Experimental toxicity of Aeromonas spp. In mouse’s small intestine: Ultrastructural aspects / Aspectos ultraestructurales de la toxicidad experimental producida por Aeromonas spp. En el intestino delgado de ratón / Aspectos ultra-estruturais da toxicidade experimental produzida por Aeromonas spp. No intestino delgado de rato

Ultrastructural aspects of mouse small intestinal tissue cultures infected with Aeromonas spp. strains are described. High resolution light and transmission electron microscopy were used to assess the bacterial pathogenic mechanism, the ultrastructural changes that take place during the colonization of the intestinal tract and the interaction of Aeromonas spp. with the host epithelium. After 24h of culture, chains of vesicles were seen on the outer surface of the Aeromonas’ membrane. The vesicles were also found on the enterocyte surface. After 48h of culture, lysis of the epithelial intestinal cells, mononuclear phagocytic cells, phagocytic eosinophils and phagocyted Aeromonas were observed.

Se describen aspectos ultraestructurales del tejido intestinal de ratón cultivado e infectado con Aeromonas spp. Se utilizó microscopía de luz de alta resolución y microscopía electrónica de transmisión para evaluar los mecanismos patogénicos bacterianos, los cambios ultraestructurales que ocurren durante la colonización del tracto intestinal por Aeromonas spp. y la interacción de éstas con el epitelio huésped. En los cultivos de 24h se observaron vesículas distribuidas en cadenas sobre la superficie de la membrana externa de las Aeromonas. Estas vesículas se observaron unidas a la superficie del enterocito. En los cultivos de 48h se observó lisis de la superficie epitelial del intestino, migración de células fagocíticas mononucleares y presencia en la cavidad intestinal de eosinófilos fagocíticos, algunos conteniendo Aeromonas en su interior.

Descrevem-se aspectos ultra-estruturais do tecido intestinal de rato cultivado e infectado com Aeromonas spp. Utilizaram-se microscopia de luz de alta resolução e microscopia eletrônica de transmissão para avaliar os mecanismos patogênicos bacterianos, As mudanças ultra-estruturais que ocorrem durante a colonização do tracto intestinal por Aeromonas spp. e a interação destas com o epitélio hospede. Nos cultivos de 24h se observaram vesículas distribuídas em cadeias sobre a superfície da membrana externa das Aeromonas. Estas vesículas se observaram unidas à superfície do enterócito. Nos cultivos de 48h se observou lise da superfície epitelial do intestino, migração de células fagocíticas mononucleares e presença na cavidade intestinal de eosinófilos fagocíticos, alguns contendo Aeromonas no seu interior.

Antibiotic resistance patterns and SDS-PAGE protein profiles of Burkholderia cepacia complex isolates from nosocomial and environmental sources in Venezuela.

BACKGROUND: Antibiotic resistance and SDS-PAGE protein patterns were determined in nosocomial and environmental isolates of the Burkholderia cepacia complex to assess similarities between them and to identify common protein bands that could be associated with resistance to certain antimicrobial agents. MATERIAL/METHODS: Antibiotic resistance patterns were determined by the disk diffusion method on Mueller-Hinton medium and minimum inhibitory concentrations were obtained by the dilution method on agar. Electrophoresis of whole-cell protein extracts and purified external membrane proteins were performed by SDS-PAGE. Based on resistance to nine antibiotics and the presence or absence of specific protein bands, dendrograms were constructed by the unweighted pair-group using the average linkage clustering method. RESULTS: Both environmental and nosocomial Bcc isolates showed resistance to multiple antibiotics; however, clinical isolates demonstrated two times higher resistance levels than environmental isolates. The Dice index similarity coefficients between environmental and nosocomial strains ranged from 72% to 91.4%. Comparative analysis between common protein bands and antibiotic resistance patterns revealed close association of Mr 135, 76, 72, 53, and 50 kDa with imipenem and aztreonam, Mr 53 and 31 kDa with trimethoprim/sulfamethoxazole, and Mr 50 kDa with ceftazidime resistance. CONCLUSIONS: 1. The environmental and nosocomial Bcc isolates showed a high degree of similarity in their protein profiles. 2. Three common protein bands, i.e. Mr of 31, 50, and 53 kDa, detected in the Bcc isolates from both clinical and natural sources could be associated with resistance to the antimicrobial agents trimethoprim/sulfamethoxazole, ceftazidime, and aztreonam, respectively.

