Aplicación de PCR a partir de cultivo en la identificación de subespecies del complejo Mycobacterium Tuberculosis / The use of culture-based PCR for the identification of subspecies of the mycobacterium tuberculosis complex

Los métodos diagnósticos clásicos de tuberculosis (TB) se basan en la utilización de baciloscopía y cultivo. La identificación del agente etiológico desde la positivización del cultivo requiere entre 10 y 15 días, mientras que el empleo de la reacción en cadena de la polimerasa (PCR) disminuye el tiempo a 24 h, lo que permite no solo identificar las subespecies del complejo Mycobacterium tuberculosis (CMTB) sino también diferenciarlas de otras especies ambientales clínicamente importantes (MOTT) facilitando el diagnóstico y tratamiento. El objetivo del presente trabajo fue determinar la utilidad de la PCR en la identificación temprana de las micobacterias pertenecientes al CMTB, a partir de cultivos positivos, de pacientes con sospecha de TB, atendidos en un hospital pediátrico de alta complejidad, durante un período de cuatro años. A cada muestra, se le realizó baciloscopía y cultivo en medio líquido. A los cultivos positivos, una inmunocromatografía lateral (TBIDR) y luego PCR. El 4,6% del total de muestras (510/11.162) pertenecientes a 198 pacientes presentó cultivos positivos. Cuatrocientos veintiseis (84%) correspondieron a muestras respiratorias. El rendimiento de la baciloscopía directa fue del 41% (194/470). Cuatrocientos treinta y ocho (86%) resultaron M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG y 44 (9%) MOTT. La utilización de medios de cultivos líquidos junto con el empleo de PCR favorecen una rápida orientación microbiológica y constituye una estrategia útil para optimizar el manejo clínico de estas infecciones, desde el punto de vista terapéutico y epidemiológico, especialmente en pediatría (AU)

Classical diagnostic methods for tuberculosis (TB) are based on the use of smear microscopy and culture. The identification of the etiological agent from positive culture requires 10 to 15 days, while the use of the polymerase chain reaction (PCR) reduces the time to 24 h, which allows not only to identify the subspecies of the Mycobacterium tuberculosis complex (MTC) but also to differentiate them from clinically important environmental mycobacteria other than tuberculosis (MOTT), facilitating diagnosis and treatment. The aim of this study was to determine the usefulness of PCR in the early identification of mycobacteria belonging to the MTC, from positive cultures of patients with suspected TB seen in a pediatric tertiary hospital over a 4-year period. For each sample, smear microscopy and culture in liquid medium was performed. Positive cultures were subjected to lateral immunochromatography (TBIDR) and then PCR. Of the total number of samples (510/11,162) belonging to 198 patients, 4.6% showed positive cultures; 426 (84%) were respiratory samples. The direct smear microscopy yield was 41% (194/470). Overall, 438 (86%) were found to be M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG, and 44 (9%) MOTT. The use of liquid culture media together with the use of PCR favors a rapid microbiological orientation and is a useful strategy to optimize the clinical management of these infections, from a therapeutic and epidemiological point of view, especially in children (AU)

Lanthanoid analysis in seawater by seaFAST-SP3™ system in off-line mode and magnetic sector high-resolution inductively coupled plasma source mass spectrometer.

