It's a small world for parasites: evidence supporting the North American invasion of European Echinococcus multilocularis.

Echinococcus multilocularis (Em), the causative agent of human alveolar echinococcosis (AE), is present in the Holarctic region, and several genetic variants deem to have differential infectivity and pathogenicity. An unprecedented outbreak of human AE cases in Western Canada infected with a European-like strain circulating in wild hosts warranted assessment of whether this strain was derived from a recent invasion or was endemic but undetected. Using nuclear and mitochondrial markers, we investigated the genetic diversity of Em in wild coyotes and red foxes from Western Canada, compared the genetic variants identified to global isolates and assessed their spatial distribution to infer possible invasion dynamics. Genetic variants from Western Canada were closely related to the original European clade, with lesser genetic diversity than that expected for a long-established strain and spatial genetic discontinuities within the study area, supporting the hypothesis of a relatively recent invasion with various founder events.

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada.

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.

A review on invasions by parasites with complex life cycles: the European strain of Echinococcus multilocularis in North America as a model.

In a fast-changing and globalized world, parasites are moved across continents at an increasing pace. Co-invasion of parasites and their hosts is leading to the emergence of infectious diseases at a global scale, underlining the need for integration of biological invasions and disease ecology research. In this review, the ecological and evolutionary factors influencing the invasion process of parasites with complex life cycles were analysed, using the invasion of the European strain of Echinococcus multilocularis in North America as a model. The aim was to propose an ecological framework for investigating the invasion of parasites that are trophically transmitted through predator­prey interactions, showing how despite the complexity of the cycles and the interactions among multiple hosts, such parasites can overcome multiple barriers and become invasive. Identifying the key ecological processes affecting the success of parasite invasions is an important step for risk assessment and development of management strategies, particularly for parasites with the potential to infect people (i.e. zoonotic).

Evaluation of an automated magnetic bead-based DNA extraction and real-time PCR in fecal samples as a pre-screening test for detection of Echinococcus multilocularis and Echinococcus canadensis in coyotes.

Efficient and sensitive diagnostic tools are essential for the study of the eco-epidemiology of Echinococcus species. We evaluated an automated magnetic bead-based DNA extraction commercial kit followed by qPCR (MB-qPCR), for the detection of Echinococcus multilocularis and Echinococcus canadensis in coyote (Canis latrans) fecal samples. The diagnostic sensitivity was determined by validating the method against the scraping, filtration, and counting technique (SFCT) for samples collected in Canada. From the 60 samples tested, 27 out of 31 SFCT positives samples for Echinococcus cestodes were positive in the MB-qPCR for E. multilocularis, with a sensitivity of 87.1% (95% CI 70.2 to 96.4%). Two samples were also positive for E. canadensis in the MB-qPCR and confirmed by morphological identification of adult worms. The agreement of the MB-qPCR and the SFCT was statistically significant with a kappa value of 0.67 (95% CI 0.48-0.85; p value < 0.001). The magnetic bead-based DNA extraction followed by qPCR proved to have a sensitivity comparable to the SFCT to detect E. multilocularis. Although the diagnostic sensitivity for E. canadensis was not estimated, MB-qPCR identified E. canadensis cases previously overlooked when using SFCT. We propose a combination of molecular and morphological identification using the MB-qPCR and the SFCT to detect both parasites, allowing for a more efficient large-scale surveillance, and detecting co-infections of Echinococcus species that can be difficult to identify when only based on morphology.

Corrigendum to 'Detecting co-infections of Echinococcus multilocularis and Echinococcus canadensis in coyotes and red foxes in Alberta, Canada using real-time PCR' [IJP: Parasites and Wildlife 7 (2018) 111-115].

[This corrects the article DOI: 10.1016/j.ijppaw.2018.03.001.].

Detecting co-infections of Echinococcus multilocularis and Echinococcus canadensis in coyotes and red foxes in Alberta, Canada using real-time PCR.