Histotypic mouse parietal cortex cultures: excitation/inhibition ratio and ultrastructural analysis.

Primary cultures of mouse parietal cortex, prepared between postnatal day 3 (P3) and P9, were studied using transmission electron microscopy and HPLC of excitatory (aspartate and glutamate) and inhibitory neurotransmitters (glycine, GABA and taurine) to determine their morphological and functional development. Relations between excitation and inhibition (E/I) were contrasted with ultrastructural features over the time course of in vitro development. After 6 days in vitro, cultured parietal cortex neurons prepared from mice at P3 had immature morphological characteristics, whereas P5 cultures showed a more developed histological structure but still with scarce synapses. The acquirement of histotypic characteristics was seen in P7 cultures, which contained numerous symmetric and asymmetric synaptic contacts. On P9, the cultures showed signs of tissue damage. In terms of neurotransmitter levels and E/I ratios, P7 cultures had relatively low E/I ratio as compared with the rest of the cultures prepared before or after P7. These results demonstrated that the development of inhibitory synaptic transmission, as indicated in the fall of E/I ratio, marked the maturation of cerebral cortical tissue and that the critical period to obtain histotypic cultures of mouse parietal cerebral cortex coincides between P5 and P7. This work provides useful information regarding the balance between excitation and inhibition as an indicative parameter for in vitro nerve cell survival, differentiation and maturation and reinforces the great value of histotypic cultures in the study of central nervous system development.

Metabolismo mitocondrial y ultraestructura de músculo esquelético de ratón con restricción calórico proteica / Mitochondrial metabolism and ultrastructure of mice's skeletal muscle with caloric proteic restriction

Para estudiar el metabolismo mitocondrial en este trabajo se utilizó el método de permeabilización del tejido muscular mediante saponina, la cual destruye el sarcolema, manteniendo las mitocondrias "in situ". Para crear un modelo de patología mitocondrial se produjo restricción calórico-proteica (RCP) en ratones adultos con un 50 por ciento de la dieta de los ratones alimentados "ad libitum" durante un período de 15 días. Se efectuaron estudios del consumo de oxígeno, relación ADP/O, así como la actividad enzimática de los Complejos I y II de la cadena respiratoria y la determinación de proteínas. Para evaluar el efecto de la saponina sobre el tejido muscular en presencia y ausencia de restricción calórica-proteica se observaron muestras del mismo al microscopio electrónico de transmisión. Los resultados revelaron en los animales una pérdida de peso, disminución en el consumo de oxígeno y de la relación ADP/O en presencia de glutamato y de succinato, por otra parte se obtuvo una disminución evidente de la actividad del Complejo I, sin modificación del Complejo II. Las observaciones obtenidas con el microscopio electrónico demuestran que la RCP en ratas tiene efecto directo sobre el aparato contráctil de las fibras musculares, produciendo desorganización de las estructuras sarcoméricas y reducción importante de los miofilamentos. La saponina, por otra parte afecta todo el sistema de citomembranas.

In this work, the method of permeabilization of muscular tissue with saponin was employed, which destroyed the sarcolemma but maintained the mitochondria "in situ". To create a model of mitochondrial pathology, a caloric-protein restriction (CPR) was produced in adult mice by reducing 50 percent the diet to fed animals for 15 days. Determinations of oxygen consumption, ADP/O ratio, total proteins, as well as enzymatic activities of respiratory chain Complexes I and II were performed. To evaluate the effect of saponin on muscular tissues under normal or CPR conditions, electron microscopy observations were done. Results of the study showed that the animals in CPR group had loss of weight, reduced total proteins, diminished oxygen consumption, lower ADP/O ratio in the presence of glutamate and succinate, and a notable decrement in the activity of Complex I but without modifications of that of Complex II. Electron microscopic observations demonstrates directs effects of CPR over the contractile apparatus of muscle fibers, such as, disorganization of sarcomeric structures and an important reduction of the myofilaments. On the other hand, saponin affected all cytomembranous systems.

