Armando Zepeda-Rodríguez

Ependymal epithelium disruption after vanadium pentoxide inhalation. A mice experimental model.

The blood-brain barrier (BBB) protects the CNS against chemical insults. Regulation of blood-brain tissue exchange is accomplished by ependymal cells, which possess intercellular tight junctions. Loss of BBB function is an etiologic component of many neurological disorders. Vanadium (V) is a metalloid widely distributed in the environment and exerts potent toxic effects on a wide variety of biological systems. The current study examines the effects of Vanadium pentoxide (V2O5) inhalation in mice ependymal epithelium, through the analysis of the brain metal concentrations and the morphological modifications in the ependymal cells identified by scanning and transmission electron microscopy after 8 weeks of inhalation, in order to obtain a possible explanation about the mechanisms that V uses to enter and alter the CNS. Our results showed that V2O5 concentrations increase from the first week of study, stabilizing its values during the rest of the experiment. The morphological effects included cilia loss, cell sloughing and ependymal cell layer detachment. This damage can allow toxicants to modify the permeability of the epithelium and promote access of inflammatory mediators to the underlying neuronal tissue causing injury and neuronal death. Thus, understanding the mechanisms of BBB disruption would allow planning strategies to protect the brain from toxicants such as metals, which have increased in the atmosphere during the last decades and constitute an important health problem.

Cd2+ resistance mechanisms in Methanosarcina acetivorans involve the increase in the coenzyme M content and induction of biofilm synthesis

To assess what defence mechanisms are triggered by Cd(2+) stress in Methanosarcina acetivorans, cells were cultured at different cadmium concentrations. In the presence of 100 μM CdCl2, the intracellular contents of cysteine, sulfide and coenzyme M increased, respectively, 8, 27 and 7 times versus control. Cells incubated for 24 h in medium with less cysteine and sulfide removed up to 80% of Cd(2+) added, whereas their cysteine and coenzyme M contents increased 160 and 84 times respectively. Cadmium accumulation (5.2 μmol/10-15 mg protein) resulted in an increase in methane synthesis of 4.5 times in cells grown on acetate. Total phosphate also increased under high (0.5 mM) Cd(2+) stress. On the other hand, cells preadapted to 54 μM CdCl2 and further exposed to > 0.63 mM CdCl2 developed the formation of a biofilm with an extracellular matrix constituted by carbohydrates, DNA and proteins. Biofilm cells were able to synthesize methane. The data suggested that increased intracellular contents of thiol molecules and total phosphate, and biofilm formation, are all involved in the cadmium resistance mechanisms in this marine archaeon.

Air-Adapted Methanosarcina acetivorans Shows High Methane Production and Develops Resistance against Oxygen Stress

Methanosarcina acetivorans, considered a strict anaerobic archaeon, was cultured in the presence of 0.4–1% O2 (atmospheric) for at least 6 months to generate air-adapted cells; further, the biochemical mechanisms developed to deal with O2 were characterized. Methane production and protein content, as indicators of cell growth, did not change in air-adapted cells respect to cells cultured under anoxia (control cells). In contrast, growth and methane production significantly decreased in control cells exposed for the first time to O2. Production of reactive oxygen species was 50 times lower in air-adapted cells versus control cells, suggesting enhanced anti-oxidant mechanisms that attenuated the O2 toxicity. In this regard, (i) the transcripts and activities of superoxide dismutase, catalase and peroxidase significantly increased; and (ii) the thiol-molecules (cysteine + coenzyme M-SH + sulfide) and polyphosphate contents were respectively 2 and 5 times higher in air-adapted cells versus anaerobic-control cells. Long-term cultures (18 days) of air-adapted cells exposed to 2% O2 exhibited the ability to form biofilms. These data indicate that M. acetivorans develops multiple mechanisms to contend with O2 and the associated oxidative stress, as also suggested by genome analyses for some methanogens.

Cadmium removal by Euglena gracilis is enhanced under anaerobic growth conditions

The facultative protist Euglena gracilis, a heavy metal hyper-accumulator, was grown under photo-heterotrophic and extreme conditions (acidic pH, anaerobiosis and with Cd(2+)) and biochemically characterized. High biomass (8.5×10(6)cellsmL(-1)) was reached after 10 days of culture. Under anaerobiosis, photosynthetic activity built up a microaerophilic environment of 0.7% O₂, which was sufficient to allow mitochondrial respiratory activity: glutamate and malate were fully consumed, whereas 25-33% of the added glucose was consumed. In anaerobic cells, photosynthesis but not respiration was activated by Cd(2+) which induced higher oxidative stress. Malondialdehyde (MDA) levels were 20 times lower in control cells under anaerobiosis than in aerobiosis, although Cd(2+) induced a higher MDA production. Cd(2+) stress induced increased contents of chelating thiols (cysteine, glutathione and phytochelatins) and polyphosphate. Biosorption (90%) and intracellular accumulation (30%) were the mechanisms by which anaerobic cells removed Cd(2+) from medium, which was 36% higher versus aerobic cells. The present study indicated that E. gracilis has the ability to remove Cd(2+) under anaerobic conditions, which might be advantageous for metal removal in sediments from polluted water bodies or bioreactors, where the O₂ concentration is particularly low.

