular approaches would have taken considerably longer. We also show how the recent effort to create an extensive database of expressed sequence tags for this parasite can promote the very rapid discovery of genes that reveal much about the biology of Toxoplasma. The example presented deals with the expression of a family of closely related surface antigens in the tachyzoite stage.

Asunto(s)

Antígenos de Protozoos , Genes Protozoarios , Polimorfismo de Longitud del Fragmento de Restricción , Toxoplasma/genética , Alelos , Secuencia de Aminoácidos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Mapeo Cromosómico/métodos , Clonación Molecular , Cruzamientos Genéticos , Cartilla de ADN , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Lugares Marcados de Secuencia , Piel , Toxoplasma/fisiología

RESUMEN

Pelvic floor hernias are rare. Three different types can be distinguished; these are, in order of decreasing frequency: obturator, perineal and sciatic hernias. We report a rare case of a perineal hernia with an intra- and extraperitoneal part. Furthermore a two-stage surgical approach (first transabdominal, then posterior) is presented.

Asunto(s)

Nalgas , Enfermedades del Colon/cirugía , Herniorrafia , Diafragma Pélvico , Perineo , Enfermedades del Recto/cirugía , Nalgas/patología , Nalgas/cirugía , Enfermedades del Colon/patología , Diagnóstico Diferencial , Femenino , Hernia/patología , Humanos , Persona de Mediana Edad , Diafragma Pélvico/patología , Diafragma Pélvico/cirugía , Perineo/patología , Perineo/cirugía , Complicaciones Posoperatorias/patología , Complicaciones Posoperatorias/cirugía , Enfermedades del Recto/patología , Reoperación

RESUMEN

Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface. Although GARP shows no sequence similarity to procyclins from T. brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes. Since T. brucei and T. congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments. As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T. brucei in which the GARP coding region has been integrated into a procyclin expression site. GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T. brucei rather than in T. congolense. The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection. We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.

Asunto(s)

Antígenos de Protozoos/biosíntesis , Antígenos de Superficie/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Proteínas Protozoarias/biosíntesis , Trypanosoma congolense/química , Animales , Animales Modificados Genéticamente , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Secuencia de Bases , Clonación Molecular , Interacciones Huésped-Parásitos , Epítopos Inmunodominantes/biosíntesis , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Separación Inmunomagnética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Microesferas , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Recombinantes de Fusión/inmunología , Selección Genética , Transfección , Trypanosoma congolense/crecimiento & desarrollo , Trypanosoma congolense/inmunología , Moscas Tse-Tse/metabolismo , Moscas Tse-Tse/parasitología

RESUMEN

Between January 1, 1986 and December 31, 1992 93 patients with irresectable carcinoma of the pancreatic head underwent surgical palliation: In group I (n = 51) a single loop biliary (BB)- and gastric bypass (GB) was performed. 34 times the gastrojejunostomy was performed prophylactically. In group II (n = 42) surgical palliation was carried out only by biliary decompression in Roux-Y technique. In 30.9% gastric outlet obstruction (GOO) developed during follow-up. Both groups were comparable according to effectiveness of biliary drainage (93% (n = 42) (I); 97% (n = 38) (II)) with median post-operative bilirubin levels of 3.21 mg/dl (I) and 4.1 mg/dl (II). Cholecysto-choledocho- and hepaticojejunostomy were equally effective. Median operative time, morbidity (19.6 (I) vs. 26.2 (II)) and postoperative hospitalization were similar. Since there is a high frequency of secondary GOO after single BB we think that GB should be performed in all patients that undergo BB because secondary gastrojejunostomy at a later stage significantly increases morbidity and mortality.

Asunto(s)

Anastomosis en-Y de Roux/métodos , Anastomosis Quirúrgica/métodos , Conductos Biliares/cirugía , Colestasis Extrahepática/cirugía , Yeyunostomía/métodos , Cuidados Paliativos/métodos , Neoplasias Pancreáticas/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Causas de Muerte , Colestasis Extrahepática/mortalidad , Colestasis Extrahepática/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Complicaciones Posoperatorias/mortalidad , Estudios Retrospectivos , Tasa de Supervivencia

RESUMEN

Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.

Asunto(s)

Genes , Intrones , Glicoproteínas de Membrana , Poli A/biosíntesis , Proteínas Protozoarias/genética , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Secuencia de Bases , Secuencia Conservada , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Pirimidinas , ARN Protozoario/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Trypanosoma brucei brucei/genética

RESUMEN

African trypanosomes that cycle between mammalian hosts and the tsetse fly vector must be poised to survive in different environments. The control of stage-specific gene expression is undoubtedly one of the keys to successful adaptation, but no regulatory elements have been defined to date. Procyclins (also known as procyclic acidic repetitive proteins) are specifically expressed on the surface of procyclic and epimastigote forms in the fly. Procyclin genes are already transcribed in bloodstream forms, but stable mRNA, and later the protein, are first detected when the parasites begin to differentiate into procyclic forms. We have now identified a region of 16 bases that forms part of a predicted stem-loop structure in the 3' untranslated regions of different procyclin mRNAs; both the sequence and the secondary structure of this 16-mer appear to be required for efficient translation of a reporter gene in procyclic forms. The level of steady-state mRNA, its polyadenylylation, and its distribution in the cell are all unaffected by the presence or absence of this element. Deletion of the 16-mer alone reduces expression more than removal or reversal of the entire 3' untranslated region and flanking region, suggesting that there are additional negative regulatory elements in the same 3' untranslated region.

Asunto(s)

Regulación de la Expresión Génica , Glicoproteínas de Membrana , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Secuencia de Bases , Cartilla de ADN/química , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Poli A/metabolismo , Proteínas Protozoarias/genética , ARN Mensajero/química , ARN Mensajero/genética

RESUMEN

The identification of procyclins as stage-specific coat proteins of procyclic forms of Trypanosoma brucei has not only provided a convenient molecular marker for the differentiation of bloodstream-form trypanosomes into procyclic forms, but has also allowed some important insights into gene regulation in trypanosomes. Here, Adrian Hehl and Isabel Roditi summarize what has been learnt in the past few years about the control mechanisms that may contribute to the stage-specific expression of procyclins.