UV-screening strategies of a lower eukaryote grown in hydrocarbon media.

In this paper, a detailed analysis of the UV-screening strategies of the fungus Fusarium alkanophyllum is offered using spectroscopic (UV-VIS, FTIR), chromatographic (TLC, HPLC) and physiological analysis methods. Fusarium alkanophyllum showed an optimum growth when exposed to UV radiation at 253.7 (inducing DNA and protein damages) or 354.5 nm (inducing photoxidative damage) in several hydrocarbon media. Further, no ultrastructural difference was seen when cultures were or not irradiated with monochromatic UV. High absorbance between 200-300 nm of F. alkanophyllum indole derivatives, viz. melanin-type pigments, suggests a protector effect for proteins and nucleic acids. The presence of sulfur linked to aliphatic groups in hydrocarbons which is itself a strong UV absorber in the region of lambda < 270 nm can explain why mineral oil and kerosene showed high absorptivity at the UVC and UVB ranges. In light hydrocarbon, high absorptivities at the UVB and UVA spectral regions could be explained due to the presence of C=O stretching vibrations corresponding to ketones linked to aliphatic groups. The occurrence of indole derivatives in modern fungi may be a significant relic of the early evolution of microbial pigmentation. Likewise, it is thought that sulfur-enriched heterogeneous hydrocarbon environments could have occurred on the surface of the early Earth and could have absorbed and scattered UV-radiation avoiding or minimizing the damage produced on the biochemical machinery of early microorganisms able to metabolize those hydrocarbons.

Effects of prenatal paraquat and mancozeb exposure on amino acid synaptic transmission in developing mouse cerebellar cortex.

The goal of this study was to analyze the effects of prenatal exposure to the pesticides paraquat (PQ) and mancozeb (MZ) on the development of synaptic transmission in mouse cerebellar cortex. Pregnant NMRI mice were treated with either saline, 10 mg/kg PQ, 30 mg/kg MZ or the combination of PQ + MZ, between gestational days 12 (E12) and E20. Variation in the levels of amino acid neurotransmitters was determined by HPLC, between postnatal day 1 (P1) and P30. Motor coordination was assessed by locomotor activity evaluation of control and experimental pups at P14, P21 and P30. Significant reductions in the levels of excitatory neurotransmitters, aspartate and glutamate, were observed in PQ-, MZ- or combined PQ + MZ-exposed pups, with respect to control, during peak periods of excitatory innervation of Purkinje cells: between P2-P5 and P11-P15. However, at P30, lower aspartate contents, in contrast with increased glutamate levels, were detected in all experimental groups. During the first two postnatal weeks, delays in GABA and glycine ontogenesis were observed in PQ- and PQ + MZ-exposed pups, whereas notable decrements in GABA and glycine levels were seen in PQ + MZ-exposed animals. Decreased taurine contents were detected at P3 and P11 in PQ- and PQ + MZ-exposed mice. Pups in different experimental groups all showed hyperactivity at P14 and then exhibited reduced locomotor activity at P30. Taken together, our results indicate that prenatal exposure to either PQ or MZ or the combination of both could alter the chronology and magnitude of synaptic transmission in developing mouse cerebellar cortex.

Daño ultraestructural del intestino medio abdominal de Lutzomyia ovallesi (Ortiz) (Diptera: Psychodidae) ocasionado por Leishmania (Leishmania) amazonensis / Ultrastrutuctural damage to the middle intestine of Lutzomyia ovallesi (Ortiz) (diptera: Psychodidae) caused by Leishmania (Leishmania) amazonensis

