Acoustophoretic Motion of Leishmania spp. Parasites.

The analysis of cell motion in an acoustic field is of interest as it can lead to new methods of cell separation, isolation and manipulation for diagnosis and treatment of diseases. Studies of the motion of different species of Leishmania parasites during exposure to ultrasonic standing waves in a microfluidic device allowed identification of acoustic responses of these parasites in their promastigote and amastigote forms. Both forms exhibited a positive acoustic contrast factor and were driven toward the pressure node established in the center of the channel by the acoustically induced radiation force (FR). Promastigotes experience calculated FR amplitudes one order of magnitude larger than those experienced by amastigotes because of the measured differences in volume. The aggregates formed at the pressure node have distinct shapes and stability conditions, for both promastigotes and amastigotes.

Morphological and electrical disturbances after split-flow fractionation in murine macrophages.

Split-flow fractionation (SPLITT) is a family of techniques that separates in the absence of labeling using very low flow rates and force fields, and is therefore expected to minimize cell damage. Although it has been documented that separation methods cause physiological changes in immune cells that are attributable to mechanical stress and antibody labeling, SPLITT has not yet been examined for possible damaging effects of hydrodynamic stress, partly because it is assumed that the low flow rates and weak forces used in this technique do not generate significant mechanical stress. The aim of this study was to investigate the effects of SPLITT on cell function of a murine macrophage cell, and to compare these effects with those induced by centrifugation. Macrophages J774.2 were cultured in RPMI-enriched media, then detached from the culture flask and resuspended for 12 h. Cell suspensions were diluted in a buffered saline solution and exposed to SPLITT (flow rates 1-10 ml/min) or centrifugation (100-1500g) for 10 min. Cell viability, diameter, membrane potential, and nitric oxide production were measured. Under the operating conditions employed, cell viability was above 98% after SPLITT and centrifugation but cells suffered immediate hydrodynamic cell damage, including decreased cell diameter and membrane hyperpolarization which was inhibitable by 4-aminopyridine; nitric oxide production was not affected. Pressure values during SPLITT and centrifugation correlated with diameter and membrane potential. Our data do not support the assumption that SPLITT is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Membrane potential and cell diameter are sensitive indicators for the evaluation of sublethal damage in different cell models, and allow identification of optimal operating conditions on different scales.

Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells.

Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1-10 ml/min) or centrifugation (100-1500 g) for 10 min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales.

Valoración optométrica y socioeconómica de personas asociadas a Corpocima de la localidad de Bosa / Optometric and socio-economic assessment of people associated

Introducción: este proyecto de investigación se inició en el 2008 y culminó en el 2010. Fue realizado por un grupo interdisciplinario conformado por profesionales del área de economía, trabajo social, psicología y optometría. Objetivo: potenciar capacidades y empoderamiento de hombres y mujeres con discapacidad visual con miras al fortalecimiento de la gobernabilidad democrática, la incidencia en las políticas públicas, consecución del desarrollo humano y desarrollo de circuitos económicos en la localidad de Bosa. Desde el punto de vista de la optometría, el objetivo específico fue generar alternativas desde la perspectiva del desarrollo humano sustentable, mediante la valoración del estado visual y el diagnóstico de personas con discapacidad visual. Metodología: se realizó una valoración visual completa que incluyó: agudeza visual, pentacam, sensibilidad al contraste, cámara retinal y refracción a 48 pacientes, en dos jornadas en la Universidad de La Salle. Resultados: se encontró un porcentaje del 8,33% de personas ciegas, frente a un 25% de personas con limitación visual o baja visión, lo que permite el conocimiento real del problemaque representa cualquier tipo de limitación, cuya consecuencia es el aumento de personas no activas laboral, económica y socialmente, ocasionando un problema serio de salud pública.