The continued monitoring of Echinococcus species in intermediate and definitive hosts is essential to understand the eco-epidemiology of these parasites, as well to assess their potential impact on public health. In Canada, co-infections of Echinococcus canadensis and Echinococcus multilocularis based on genetic characterization have been recently reported in wolves, but not yet in other possible hosts such as coyotes and foxes. In this study, we aimed to develop a quantitative real-time PCR assay to detect E. multilocularis and E. canadensis and estimate the occurrence of co-infections while inferring about the relative abundance of the two parasites within hosts. We tested DNA extracted from aliquots of Echinococcus spp. specimens collected from intestinal tracts of 24 coyote and 16 fox carcasses from Alberta, Canada. We found evidence of co-infections of E. multilocularis and E. canadensis in 11 out of 40 (27%) samples, with 8 out of 24 (33%) in coyote samples and 3 out of 16 (19%) in red fox samples. DNA concentrations were estimated in three samples with Cq values within the range of the standard curve for both parasites; two of them presented higher DNA concentrations of E. multilocularis than E. canadensis. The use of qPCR aided detection of co-infections when morphological discrimination was difficult and quantification of DNA for samples within the standard curve. This is the first molecularly confirmed record of E. canadensis in coyotes and the first evidence of co-infections of E. multilocularis and E. canadensis in coyotes and red foxes.

Panorámica sobre la formación de especialista en 2007: retos y oportunidades / No disponible

No disponible

Consumo y actitudes hacia el tabaco de los profesionales de la medicina y enfermería del Hospital Clínico San Carlos de Madrid / No disponible

Objetivo Valorar la prevalencia de consumo de tabaco de Licenciados en Medicina (LM) y Diplomados Universitarios en Enfermería (DUE), y sus opiniones, actitudes y hábitos (personales y profesionales) en relación con el consumo de tabaco en un centro hospitalario. Métodos Estudio transversal, muestreo estratificado con asignación proporcional. Realización de 426 entrevistas directas, con encuesta validada, entre abril-mayo de 2004 a profesionales adscritos mayoritariamente al Hospital Clínico San Carlos (HCSC). Resultados Se obtuvieron 377 encuestas válidas. La prevalencia de tabaquismo es de un 38,5%. Mujeres (42,7%), menores de 35 años (51,2%) y DUE (44,5%) son los subgrupos con mayor prevalencia de fumadores. Ser fumador se asocia significativamente con menor preocupación por los efectos del consumo de tabaco y del humo ambiental sobre la salud; también están menos convencidos de que el hospital ha de ser un espacio sin humo. Un 45,3% fuman en zonas no permitidas. Los fumadores promueven menos el cumplimiento de la normativa y dan consejo antitabaco con menor frecuencia. Más del 50% del total no piensa que los profesionales sanitarios desempeñen un papel de modelo social; alrededor del 20% cree que los fumadores no tienen en cuenta sus recomendaciones y un 85% considera necesaria una formación específica. Conclusiones El hecho de fumar condiciona opiniones y comportamientos relacionados con el tabaco, influyendo en el desempeño profesional. Existe una necesidad sentida y objetiva de formación sobre el abordaje del paciente y del compañero fumador, y un clima de escepticismo en la efectividad de las acciones de los profesionales para reducir el consumo de tabaco y conseguir el cumplimiento de las restricciones dentro de los centros sanitarios. Aunque tras los años de actividades preventivas existe un estado de opinión que apoya la transformación del hospital en un centro sin humo, sigue siendo necesario intervenir con los profesionales, hasta conseguir la resolución de este problema de salud pública

Objectives To assess smoking prevalence among Medicine and Nursery graduates at the Clinical Hospital San Carlos (HCSC), and to get some information about their opinions, personal and professional attitudes, as well as their habits related to the tobacco consumption within the Hospital. Methods A prevalence study was performed within a 426 representative sample of health professionals (doctors and nurses from secondary and third health assistance levels), who were interviewed with a Spanish validated questionnaire. Period: April to May 2004. Spss 12.0 was used to analyse results. Results Smoking prevalence among HCSC health professionals, was 38,5%. The most affected groups were nurses (44,5% smokers), < 35 years and women (42,7%). The smoker condition is significantly associated to less concern about both the fact of smoking and the enviromental smoke effects against health. 45,3% of them smoke in not allowed places, and they have a more tolerant attitude in general regarding this issue. Only 57,3% of professionals usually pro mote the fulfilment of tobacco control rules amid patients and relatives, and smokers do it even less often. Near 20% of total sample think that the smokers do not consider their recommendations and 85% think that specific formation is required. Conclusions The smoker condition is a determinant factor for opinions, attitudes and behaviour related to smoking matters, and it has a c1ear influence on the professional activities. There is a lack of training about professional health promotion approach to smokers (both patients and colleagues). There is also a sceptic view of the environmental tobacco regulation effectiveness. Despite of the advances produced in these past years, it is still necessary to take part and dedicate resources to this public health problem