Analysis of lanthanoids in seawater is challenging due to the complex matrix (∼35 g L-1 TDS) and low dissolved concentrations (in ng L-1). A 4-step strict analytical protocol and state-of-the-art technology were implemented and validated in this study. The 4-steps method involves the 1) sample filtration and acidification (pH<2); 2) pre-concentration by the matrix separation system, 3) off-line injection of the eluted sample; and 4) determination of lanthanoids by high-resolution inductively coupled plasma mass spectrometer (HR-ICP-MS). Since there are no certified values for lanthanoids in seawater are available, the method validation was done by analyzing SLEW-3 (estuarine water reference samples) and comparing with other reports and artificial seawater (100 ng L-1 lanthanoid multi-element standard solutions). SLEW-3 recovery varied from 78.6% to 106% and in artificial samples it ranged from 87 to 110%. Low recovery can be explained by complex organic in seawater, because the UV oxidation was not performed in the acidified samples. The variation was ≤10%, except for Gd, Tb, and Yb (11-13.75%). Blanks varied between 0.01 and 0.07 ng L-1, except for La and Ce (0.13-0.21 ng L-1). Blanks represent <5% SLEW-3 values and <1% synthetic seawater. The procedural detection limit varied from 0.01 to 0.03 ng L-1.•Lanthanoids as geochemical tracers in seawaters•A 4-step strict analytical protocol and state-of-the-art technology for lanthanoids analyses in seawaters•Sample pre-concentration system for matrix separation for the detection of ultra-low lanthanoids levels.

Geogenic lanthanoid signature in coastal and marine waters from the southern Gulf of California.

Lanthanoids in the southern Gulf of California (GC) seawater are reported for the first time. Lanthanoids showed differences between peninsular and continental coastline, coastal or marine ecosystems, and dry or rainy season. The chondrite-normalized values showed high variability but followed a same pattern. Light lanthanoids were more enriched than heavy ones. Values of ∑Ln and La/Lu were higher in continental than peninsular coastlines, coastal than adjacent marine ecosystems, and rainy than dry season. Differences were related to the lithology and perturbation degree of the ecosystem watersheds. The chondrite-normalized patterns are typical of geological origin. Slightly negative Ce anomaly was related to the low levels of oxygen in water for the oxidation of Ce (III) to Ce (IV) and its posterior scavenging. Negative δEu anomaly is explained by an influx of fluvial and eolian materials from the upper continental, while a positive Eu anomaly related to hydrothermal vent inputs was non-evidenced.

Dominance patterns in macroalgal and phytoplankton biomass under different nutrient loads in subtropical coastal lagoons of the SE Gulf of California.

Nine macroalgal blooms were studied in five coastal lagoons of the SE Gulf of California. The nutrient loads from point and diffuse sources were estimated in the proximity of the macroalgal blooms. Chlorophyll a and macroalgal biomass were measured during the dry, rainy and cold seasons. Shrimp farms were the main point source of nitrogen and phosphorus loads for the lagoons. High biomasses were found during the dry season for phytoplankton at site 6 (791.7±34.6 mg m(-2)) and during the rainy season for macroalgae at site 4 (296.0±82.4 g m(-2)). Depending on the season, the phytoplankton biomass ranged between 40.0 and 791.7 mg m(-2) and the macroalgal biomass between 1 and 296.0 g m(-2). The bulk biomass (phytoplankton+macroalgal) displayed the same tendency as the nutrient loads entering the coastal lagoons. Phytoplankton and macroalgal biomass presented a significant correlation with the atomic N:P ratio.

Isolation and nucleotide sequence analysis of the of Rhinella arenarum ß-catenin: an mRNA and protein expression study during the larval stages of the digestive tract development.

ß-catenin interacts with several proteins mediating key biological processes, such as cadherin-mediated cell-cell adhesion as well as signal transduction. This work was done to establish the molecular basis and regulation of the formation pattern of cadherin/ß-catenin-mediated adherens junctions, using an animal model of unknown gene sequence, the toad Rhinella arenarum. A Rhinella arenarum ß-catenin homolog was isolated from larval tissue, their sequence compared and analyzed with those of eight other vertebrates using bioinformatics tools. The mRNA and protein expression levels of ß-catenin were determined during the development of Rhinella arenarum digestive tract both by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry-morphometry respectively. Using Xenopus laevis frog specific primers, a fragment 539 bp of Rhinella arenarum toad ß-catenin cDNA was obtained and sequenced. The resulting putative sequence of 177 amino acids showed high similarity at the amino acid level (97%) when compared to other six vertebrates (Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus, Bos taurus and Homo sapiens), with sequences and structural domains characteristic of catenins. Subsequently, using primers specifically designed for Rhinella arenarum nucleotide sequence, ß-catenin-mRNA increasing levels were found during the Rhinella arenarum metamorphosis. Finally, increasing ß-catenin protein expression during development has confirmed the specificity the detection of Rhinella arenarum ß-catenin. Summarizing, we have isolated and sequenced a ß-catenin-homologue sequence from the Rhinella arenarum toad, which is highly conserved between species, and following we have detected ß-catenin mRNA and protein levels during their digestive tract development.