Introducción: este proyecto de investigación se inició en el 2008 y culminó en el 2010. Fue realizadopor un grupo interdisciplinario conformado por profesionales del área de economía, trabajo social, psicología y optometría. Objetivo: potenciar capacidades y empoderamiento de hombres ymujeres con discapacidad visual con miras al fortalecimiento de la gobernabilidad democrática, la incidencia en las políticas públicas, consecución del desarrollo humano y desarrollo de circuitos económicos en la localidad de Bosa. Desde el punto de vista de la optometría, el objetivo específico fue generar alternativas desde la perspectiva del desarrollo humano sustentable, mediante la valoración del estado visual y el diagnóstico de personas con discapacidad visual. Metodología: se realizó una valoración visual completa que incluyó: agudeza visual, pentacam, sensibilidad al contraste, cámara retinal y refracción a 48 pacientes, en dos jornadas en la Universidad de La Salle. Resultados: se encontró un porcentaje del 8,33% de personas ciegas, frente a un 25% de personas con limitación visual o baja visión, lo que permite el conocimiento real del problemaque representa cualquier tipo de limitación, cuya consecuencia es el aumento de personas no activas laboral, económica y socialmente, ocasionando un problema serio de salud pública.

Introduction: This research project began in 2008 and culminated in 2010. It was carried out by an interdisciplinary group made of professionals in the areas of economics, social work, psychology and optometry. Objective: To improve the skills and empowerment of men and women with vision impairment, aiming to strengthen democratic governance, incidence in public policies,attainment of human development and development of economic circuits in the Bosa locality.From the point of view of optometry, the specific purpose was to generate alternatives from aperspective of sustainable human development through the assessment of visual condition and the diagnosis of people with vision impairment. Methodology: A complete visual assessment was made that included: visual acuity, pentacam, contrast sensitivity, retinal camera and refraction to 48 patients during two days at La Salle University. Results: A percentage of 8.33% of blind people was found, next to a 25% of people with visual limitation or low vision. This makes it possible to get a real knowledge of the problem represented by any type of limitation, the consequence of which is a higher number of non-active people in the work market, as well as in the economic and social world, thus generating a serious public health problem.

Mecanismos de sllida de parásitos intracelulares de su célula hospedrra / Exit Mechanisms of Intracellular Parasites from their Host Cells

Algunos parásitos intracelulares durante la infección en hospederos vertebrados se localizan al interior de sus células hospederas en un compartimiento intracelular rodeado por membrana denominado vacuola parasitófora. Para el sostenimiento e incremento de las infecciones causadas por estos parásitos es necesario que se dé un evento de liberación/salida de las formas infectivas, para que estas reinicien la infección en nuevas células. Para dicho fenómeno de liberación se han planteado dos mecanismos básicos: 1. la salida se da por eventos de ruptura de la membrana de la vacuola parasitófora (MVP) y de la membrana plasmática de la célula hospedera y/o 2. la salida se da por un proceso de fusión entre la MVP y la membrana de la célula hospedera, de forma que la luz de la VP y el espacio extracelular se hacen continuos, permitiendo la liberación del parásito. En esta revisión se presenta la evidencia que apoya estos modelos en bacterias y protozoarios intracelulares obligatorios, con especial énfasis en la salida de Leishmania.

Some intracellular parasites inhabit intracellular compartments known as parasitophorous vacuoles. To maintain and amplify infection, infective forms of the parasite must exit from the host cell to infect new cells. During parasite egress, two main mechanisms have been proposed: lysis of the membranes of the parasitophorus vacuole and the plasma membrane of the cell host, or fusion of these two membranes providing continuity between the lumen of the parasitophorous vacuole and the extracellular compartment. In this review we present evidence supporting these models for intracellular parasites of human importance with particular emphasis on Leishmania exit.

Cambios en la relación acomodación convergencia AC/A en el valor de las forias después de cirugía refractiva Lasik / Changes of the convergence accomodation relation (A/CA) and the forias in patiens before and after refractive surgery Lasik