Estreptococo del grupo A: un estudio sobre el estado de portador. Implicaciones en el valor diagnóstico del cultivo faríngeo / Group A Streptococcus: a study of carrier status. Implications for the diagnostic value of throat culture

Fundamento: El estreptococo del grupo A o Streptococcus pyogenes (SP) es la causa más importante de faringitis aguda bacteriana en la población pediátrica. Algunos pacientes infectados pueden permanecer asintomáticos como portadores sanos. Objetivo: Analizar la tasa de portadores de SP y su influencia sobre el cultivo faríngeo (CF) para el diagnóstico etiológico de faringitis estreptocócica . Pacientes y métodos: Se estudió la presencia de SP en muestras de garganta de niños sanos de edades comprendidas entre 6 meses y 14 años, en 185 individuos en verano y 228 en invierno de entre la población adscrita a un centro de atención primaria. De forma simultánea, se estudiaron, de entre esa misma población, 120 individuos con clínica de faringitis en verano y 162 en invierno. Se les efectuó una toma de exudado faríngeo, cultivándose en agar-sangre e identificando S. pygenes mediante el test de la bacitracina. Se compararon las tasas de SP en sanos y enfermos en estas dos épocas del año. Resultados: La incidencia de S. pyogenes en individuos sanos fue menor en verano que en invierno (5,9-17,1%) (p <0,0001); de igual forma, su incidencia en niños con faringitis fue menor en verano que en invierno (16,6-29%) (p <0,05). En ambos casos, la mayoría de los positivos se encontró en el grupo de 3-14 años. La tasa de portadores se alejó más de la de enfermos con CF positivo a S. pygenes en verano que en invierno (5.7-16,6% frente a 17,1-29%). Conclusión: La incidencia de S. pyogenes en niños sanos varía en relación con la estación del año y la edad. También varía la tasa de enfermos de faringitis con cultivo faríngeo positivo a este microrganismo. Ello puede suponer implicaciones para la valoración clínica del CF, ya que será de menos valor cuanto más se aproximen ambas tasas. Sin embargo, estas implicaciones no invalidan el cultivo faríngeo como método diagnóstico para el manejo de las faringitis

Background. Group A streptococcus, Streptococcus pyogenes (SP). is the major cause of acute bacterial pharyngitis in children. Some infected patients remain asymptomatic, acting as healthy carriers. Objective. Our objective was to examine the carrier rate of SP and its influence on the diagnostic value of throat culture for streptococcal pharyngitis. Patients and methods. The presence of SP in throat specimens from healthy children ranging in age from 6 months to 14 years in a primary health care center was studied in the summer (n= 185) and in the winter (n= 228). At the same time, children from the same population with clinical signs of pharyngitis were studied in summer (n= 120) and in winter (n= 162). Throats specimens were obtained and cultured in blood agar, and SP was identified by the bacitracin test. The rates of SP in healthy and sick children during the two seasons were compared. Results. The incidence of SP in healthy individuals was 10wer in the summer than in winter (5.9% versus 17.1 %; p<0.000l). The incidence of SP in children with clinical pharyngitis was also lower in summer than in winter (16.6% versus 29%; p <0.05). Most of the positive cultures in both healthy and ill groups corresponded to the group comprised of 3 to 14year-olds. The difference between the rate of positivity in healthy carriers and the incidence of positive throat cultures in children with clinical signs of pharyngitis was greater in summer than in winter (5.7%/16.6% versus 17.1 %/29%). Conclusions. The incidence of SP in healthy and ill individuals varies according to season and age. Implications for the diagnostic value of throat culture may exist. since said value decreases as the difference between the two rates is reduced. However, this circumstance does not invalidate the clinical usefulness of throat culture