Gender differences in ancestral contribution and admixture in Venezuelan populations.

The origin of the contribution of uniparental heritage were analyzed in 615 samples of individuals proceeding from 13 towns classified according to historic differences in their emergence and development as African-derived, European-derived, and admixed/urban. Mitochondrial and Y-chromosome haplogroups were identified by PCR-restriction fragment length polymorphism. The results were compared with previous estimates of admixture made with autosomal markers and with historic aspects. The results show a predominantly indigenous genetic contribution through the female, being more prevalent in urban populations; the African contribution, although dispersed, presents a larger concentration in the African-derived towns, whereas the European contribution is limited to populations with this origin, reflecting isolation and the conservation of the distribution pattern of genes of the Colonial era. With regard to admixture through males, it is almost exclusively of European origin, whereas the African contribution is basically concentrated in the African-derived towns, and the Amerindian lineages are almost nonexistent. The genome of paternal heredity, as opposed to the autosomal and the mitochondrial, shows a homogeneous pattern of admixture that is independent of the origin of the population studied, suggesting that European genes have been introduced into the Venezuelan population through male immigrations, whereas the indigenous contribution has been preserved in the Venezuelan genetic pool through the women. These results provide evidence of the heterogeneity in the genetic origin of the Venezuelan population, which should be taken into account in forensic and epidemiologic genetic studies.

T3 regulates E-cadherin, and ß- and α-catenin expression in the stomach during the metamorphosis of the toad Rhinella arenarum.

The metamorphosis of Rhinella arenarum was induced precociously for 5 days, then blocked for 3 months to evaluate the role of thyroid hormones as modulators of morphoregulatory molecules such as E-cadherin, and ß- and α-catenin during epithelium remodeling. We then performed an immunohistochemical and morphometric study of these molecules in the larval stomach. We show that 3,5,3'-triiodothyronine exerts a positive regulatory effect on E-cadherin and ß- and α-catenin expression in stomach epithelium. This suggests continuous synthesis of E-cadherin, and ß- and α-catenin; synthesis essentially is thyroid hormone-independent during premetamorphosis and early prometamorphosis, but it becomes thyroid hormone-dependent during metamorphic climax.

Role of E-cadherin in epithelial architecture maintenance.

Morphogenesis and architecture of a developing epithelium is controlled by both cell shape and contacts, mediated by spatially and temporally regulated cell adhesion molecules. The authors study if E-cadherin functions as a key factor of epithelial adhesion and epidermal architecture in vivo. They apply whole-mount digital deconvolution microscopy to evaluate three-dimensional (3D) E-cadherin expression during skin morphogenesis of Rhinella arenarum and in a cell adhesion alteration model. Results show morphogenetic changes in the 3D E-cadherin spatiotemporal expression pattern correlated with the increase of E-cadherin and in the number of cells with hexagonal geometry. Alterations in junction-protein phosphorylation showed drastic loss of E-cadherin and beta-catenin in cell-cell contacts and the increase of cytoplasm and nuclear beta-catenin in epidermis, suggesting the activation of the beta-catenin signal pathway. Surprisingly, no changes in cell shape and skin architecture were registered, suggesting that epidermal E-cadherin appears to be involved in signaling rather than cell contact maintenance in vivo.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae).