Objetivo: determinar cambios significativos de la relación acomodación convergencia (AC/A) y las forias en pacientes antes y después de cirugía refractiva con técnica Lasik. Metodología: se realizó un estudio prospectivo para una muestra poblacional de veinte pacientes (cuarenta ojos) sometidos a cirugía refractiva, evaluados ortópticamente en Optiláser en Bogotá. Se determinó como criterio de inclusión haber sido valorado ortópticamente antes y después de la cirugía refractiva. Se evaluaron condiciones motoras, sensoriales, signos, síntomas, agudeza visual, AC/A, forias con cover test y con varilla de maddox. Resultados: en la medida de las forias se observa qué tanto para Cover Test como para Varilla de Maddox no se presentan cambios estadísticamente significativos entre el pre y el posquirúrgico. La relación AC/A calculada con el método de la Heteroforia tuvo valores similares antes y después de la cirugía refractiva; estadísticamente, no se observan cambios significativos, pues se mantuvieron los valores normales en un promedio de 55 por ciento (once pacientes) y 65 por ciento (trece), respectivamente. En cuanto a los pacientes con ACA bajo, se observó que antes de la cirugía había 45 por ciento (nueve pacientes) y después de la cirugía 65 por ciento (seis pacientes). Cabe resaltar que un paciente (5 por ciento) que presentaba foria antes de la cirugía se descompensó en el posquirúrgico, lo que generó tropia en visión próxima y, por ende, imposibilidad en la realización de la valoración de ACA. Conclusiones: la relación acomodación convergencia en pacientes sometidos a cirugía refractiva no presenta cambios significativos, con respecto a su condición inicial. El cover test y la varilla de Maddox reportan diferencias, encontrándose mayor alteración en los valores de las forias en la varilla de Maddox; es decir, que con el método disociante se evidenció un mayor valor de ésta y se encontró una variación significativa con respecto al valor encontrado con el método habitual.

The objetive of the study was to determine significant changes of the convergence accomodation relation (A/CA) and the forias in patiens before and after refractive surgery. Methodos: 40 eyes was submitted to refractive surgery, evaluated by ortoptic in a refractive center OPTILASER. Conditions such as motored, sensorial, signs, symptoms, visual a harpness, AC/A, binocular vision were evaluated with cover test and with maddox rod. Results: in the measures of forias, it is observed that so much for cover test as for maddox rod, there are not significant statistically changes between the pre and the postsurgical pursuit.The relation AC/A calculated with the method of the heteroforia, had similar values before and after the refractive surgery, statistically there are not significant changes observed; therefore the normal values were maintained in an average of 55 percent (11 patients) and 65 percent (13) respectively. As for the patiens with ACA is observed that before the surgery there was 45 percent ( 9 patients) and after the surgery 65 percent (6 patients). It fits to stand out that 1 patient (5 percent) that presented foria before the surgery, is descompensated in the postsurgical treatment generating strabismus in near vision and therefore impossibility in the execution of the appraisal of ACA. Conclusions: the convergence accomodation (AC/A) relation in patients submitted to refractive surgery does not present significant changes, with respect to its initial condition. The cover test and the Maddox rod report differences finding greater alteration in the values of the forias in the maddox rod; that is to say that withthe dissociate method was shown a greater value of the same one, finding a significant variation with respectto the value found with the habitual method.

Changes in macrophage membrane properties during early Leishmania amazonensis infection differ from those observed during established infection and are partially explained by phagocytosis.

Understanding the impact of intracellular pathogens on the behavior of their host cells is key to designing new interventions. We are interested in how Leishmania alters the electrical function of the plasma membrane of the macrophage it infects. The specific question addressed here is the impact of Leishmania infection on macrophage membrane properties during the first 12h post-infection. A decrease of 29% in macrophage membrane capacitance at 3h post-infection indicates that the phagolysosome membrane is donated on entry by the macrophage plasma membrane. Macrophage membrane potential depolarized during the first 12h post-infection, which associated with a decreased inward potassium current density, changed in inward rectifier conductance and increased outward potassium current density. Decreased membrane capacitance and membrane potential, with no changes in ion current density, were found in macrophages after phagocytosis of latex beads. Therefore we suggest that the macrophage membrane changes observed during early Leishmania infection appear to be associated with the phagocytic and activation processes.

Separation of Leishmania-infected macrophages by step-SPLITT fractionation.