Formación MIR en Medicina Preventiva y Salud Pública: Aproximación a un mapa de rotaciones de la especialidad / No disponible

La especialidad de Medicina Preventiva y Salud Pública requiere una amplia formación multidisciplinar en diferentes áreas. Dicha formación se organiza en varios períodos de duración variable, cada uno de los cuales se realiza en distintas unidades, servicios y/o instituciones, por las que se va rotando. Dentro de las directrices marcadas por el programa oficial de la especialidad, cabe un amplio abanico de opciones no siempre bien conocidas por todas las unidades docentes y los especialistas en formación. Ante la escasez de información sobre todas las rotaciones posibles, nos planteamos conocer cuáles son las unidades en las que se forman los residentes en España. Se envió una encuesta vía correo electrónico durante los meses de Julio a Agosto de 2004 dirigida a MIR (2°-4° año) y a especialistas de las 2 últimas promociones, que recogía el nombre completo de las unidades e instituciones donde habían realizado algún período formativo, tipo de institución y provincia. Se recibieron 55 encuestas (Tasa de respuesta: 31,8%), con un total de 245 rotaciones, que se habían realizado en 152 unidades diferentes. El 47,2% de ellas se habían llevado a cabo en Madrid y un 15,5% en Barcelona. Un 52% de ellas tuvieron lugar en hospitales. Se presenta un listado con las unidades donde se formaron los encuestados. Este "mapa de rotaciones" supone un importante primer paso en el conocimiento de la oferta formativa de la especialidad, y pretende ser un documento orientativo que residentes y tutores puedan consultar antes de elaborar conjuntamente su plan de formación

Preventive medicine and public health residency programme requires a multidisciplinary training in different fields. This specialized formation is organized in several periods of variable duration, performed in such different units, services and institutions related to the various fields; so is a rotary practical formation system. There is a wide range of possibilities permitted by the official specialization program framework, and often not well known by both the training units and the specializing physicians. Due to this lack of information regarding where residents are able to be trained, the objective of this study was to describe in which units, services and institutions are the future specialists in Spain adquiring their competencies and skills. A survey was delivered by e-mail from July to October 2004 to residents (2nd to 4th year) and specialists graduated in 2003 and 2004. The questionnaire identified the rotations through the unit's full name, institution, institution type and region. Filled-in surveys were received from 55 residents (overall response rate 31,8%). A total number of 245 rotations were identified from 152 different units. 47,2% of the rotations had taken place in Madrid and 15,5% in Barcelona. 52% of them belonged to hospital departments. A complete list with all the formation locations is annexed. We believe this "rotation map" means an important achievement within the knowledge of the training offer in this specialty, and aims to be a guide for residents and tutors when planning together the individual training programme

Diagnóstico de la faringitis estreptocócica en la consulta de atención primaria: una antigua solución reconsiderada / Diagnosis of streptococcal pharyngitis in the office: an old solution reconsidered

Introducción: La faringitis aguda es muy prevalente en pediatría primaria. La etiología más frecuente es viral (70%-80%), siendo Streptococcus pyogenes (SP) la causa bacteriana más importante. El objetivo de este trabajo es evaluar tres scores clínicos publicados para predecir la faringitis estreptocócica, en comparación con el resultado del cultivo faríngeo (CF) realizado en la consulta. Material y métodos: Se estudiaron 697 niños de entre 6 meses y 14 años de edad, con clínica de faringitis. En todos los casos se efectuó un CF mediante siembra del exudado faríngeo en agar-sangre e identificación de SP por la prueba de la bacitracina. Se calculó el coste y el tiempo invertido en cada determinación. Se valoraron tres scores, comparando cada uno de ellos con el resultado del CF. Resultados: Un total de 174 (25%) de los niños estudiados tuvo un cultivo faríngeo positivo para SP. El valor predictivo positivo (VPP) en los tres scores es inferior al 40% y su sensibilidad (S) varía entre un 25 y un 39%. El coste de cada CF fue de 0,50 euros. El tiempo medio invertido en cada CF fue de 4 minutos. Conclusiones: Según los resultados obtenidos, los bajos VPP y la escasa S sugieren que no existen criterios clínicos fiables que permitan el diagnóstico de la faringitis estreptocócica en la mayor parte de los casos. La triple disyuntiva que supone optar por realizar CF en los laboratorios de bacteriología, usar métodos rápidos o emplear criterios clínicos queda superada por la realización del CF en la consulta. En nuestra experiencia, la práctica del CF y la prueba de la bacitracina como técnica diagnóstica es idónea en la consulta de atención primaria, por ser segura, poco costosa, práctica y de fácil realización