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, beta-and alpha-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of beta-and alpha-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of beta-and alpha-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, beta-catenin and alpha-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae) / Expressão das moléculas de adesão celular no desenvolvimento normal e com a inibição do hormônio tireoidea do rim nas larvas do Bufo arenarum (Amphibia, Anura, Bufonidae)

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, β-and α-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of β-and α-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of β-and α-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, β-catenin and α-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Moléculas de adesão celular atuam como tradutores do ambiente extracelular para o citoesqueleto e o núcleo e, conseqüentemente, induzindo mudanças no padrão da expressão das proteínas estruturais. Neste estudo, observamos os efeitos da inibição do hormônio tireóidea (TH) e detenção da metamorfose na expressão da E-caderina, β- e α- catenina no desenvolvimento do rim do Bufo arenarum. As moléculas de adesão celular durante o desenvolvimento têm uma expressão temporal e espacial seletiva, sugerindo um papel específico na nefrogênese. Com o propósito de estudar os mecanismos de controle da expressão das moléculas de adesão durante o desenvolvimento renal, bloqueou-se a metamorfose do B. arenarum com uma substancia goitrogênica que bloqueia a síntese de TH. A expressão da E-caderina nos tubos proximais é independente do controle da tireóide. Entretanto, o bloqueio da síntese de TH provoca uma sobre elevação da E-caderina nos dutos coletores, nos tubos distais e nos glomérulos. A expressão da β- e α-catenina nos dutos coletores, nos tubos distais, nos glomérulos e no mesênquima mesonéfrico é independente da TH. O bloqueio da TH causa uma sobre-regulação da β- e α-catenina nos tubos proximais. Em contraste com a E-caderina, a expressão da caderina desmossomal demogloína 1 (Dsg-1) é ausente no controle durante a metamorfose da fase larval dos rins e se expressa em algumas células intersticiais nas larvas tratadas com KClO4. De acordo com este trabalho, a expressão Dsg-1 é subregulada pela TH. Demonstramos que a expressão da E-caderina, Dsg-1, β-catenina e α-catenina são afetadas de forma diferencial pelos níveis de TH, sugerindo um dependência hormonal destas proteínas na metamorfose renal do B. arenarum.

3D automatic quantification applied to optically sectioned images to improve microscopy analysis.

New fluorescence microscopy techniques, such as confocal or digital deconvolution microscopy, allow to easily obtain three-dimensional (3D) information from specimens. However, there are few 3D quantification tools that allow extracting information of these volumes. Therefore, the amount of information acquired by these techniques is difficult to manipulate and analyze manually. The present study describes a model-based method, which for the first time shows 3D visualization and quantification of fluorescent apoptotic body signals, from optical serial sections of porcine hepatocyte spheroids correlating them to their morphological structures. The method consists on an algorithm that counts apoptotic bodies in a spheroid structure and extracts information from them, such as their centroids in cartesian and radial coordinates, relative to the spheroid centre, and their integrated intensity. 3D visualization of the extracted information, allowed us to quantify the distribution of apoptotic bodies in three different zones of the spheroid.

Causas de muerte en pacientes con aneurisma de aorta abdominal quirúrgico no tratado de forma electiva / Causes of death in patients with an abdominal aortic aneurysm that was not treated with elective surgery