After a primary infection protocol of macrophages with Leishmania amazonensis, the percentage of infection drops as infection progresses and the uninfected population of macrophages mask the effects of infection for electrophysiological studies. In order to increase or maintain the infection percentage, we introduce an enrichment process after primary infection, which increases the possibility of following the infection longer times than any known process. A membraneless separation technique, step-SPLITT fractionation, implying flow and transverse gravity field in a ribbon-like channel, was used for enriching samples of macrophages infected with particles and with L. amazonensis. We demonstrate the capability of the s-SPLITT of generating, from a mixture resulting from a primary infection, an enriched and a depleted fraction with infected cells, without using any selective labeling pre-processing. It is also shown that a continuous sorting is possible without damaging cells and the losses of matter into the separation chamber is minimal.

Caracterizaciónbbiológica de variantes de placade la cepa vacunal 17d contra lla fiebre amarilla / Biological charccterisation of yellow fever 17d strain plaque variatss / Caracteriação biiológica das variantes daplaca cepa da vacina 17d contraa a febre amarela

La vacuna colombiana 17D contiene por lo menos cuatro fenotipos, denominados pequeño (0,3-1,2 mm), mediano (1,3 -2,1 mm), grande (2,2 - 3,0 mm) y extra-grande (>3,1 mm). La composición y distribución porcentual de esos fenotipos variaron entre lotes y entre ampolletas de un mismo lote. Cada variante fue clonada por dilución de la vacuna y su efecto virulento fue analizado en ratones; el fenotipo de placa pequeño estuvo ligeramente sub representado en los lotes analizados y mostró una virulencia similar a la de la cepa silvestre neurotrópica francesa (DL50 >10-6), mientras que el fenotipo predominante y más atenuado fue el mediano (DL50:10-4). Los fenotipos grande y extragrande mostraron una virulencia intermedia (DL 50:10-5) con relación a los anteriores. Los análisis de secuencia de las variantes sobre una región comprendida entre el extremo 3'NS5 y el inicio de 3'NCR mostró la cercanía entre aquellas variantes con algún grado de virulencia, y entre la variante atenuada y la vacuna colombiana. La heterogeneidad de la vacuna 17D constituye una evidencia de la estructura de cuasiespecies propia de los virus RNA, y señala cómo los casos de reacciones pos vacunales adversas pueden estar asociados con la aplicación de vacunas fabricadas a partir de cepas virales atenuadas.

The Colombian 17D vaccine contains at least four phenotypes denominated small (0.3 - 1.2 mm), medium (1.3 - 2.1 mm) large (2.2 - 3.0 mm) and extra-large (>3.1 mm). These phenotypes composition and percentage distribution vary between different production lots and between ampoules from a particular lot. Each variant was cloned by diluting the vaccine and its virulent effect was analysed in young mice. The small plaque phenotype was slightly under represented in the lots analysed and showed similar virulence to the neurotropic French wild strain (LD50 >10-6), whilst the medium- sized phe-notype predominated and was the most attenuated (LD50:10-4). The large and extra-large phenotypes showed intermediate virulence (LD50:10-5) regarding the others. Sequence analysis of variants within the region between the 3'NS5 end and the beginning of 3'NCR showed the closeness between those variants having some degree of virulence and between the attenuated variant and the Colombian vaccine. The 17D vaccine's heterogeneity constitutes evidence of RNA virus quasi-specie structure and shows how adverse yellow fever vaccine reaction can be associated with applying vaccines produced from attenuated viral strains.

A vacina colombiana 17D, contém pelo menos quatro fenótipos, denominados pequeno (0.3 - 1.2 mm), médio (1.3 -2.1mm), grande (2.2 - 3.0mm) e extra grande (>3.1 mm). A composição e distribuição percentual destes fenótipos, variou entre lotes e ampolas do mesmo lote. Cada variante foi clonada por diluição da vacina e seu efeito viral foi analisado em ratos; o fenótipo da placa pequena, esteve ligeiramente sub-representado nos lotes analisados e mostrou uma virulência similar a da cepa silvestre neurotrópica francesa (LD50 >10-6), enquanto que, o fenótipo predominante e mais atenuado foi o médio (LD50:10-4). Os fenótipos grande e extra grande mostraram um estado viral intermediário (LD50:10-5) com relação aos anteriores. As análises da seqüência das variantes sobre uma região compreendida entre o extremo 3'NS5 e o início de 3'NCR, mostrou a proximidade entre aquelas variantes com algum grau de virulência, e entre a variante atenuada e a vacina colombiana. A heterogeneida-de da vacina 17D, constitui uma evidência da estrutura de qualquer espécie própria do vírus RNA e assinala como os casos de reações pós vacinação adversas, podem estar associadas com a aplicação das vacinas fabricadas a partir das cepas virais atenuadas.