Se estudió el intestino medio abdominal de Lutzomyia ovallesi infectada con Leishmania (Leishmania) amazonensis. Las hembras se observaron a los siete días post- infección, utilizando microscopía de luz de alta resolución y microscopía electrónica de transmisión. Se distinguieron dos tipos de células epiteliales, células digestivas y células secretoras, en el intestino medio abdominal de L. ovallesi, tanto en los insectos controles como en flebotominos infectados. Los resultados muestran además, que L. ovallesi presentó alteraciones en la citoar-quitectura celular del intestino medio abdominal producto de la infección con L. (L.) amazonensis, observándose gran distensión del diámetro de la luz intestinal, degeneración de sus células, pérdida parcial o total de las microvellosidades y engrosamiento de la capa basal de toda la porción del intestino. Las células epiteliales presentaron degeneración vacuolar y mitocondrial y en la luz intestinal se observaron desechos epiteliales. El daño celular observado en el intestino medio abdominal de L. ovallesi, pueden tener relación con la secreción de lectinas y con la formación del gel observado, semejante a una matriz, en el lumen del intestino. Es necesario continuar los estudios para dilucidar aspectos importantes de la interrelación Leishmania-vector.

Prenatal and postnatal contents of amino acid neurotransmitters in mouse parietal cortex.

This study documents the variation in the amino acid neurotransmitter contents during mouse parietal cortex development, from embryonic day 13 (E13) until young adulthood, between postnatal day 21 (P21) and P30. Taurine, an inhibitory neurotransmitter and neuromodulator, is the most abundant neurotransmitter in the developing neocortex, whereas, at the adult stage, glutamate is the more prominent neurotransmitter playing an excitatory role, and GABA is the major inhibitory neurotransmitter. During the proliferative stage of neurogenesis in the mouse cerebral cortex, between E13 and E17, relatively high levels of glutamate, aspartate, taurine and glycine were detected, consistent with a possible trophic influence of these neurotransmitters during cortical development prior to synaptogenesis. Between E17 and E19, a significant decline in the contents of these neurotransmitters was observed, consistent with earlier reports of cell death in the ventricular and subventricular zones during this stage of development. During the perinatal period, a progressive increment in glutamate level was seen between E21 and P5, and then the values remained constant until the second postnatal week. Glutamate also decreased by about 25% between P11 and P15, on the other hand, aspartate diminished by about 20% between P7 and P9. These results were consistent with previous reports of histogenetic cell death during the first 2 postnatal weeks in mouse neocortex. GABA increased from the embryonic period until young adulthood, in contrast, the glycine content decreased; thus, in the adult parietal cortex, the GABA content was about 2.6-fold higher than that of glycine. During the first postnatal week, the concentrations of glutamate and GABA showed significant increments between P0 and P5, while those of aspartate and glycine remained constant. During this period, amino acids are predominantly excitatory and the cerebral cortex is vulnerable to epileptiform activity; the significant increment in taurine content between P0 and P3 suggests a neuroprotective action of taurine against excitotoxicity. At P15, coinciding with the period of maximum cortical synaptogenesis, significant increments in GABA and glycine contents were observed which could be related to the maturation of inhibitory synaptic transmission. At the young adult stage, there was a rise in the levels of both excitatory neurotransmitters, glutamate and aspartate, and a significant reduction in the contents of all three inhibitory neurotransmitters, GABA, glycine and taurine.

Levels of amino acid neurotransmitters during neurogenesis and in histotypic cultures of mouse spinal cord.

The development of spinal cord interneurons and the formation of interneuronal synaptic connections has received little attention; the most comprehensively studied developing circuit has been the connection between motoneurons and the muscle they innervate. All motoneurons are cholinergic whereas spinal interneurons are mostly glutamatergic, glycinergic or GABAergic neurons. In this study, we show quantitative data, obtained by high-pressure liquid chromatography (HPLC), on the levels of amino acid neurotransmitters during mouse spinal cord neurogenesis, from embryonic day (E) 12 until postnatal day (P) 30. At E12, high levels of glutamate, glycine and taurine were already detected but between E16 and P3, significant increments in their contents were observed, indicating the occurrence of maximum synaptogenesis during this period. Important reductions in their contents were also observed in two stages: between E12-E16 and P3-P7. These results suggest that the apoptotic death of interneurons and motoneurons in the developing brain or the synapse refinement of neural circuitry during maturation reduced the number of synapses, thereby decreasing the levels of neurotransmitters. The contents of these neurotransmitters were also analyzed in primary cultures of mouse spinal cord prepared from embryos between E13 and E19. As deduced from light microscopy, ultrastructural studies, as well as results from HPLC analysis, the cultures derived from E15-E16 embryos showed the highest degree of histotypic features and neurotransmitter contents comparable with those obtained in situ. Although glycine, GABA and taurine levels reached about 80-90% of normal in situ values, the contents of aspartate and glutamate were lower by about 40%, which could be mainly due to deafferentation of both sensory and supraspinal afferent axon terminals. These results indicate that intrinsic synaptic circuits can be maintained in histotypic spinal cord cultures prepared from E15-E16 mouse embryos. Histotypic cultures of the spinal cord will serve as a good model for studies on the pathophysiology of amino-acid-based neurotransmission and repair strategies in many CNS disorders.