Visualization and 3D Reconstruction of Flame Cells of Taenia solium (Cestoda)

Background Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function. Methodology/Principal Findings Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells. Conclusions/Significance We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

Ultrastructure and motility of the caudal epididymis spermatozoa from the volcano mouse (Neotomodon alstoni alstoni Merriam, 1898)

The volcano mouse Neotomodon alstoni alstoni is a genus endemic to the higher elevations of the Mexican transvolcanic belt. In the present study we examined for the first time the morphological features of the spermatozoa taken from the caudal epididymis of this species by transmission and scanning electron microscopy. Spermatozoan motility was studied in sucrose and bicarbonate solutions; vitality and morphology were observed by light microscopy. Transmission electron microscopy shows that the head of spermatozoon is asymmetric and possesses a large and curved hook. The axoneme of the spermatozoan tail is highly developed at fibers 1, 5, and 6. Absolute and relative measurements of the length of the head, the midpiece, and the rest of the tail were also obtained. N. alstoni alstoni spermatozoa were hyperactive in the presence of 290 mM sucrose and 10 and 20 nM bicarbonate solutions exhibited high motility (180-190 microm/sec), and high flagellum beating frequency (10-12 Hz). In contrast, the spermatozoa in 310 mM sucrose solution showed scarce motility (13.5 +/- 3.8 microm/sec) and low beating frequency (1.5 +/- 0.4 Hz). It is proposed that the volcano mouse spermatozoa possess some features very similar to other mammalian spermatozoa and that bicarbonate triggers caudal epididymal sperm motility of this species. J. Exp. Zool. 287:316-326, 2000.

Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

Phenotypic characteristics of isolates of Aspergillus section Fumigati from different geographic origins and their relationships with genotypic characteristics

BackgroundEpidemiological studies worldwide have shown that A. fumigatus exhibits important phenotypic and genotypic diversity, and these findings have been of great importance in improving the diagnosis and treatment of diseases caused by this fungus. However, few studies have been carried out related to the epidemiology of this fungus in Latin America. This study´s aim is to report on the epidemiology of the fungus by analyzing the phenotypic variability of Aspergillus section Fumigati isolates from different Latin American countries and the relationship between this variability, the geographical origin and genotypic characteristics.MethodsWe analyzed the phenotypic characteristics (macro- and micromorphology, conidial size, vesicles size, antifungal susceptibility and thermotolerance at 28, 37 and 48°C) of A. section Fumigati isolates from Mexico (MX), Argentina (AR), Peru (PE) and France (FR). The results were analyzed using analysis of variance (ANOVA) and Tukeys multiple comparison test to detect significant differences. Two dendrograms among isolates were obtained with UPGMA using the Euclidean distance index. One was drawn for phenotypic data, and the other for phenotypic and genotypic data. A PCoA was done for shown isolates in a space of reduced dimensionality. In order to determine the degree of association between the phenotypic and genotypic characteristics AFLP, we calculated the correlation between parwise Euclidean distance matrices of both data sets with the nonparametric Mantel test.ResultsNo variability was found in the macromorphology of the studied isolates; however, the micromorphology and growth rate showed that the PE isolates grew at a faster rate and exhibited the widest vesicles in comparison to the isolates from MX, AR and FR. The dendrogram constructed with phenotypic data showed three distinct groups. The group I and II were formed with isolates from PE and FR, respectively, while group III was formed with isolates from MX and AR. The dendrogram with phenotypic and genotypic data showed the same cluster, except for an isolate from FR that formed a separate cluster. This cluster was confirmed using PCoA. The correlation between the phenotypic and genotypic data of the isolates revealed a statistically significant association between these characteristics.ConclusionsThe PE isolates showed specific phenotypic characteristics that clearly differentiate them from the rest of the isolates, which matches the genotypic data. The correlation between the phenotypic and genotypic characteristics showed a statistically significant association. In conclusion, phenotypic and genotypic methods together increase the power of correlation between isolates.

Isolation of human mesenchymal stem cells and their cultivation on the porous bone matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Observaciones al Microscopio Electrónico de Barrido del Interior de un Proglótido de un Parásito Adulto de Taenia solium

No existen, hasta el momento, imagenes que muestren la disposicion de la citoarquitectura de parasitos adultos de Taenia solium, parasitos los cuales se encuentran en el intestino de portadores humanos asintomaticos. Las causas de ello podrian tener como base el que cuando se recuperan los parasitos, ellos han sufrido alteraciones debidas a la respuesta inmune de sus hospederos o bien, por el efecto que han producido en los parasitos los farmacos antihelminticos que hayan sido usados en el tratamiento de los pacientes. Una de las alternativas que se han encontrado para la obtencion de parasitos adultos, es la obtencion de tenias a partir del modelo de teniosis experimental en hamsteres dorados e inmunosuprimidos y que gracias a este modelo se han podido efectuar diferentes tipos de estudios de los parasitos de esta fase infectiva. El proposito de este reporte es presentar imagenes de ultraestructura, obtenidas mediante Microscopia Electronica de Barrido, de un corte transversal obtenido de un proglotido de una tenia recuperada de una infeccion experimental. Las imagenes se obtuvieron a diferentes aumentos y muestran aspectos relacionados con la superficie tegumentaria, el tegumento sincicial continuo, la capa germinal que incluye el soma de algunas celulas subtegumentarias y los ductos del sistema protonefridial tanto vacios como llenos con corpusculos calcareos. Las imagenes ultraestructurales obtenidas muestran una forma de observacion de la anatomia microscopica de los parasitos en estudio y ello contribuye a ampliar el conocimiento de los mismos en relacion a aspectos de su biologia celular y su fisiologia.