Macrophage ion currents are fit by a fractional model and therefore are a time series with memory.

We studied macroscopic ion currents from macrophages and compared their patterns of behavior using classical and fractal analysis. Peak and steady state currents were measured respectively at the beginning and end of a voltage-clamp pulse. Hurst coefficients H and fractional dimensions were calculated for the current fluctuations (I(H)) during the intervening interval; these fluctuations are usually assumed to be white noise. We show that I(H) is different from 0.5 and that the increments are stationary, indicating that the dynamic model has memory and that the intervening current fluctuations cannot be considered as white noise. I(H) is less than 0.5, implying an antipersistent pattern. In addition, we show that the relation between inactivation and I(H) versus voltage V fit an equation I(H)(V) = f(V, alpha, m, d), where alpha is associated with fractional calculus and m and d are free parameters. Fitting by a fractional model confirms that the phenomenon has memory.

Determinación del efecto de la valoración ortóptica y entrenamiento visual antes y después de cirugía refractiva corneal con láser (Lasik) / Determination of the effect of the assessment orthoptics and visual training before and after of laser corneal refractive surgery (lasik)

Objetivo: establecer la importancia de la valoración ortóptica y el entrenamiento visual antes y después de la cirugía refractiva corneal con láser, técnica Lasik.Resultados: en la evaluación pre y posquirúrgica, 6 pacientes (13.63 por ciento) no presentaron alteración oculomotora,y 37 (84.09 por ciento) reportaron algún tipo de anomalíaacomodativa o motora, de los cuales 11 pacientes (29.72 por ciento) tuvieron alteraciones combinadas, 16 (43.24 por ciento) acomodativas y 10 (37.03 por ciento) motoras. En el pre y postoperatorio las frecuencias de Hirshberg tuvieron comportamiento idéntico. En la Amplitud y la flexibilidad de acomodación no se encontraron diferencias estadísticamente significativas (p > 0.05) antes y después del Lasik, aunque desde el punto de vista clínico se evidenció un aumento en las alteracionesaltas y moderadas de la amplitud de acomodacióndespués de la cirugía refractiva, al igual que se evidenció la disminución de los valores normales de la misma, en flexibilidad disminuyeron los ojos con valores de normalidad y aumentaron las alteraciones en los niveles altos y leves. La mayoría de los pacientestuvo valores de alteración de las reservas fusionalespositivas en visión lejana entre altos y moderados. Conclusiones: clínicamente, la valoración ortóptica es necesaria en todos los pacientes que son sometidosa cirugía refractiva; aunque no se observan diferenciassignificativas entre las alteraciones motoras y acomodativas, clínicamente se observó aumento de la descompensación en la magnitud de las variablesmedidas y mayor sintomatología.

Objective: To establish the importance of valuation orthoptics and visual training before or after of the corneal refractive laser surgery, Lasik.Results: In assessing pre-and post-surgical 6 patients (13.63 percent) showed no eye motor alteration and 37 (84.09 percent) reported some kind of anomaly accommodatesor motor, of whom 11 patients (29.72 percent) had abnormalitiescombined, 16 (43.24 percent) accommodative and 10 (37.03 percent) motor. In the pre-and post-operative frequencies Hirshberg had identical behavior. In the amplitude and flexibility of accommodation don’t found statistically significant differences (p> 0.05), before and after Lasik, albeit from a clinical point of view was evident increase in the alterations of high and moderate amplitude of accommodation after the refractive surgery, as is evidenced by the decrease normal values of the same, decreased flexibility in the eyes with normal values and increased disturbancesat the higher levels and mild. Most patients had values altering of the fusional reserves positive for vision near and far between High Moderate. In the values of stereopsis, the ranges of normality were stable and high and moderate alterations were correlatedwith patients with strabismic or preexisting ocular abnormalities. The next point of convergence,the cover test and the status of Correspondence Sensory did not change. 100 percent of the patients who performed visual training reported improvement in their values as in clinical symptoms.Conclusions: Clinically Orthoptics the valuation is required in all patients who undergo refractive surgery,although no significant differences between the motor impairment and accommodating, clinicallyobserved increase in the decompensation in the magnitude of the variables measured and increased symptoms.