Introducción. La evolución natural de los aneurismas de aorta abdominal (AAA) grandes es su rotura si no se resecan en el momento oportuno. Objetivo. Conocer las causas de muerte en los pacientes con un AAA quirúrgico que no han sido operados mediante reparación abierta. Pacientes y métodos. Se estudia de forma retrospectiva a 128 pacientes con un AAA a los que no se realizó reparación abierta de forma electiva, en 38 casos (29,7%) por negativa del paciente a ser intervenido, en 64 (50%) por tener diversos factores de riesgo que aumentaban la mortalidad hospitalaria de forma significativa y en 26 (20,3%) por estar contraindicada la cirugía. Resultados. La edad media de los pacientes fue de 78 años y 107 eran varones (83,6%) (53-96). Se llevó a cabo un seguimiento medio de 32,7 meses (rango: 0,1-146 meses). Fallecieron 107 pacientes (83,6%), de los cuales 27 (25,2%) lo hicieron por causa cardíaca y 19 (17,8%) por rotura del AAA. De los 38 pacientes que rechazaron la cirugía, fallecieron 30 (78,9%), 9 de ellos (30,0%) por rotura del AAA. De los 64 pacientes no operados por riesgo quirúrgico elevado, fallecieron 51 (79,7%), pero sólo en 6 de ellos (9,4%) la causa fue la rotura aórtica. Finalmente, todos los pacientes no operados por contraindicación fallecieron, el 15,4% por rotura del aneurisma. Conclusiones. En este trabajo, la principal causa de muerte en los pacientes que rechazaron la cirugía electiva fue la rotura del AAA, mientras que en el resto, su fallecimiento se debió a la patología de base

Introduction. Large abdominal aortic aneurysms (AAA) naturally progress towards rupture if they are not excised in time. Aim. To determine the causes of death in patients with a surgical AAA who did not undergo open repair surgery. Patients and methods.We conducted a retrospective study of 128 patients with an AAA in whom open repair was not performed electively, in 38 cases (29.7%) because the patient refused to undergo surgery, in 64 (50%) due to their having a number of risk factors that significantly increased the hospital mortality rate and in 26 (20.3%) because surgery was contraindicated. Results. The mean age of the patients was 78 years and 107 were males (83.6%) (53-96). Mean follow-up time was 32.7 months (range: 0.1-146 months). Altogether 107 patients (83.6%) died, 27 (25.2%) of whom did so due to cardiac causes and 19 (17.8%) because of rupture of the AAA. Of the 38 patients who refused surgery, 30 (78.9%) died, 9 of them (30.0%) due to rupture of the AAA. Of the 64 patients who were not operated on because of a high surgical risk, 51 (79.7%) died but death was caused by aortic rupture in only 6 cases (9.4%). Finally, all the patients who did not undergo surgery because it was contraindicated died, in 15.4% of cases due to rupture of the aneurysm. Conclusions. In this work the main cause of death in the patients who refused elective surgery was rupture of the AAA, whereas in the others their deaths were due to their underlying conditions

Impact of antibiotic treatment on bacterial resistance rates from patients with hospital-acquired infection.

We assessed the impact of antibiotic administration prior to sample collection on the bacterial resistance rates from patients with nosocomial infection. Every individual susceptibility report was assessed in real time at the bedside of the patient by a team composed of infectious diseases and internal medicine specialists as well as clinical microbiologists for clinical significance and appropriateness of the specimen. The report also stated the kind, source and origin of the infection, history of administration of any antibiotic during the last month prior to sample collection. To evaluate the impact of previous antibiotic administration, resistance rates were calculated separately among the group of patients with and without history of antibiotic treatment. A crude univariate analysis was performed to assess the significance of the differences between groups for every species-antibiotic pair. Patients who had received ciprofloxacin showed significantly higher rates of Escherichia coli resistant to ciprofloxacin, broad-spectrum cephalosporins and gentamicin. A higher rate of methicillin-resistant Staphylococcus aureus was observed in patients who were given gentamicin. A stratified analysis showed that the previous antibiotic administration continued to be a risk factor for increased resistance rates regardless of the hospital ward or the source of the infection. This study demonstrates the influence of previous antibiotic administration on bacterial resistance rates although this fact is barely taken into account by the laboratory when constructing the cumulative susceptibility data. Real time clinical validation of the individual susceptibility reports, performed by a multidisciplinary team prior to the data entering, might be a suitable approach to get more reliable susceptibility rates to guide the rational selection of antimicrobial empirical therapy in patients with hospital-acquired infections who have been given antimicrobial treatment prior to specimen collection.

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin.

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the biocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the bbiocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the bbiocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.(AU)

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin.

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 mug CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.