Introduction. Acute pharyngitis is highly prevalent in pediatric primary careo It is usually viral in origin (70%-80%), Streptococcus pyogenes being the leading bacterial cause. The objective of this report is to evaluate three published clinical scoring systems for the prediction of streptococcal pharyngitis, comparing them with the results of throat culture performed in the office. Material and methods. We studied 697 children between the ages of 6 months and 14 years with the clinical signs and symptoms of acute pharyngitis. Throat swab samples from each child were inoculated on blood agar plates and incubated, using the bacitracin test for the identification of S. pyogenes. The cost and time spent for each determination were calculated. Three scores were assessed and compared with the results of the throat culture. Results. One hundred seventy-four (25%) of the children studied had throat cultures positive for S. pyogenes. The positive predictive value (PPV) of the three scores was under 40%, and their sensitivity ranged between 25% and 39%. Each throat culture cost 0.50 euros and required a mean time of 4 minutes. Conclusions. According to our findings, the low PPV and sensitivity suggest the nonexistence of reliable clinical criteria for the diagnosis of streptococcal pharyngitis in the majority of cases. The performance of throat culture in the microbiology laboratory, the use of "rapid methods" and the application of clinical criteria are less advantageous than the performance of throat culture in the office. In our experience, throat culture using the bacitracin test is safe, inexpensive and easy to perform and, thus, is the diagnostic technique of choice in the primary care office setting

Apoptosis induced by different doses of caffeine on Chinese hamster ovary cells.

Caffeine has been investigated for its potential mutagenic activity to bacteria, fungi and mammalian cells in culture, and at high concentrations it is also an inducer of apoptosis. Caffeine can exert acute cellular toxicity, including inhibition of cell growth and cell death, in Chinese hamster ovary cells. The aim of this study was to evaluate the cell survival and apoptotic or non-apoptotic effects of caffeine to different concentrations in Chinese hamster ovary cells (CHO-K1). These effects were evaluated by measuring cell viability, caspase 8 activity and fragmented DNA. This study suggests that the concentration of caffeine is of critical importance because high doses of caffeine induce apoptosis and low concentrations can act as an antioxidant. Previously, the cytotoxicity of caffeine was evaluated using a wide range of concentrations by the neutral red test. From this screening, adequate doses were selected to perform the caspase activity and fragmentation DNA studies. The potential antioxidant effect of caffeine was studied using tert-butyl-hydroperoxide as a free-radical generator. The repeatability was checked through three separate tests with the same concentration.

In vitro toxicity of stimulant soft drinks.

Some drinks containing natural products with stimulant properties are highly consumed among young adults. The market for these drinks has increased in past years around the globe, and, although they might be harmless, overdoses or combination of these with other drinks could be harmful to the health of some consumers in certain circumstances. Samples were obtained at food shops and different popular brands were chosen. Cytotoxicity tests used were neutral red uptake, total protein content, and tetrazolium assay on Chinese hamster ovary cells. Results revealed that tested samples were not cytotoxic; however, studies have demonstrated the toxicity of high concentrations of some of these products. For this reason, the authors considered it to be of critical importance to carryout an in vitro toxicity screening of stimulant soft drinks that are highly consumed.