Leishmania amazonensis infection may affect the ability of the host macrophage to be activated by altering their outward potassium currents.

Understanding the impact of intracellular pathogens on the behaviour of their host cells is key to designing new interventions. We are interested in how Leishmania alters the electrical functioning of the plasma membrane of the macrophage it infects. The specific question addressed here is whether Leishmania amazonensis infection alters the macrophage's outward currents and what the consequences of such changes might be. Using the whole cell configuration of the patch clamp technique, we show that outward peak current density remains constant over the period studied but that time to peak and sensitivity to inhibitors vary during infection. Infected cells take 40% longer to activate and are more sensitive to the potassium channel inhibitor tetraethyl ammonium, compared to control cells, indicating increased potassium outward current activity. Activation of macrophages is associated with increases of nitric oxide production and membrane area, depolarization of the macrophage membrane, down regulation of inward potassium and up regulation of outward currents. After Leishmania infection, macrophage activation is characterised by a reduction of nitric oxide production and of outward current density. We therefore suggest that this reflects a weaker activation.

Leishmania amazonensis: Anionic currents expressed in oocytes upon microinjection of mRNA from the parasite.

Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents.

Papel de la vacuola parasitófora de macrófagos de ratón infectados por Leishmania amazonensis en la adquisición de de moléculas / Role of the parasitophorous vacuole of murine macrophages infected with Leishmania amazonensis in molecule acquisition

Introducción. Leishmania son parásitos intracelulares de macrófagos, confinados en compartimentos denominados vacuolas parasitóforas. La permeabilidad de este compartimento depende de su interacción con el tráfico vesicular y transportadores presentes en su membrana. Objetivo. En este trabajo se estudió la permeabilidad de la membrana de la vacuola parasitófora en la línea celular J774.A1 infectada con Leishmania amazonensis, in situ y en compartimentos aislados. Materiales y métodos. El aislamiento de vacuolas parasitóforas se hizo por gradiente de densidad. La permeabilidad de la membrana de estas se valoró por distribución de sondas fluorescentes y electrofisiología. Para establecer indirectamente el transporte de protones se usó naranja de acridina. La presencia de transportadores ABC sensibles a probenecid se estableció con amarillo lucifer y calceína. Por primera vez con la técnica de patch-clamp se registraron corrientes en la membrana de este compartimento aislado. Resultados. La vacuola parasitófora colorea de rojo con naranja de acridina indicando un pH ácido. Concentra amarillo lucifer a través de un transportador sensible a probenecid, pero excluye la sonda calceína. Vacuolas aisladas se marcan de rojo con naranja de acridina y concentran amarillo lucifer a través de un transportador sensible a probenecid. Estas vacuolas excluyeron calceína y presentaron en su membrana una corriente iónica que se activa a diferencias de potencial cercanas a 60 mV, con una conductancia de 46 ± 3 pS. Conclusiones. Se pueden aislar vacuolas parasitóforas con propiedades de permeabilidad que preservan mecanismos de transporte similares a los encontrados in situ. Se registra por primera vez la presencia de una corriente iónica poco selectiva en la membrana de este compartimiento.