Efecto de la desnutricion calorico-proteica y del estres hipoxido sobre la ultraestructura del musculo cardiaco de rata / Protein-energy malnutrition effect and hypobaric stress on ultrastructure of the heart muscle in rats

El propósito del presente trabajo fue estudiar los efectos de la restricción calórico-proteica y del estrés hipóxico agudo en el miocardio de rata. Se utilizaron ratas machos adultas de cepa Wistar distribuidas en 4 grupos: A1, A2, B1 y B2. Los grupos A1 y A2 controles, fueron alimentados ad libitum con una dieta consistente de 16 por ciento proteínas, 43 por ciento carbohidratos y 6 por ciento lípidos; y los grupos B1 y B2 con un 70 por ciento de restricción alimentaria con respecto a los grupos anteriores. Ambos grupos fueron alimentados por un período de 15 días, al cabo de los cuales los grupos A2 y B2 fueron sometidos a estrés hipóxico en una cámara de descompresión durante unas 2 horas, a una altura simulada de 3.500 m. Se obtuvieron muestras de miocardio para aislar mitocondrias y para el análisis ultraestructural. En el grupo con restricción calórico-protéica, hubo una significativa disminución del peso corporal y del músculo cardíaco, así como de las proteínas totales. Las proteínas mitocondriales en cambio, presentaron un discreto aumento. Estas observaciones se correlacionan con los hallazgos ultraestructurales en el mismo grupo experimental, en el cual se apreció disminución del número de miofilamentos contráctiles, una configuración ondulante de las crestas mitocondriales e incremento del número de mitocondrias. Las alteraciones fueron más evidentes en el grupo sometido a estrés hipóxico, observándose daño en las estructuras contráctiles, con desparición casi total de las mismas. Se sugiere que la desnutrición calórico-protéica, acompañada de hipoxia, es probablemente uno de los factores que inciden más frecuentemente en la aparición de patología cardíaca en las zonas altas andinas.

Nuevos conceptos en la etiopatogenia de la artrosis / New concepts in the etiopatogenic of the osteoarthritis

Son múltiples los agentes nocivos que pueden desencadenar una lesión artrósica, pero no en forma aislada y sin un terreno que predisponga al paciente. Sin embargo, nuestros estudios estructurales del cartílago indican que el centro de la lesión artrósica es el condrocito, y que independientemente de cuáles sean los factores etiopatogénicos, debe ocurrir una alteración de la interfase hueso-cartílago para que se produzcan daños en este tejido, y se altere, rompiéndose el equilibrio cuya consecuencia son las alteraciones descritas

Bursitis calcificada del hombro / Shoulder calcified bursitis

El autor efectúa un enfoque de la bursitis calcificada del hombro, en pacientes que concurrieron a la Consulta de Ortopedia y Traumatología del Hospital Universitario de Los Andes. En los casos en que se practicó resección quirúrgica de la lesión se procedió a analizar el material con microscopio de luz y electrónico en el centro de Microscopía Electrónica de la Universidad de Los Andes. Los hallazgos permiten concluir al autor que se trata de un proceso de metaplasia de tejido fibroblástico en tejido óseo bajo algunas condiciones especiales, es decir, que la bursitis calcificada de hombro no es un simple depósito de material cálcico entre las fibras conjuntivas