Comparación de la citotoxicidad del amaranto en dos líneas celulares de mamífero utilizando bioensayos colorimétricos rápidos / Comparison of amaranth cytotoxicity in two mammalian cell lines using rapid colorimetric bioassays

El amaranto es un colorante de color rojo ampliamente utilizado en alimentación y cosmética. En la bibliografía consultada no se han encontrado referencias sobre la citotoxicidad de este colorante por lo que se ha creído oportuno abordar su estudio. Este estudio se ha llevado a cabo en dos líneas celulares de naturaleza fibroblástica: línea celular 3T3 (fibroblastos de origen embrionario humano). La citotoxicidad fue evaluada mediante dos bioensayos: a) ensayo de rojo neutro (RN); b) ensayo de proteína total (PT). Se han calculado las IC50 del amaranto en ambas líneas celulares, observándose mayor sensibilidad al colorante con las células 3T3 que con los fibroblastos humanos (AU)

In vivo functional analysis of ezrin during mouse blastocyst formation.

During mouse blastocyst formation, a layer of outer cells differentiates in less than 48 h into a functional epithelium (the trophectoderm). Ezrin, an actin-binding structural component of microvilli in epithelial cells, is also involved in signal transduction and ionic pump control. In the mouse embryo, ezrin becomes restricted to the apical cortex of all blastomeres at compaction and of outer cells at later stages. Here we investigated the function of ezrin in living embryos during epithelial differentiation using mutant forms of ezrin tagged with green fluorescent protein (GFP). GFP-tagged wild-type ezrin (Ez/GFPc) behaved like endogenous ezrin and did not interfere with development. Deletion of the last 53 amino acids (Delta53/GFP) changed the localization of ezrin: after compaction, Delta53/GFP remained associated with the apical and basolateral cortex in all blastomeres, and its expression slightly disturbed the cavitation process. Finally, full-length ezrin with GFP inserted at position 234 (Ez/GFPi) was localized all around the cortex throughout development, although it was concentrated at the apical pole after compaction. In embryos expressing Ez/GFPi, the duration of the 16-cell stage was reduced, while the onset of cavitation was delayed. Moreover, cavitation was abnormal, and the blastocoele was small and retracted almost completely several times as if there were major leakages of blastocoelic fluid. Our results suggest that, in addition to its role in microvilli organization, ezrin is involved in the formation of a functional epithelium through a still unknown mechanism.

Sister chromatid exchange induced by several types of coffees in Chinese hamster ovary cells.

Different brands of commercial caffeinated and decaffeinated coffees (roasted, high roast, blend ground, and instant coffees) were studied. These coffees were tested for their ability to induce sister chromatid exchanges (SCE) in CHO-K1 cells. Tests were performed in the presence and in the absence of a metabolic activation system (S-9 mix). Results were compared to the roasting procedure because genotoxic products could be formed from these processes. Our results indicate that caffeinated instant coffees showed higher genotoxic activity than decaffeinated coffees. Non-significant genotoxic activity was detected with the green coffee (unroasted). The highest increase of the frequency of SCE occurred when the caffeinated instant coffee was tested in the absence of metabolic activation system. The repeatability of the test was checked through three assays with the same sample.

A major posttranslational modification of ezrin takes place during epithelial differentiation in the early mouse embryo.

The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.

Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.

We have studied the fate of the nuclear envelope (NE) in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), we observed a selective and conserved cleavage of LB, LAP2 and Nup153. In lymphoid cells, the rate of cleavage of these markers was independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 was probably caspase 3, since (1) cleavage of both proteins was specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. As LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, we suggest that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study.

Evaluation of the toxicity of guarana with in vitro bioassays.

A natural stimulant, Paullinia cupana, commonly called guarana, was tested for its ability to induce in vitro toxicity in Chinese hamster ovary (CHO) cells and bacterial cells (Photobacterium phosphoreum). The cytotoxic effects of aqueous guarana extracts were evaluated by three endpoint systems: neutral red (NR) uptake assay, total protein content [kenacid blue (KB)] assay, and tetrazolium (MTT) assay. The Microtox test was also used. Results indicated that the lowest concentration of guarana tested was not toxic and that the IC50 values calculated with the NR, KB, and MTT assays were lower than the highest concentration tested (40 mg/ml). There was no significant difference in cytotoxicity between the three test systems. The EC50 values obtained with the Microtox assay were consistent with these data. The present in vitro analysis suggests that the concentration of guarana is of critical importance in its cytotoxic activity and high doses could be harmful to human health.