Introduction. Leishmania are intracellular parasites of macrophages, confined into compartments known as parasitophorous vacuoles. The permeability of this compartment depends on its interaction with the endocytic pathway and transport proteins present on its membrane. Objective. The membrane permeability of the parasitophorous vacuole was studied in J774.A1- macrophage like cells infected with Leishmania amazonensis, in situ and on isolated compartments. Materials and methods. The parasitophorous vacuoles were isolated by density gradients. Fluorescent probe distribution and electrophysiological recordings were used to determine parasitophorous vacuole membrane permeability. Proton transport was evaluated indirectly by acridine orange staining. Probenecid sensitive ABC transporters were detected using the fluorescent probes lucifer yellow and calcein. For the first time ion currents were recorded on the membrane of isolated parasitophorous vacuoles using the patch clamp technique. Results. The parasitophorous vacuole stains red with acridine orange indicating an acidic compartment. It concentrates lucifer yellow by means of a probenecid sensitive transporter but excludes calcein. Isolated vacuoles stained red with acridine orange and concentrated lucifer yellow by means of a probenecid sensitive transporter. These vacuoles excluded calcein and showed an ion current in their membrane which is activated at potentials close to 60 mV with a mean conductance of 46 ± 3 pS. Conclusions. Isolated parasitophorous vacuoles with permeability properties preserving transport mechanisms similar to those found in situ can be purified. A poorly selective ion current on the parasitophorous vacuole membrane is reported for the first time.

[Role of the parasitophorous vacuole of murine macrophages infected with Leishmania amazonensis in molecule acquisition]. / Papel de la vacuola parasitófora de macrófagos de ratón infectados por Leishmania amazonensis en la adquisición de moléculas.

INTRODUCTION: Leishmania are intracellular parasites of macrophages, confined into compartments known as parasitophorous vacuoles. The permeability of this compartment depends on its interaction with the endocytic pathway and transport proteins present on its membrane. OBJECTIVE: The membrane permeability of the parasitophorous vacuole was studied in J774.A1-macrophage like cells infected with Leishmania amazonensis, in situ and on isolated compartments. MATERIALS AND METHODS: The parasitophorous vacuoles were isolated by density gradients. Fluorescent probe distribution and electrophysiological recordings were used to determine parasitophorous vacuole membrane permeability. Proton transport was evaluated indirectly by acridine orange staining. Probenecid sensitive ABC transporters were detected using the fluorescent probes lucifer yellow and calcein. For the first time ion currents were recorded on the membrane of isolated parasitophorous vacuoles using the patch clamp technique. RESULTS: The parasitophorous vacuole stains red with acridine orange indicating an acidic compartment. It concentrates lucifer yellow by means of a probenecid sensitive transporter but excludes calcein. Isolated vacuoles stained red with acridine orange and concentrated lucifer yellow by means of a probenecid sensitive transporter. These vacuoles excluded calcein and showed an ion current in their membrane which is activated at potentials close to 60 mV with a mean conductance of 46 +/- 3 pS. CONCLUSIONS: Isolated parasitophorous vacuoles with permeability properties preserving transport mechanisms similar to those found in situ can be purified. A poorly selective ion current on the parasitophorous vacuole membrane is reported for the first time.

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis.

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization.

Los abusos de la libertad de prensa y la agonía de la intimidad: análisis de los "diarios serios" el deber, el mundo y el nuevo día de santa cruz de la sierra

El propósito de la investigación es analizar las transgresiones al derecho a la intimidad que se dan en el contenido de los periódicos El Deber, El Mundo y El Nuevo Día, amparados en el derecho a la libertad de prensa; así mismo identifica el grado de conocimienro que la sociedad tiene del derecho a la intimidad. La dualidad: libertad de prensa y derecho a la intimidad, fué el eje central de la revisión teórica y jurídica, la cual tomó como soportes importantes el consumo cultural de los medios y la legislación boliviana, elementos desde los que se identificaron los límites y las responsabilidades que deben asumir periodistas, medios y sociedad.

Los abusos de la libertad de prensa y la agonía de la intimidad

El propósito de la investigación es analizar las transgresiones al derecho a la intimidad que se dan en el contenido de los periódicos El Deber, El Mundo y El Nuevo Día, amparados en el derecho a la libertad de prensa; así mismo identifica el grado de conocimienro que la sociedad tiene del derecho a la intimidad.