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Int. j. morphol ; 38(6): 1639-1644, Dec. 2020. graf
Article in English | LILACS | ID: biblio-1134491


SUMMARY: Previous studies from our group described the consequences of using ethanol on penile erection. Nevertheless, the molecular mechanisms surrounding microRNAs, apoptosis process and their relationship with erectile dysfunction associated with alcohol consumption are still poorly understood. The objective of this analysis was to evaluate the mechanism of apoptosis by the expression of AIF and PARP, as well as their regulatory microRNAs: miR-145, miR-210 and miR-486, in the corpus cavernosum of rats submitted to a semivoluntary alcoholism model. For this study 24 Wistar rats were divided into two groups: control (C) and treated with 20 % ethanol (A) for seven weeks. The corpus cavernosum samples were prepared for immunohistochemical analysis of AIF and PARP protein expression, and microRNAs miR-145, miR-210, miR-486 gene expression in cavernous tissue was performed by real time PCR. The immunohistochemical analysis showed little nuclear positive labeling for the protein PARP and AIF in the corpus cavernosum of control and ethanol treated animals. After analysis of miR-145, -210 and -486 microRNA expression in the 12 animals studied, no results were found with significant statistical difference between the control and alcoholized groups. The expression of AIF and PARP and their regulatory microRNAs involved in apoptotic process (miR-145, miR-210 and miR-486) were not altered in the corpus cavernosum of rats submitted to semivoluntary alcoholism.

RESUMEN: Estudios previos de nuestro grupo describieron las consecuencias del uso de etanol en la erección del pene. Sin embargo, los mecanismos moleculares que rodean a los microARN, el proceso de apoptosis y su relación con la disfunción eréctil asociada con el consumo de alcohol aún no se conocen bien. El objetivo de este análisis fue evaluar el mecanismo de apoptosis mediante la expresión de AIF y PARP, así como sus microARN reguladores: miR-145, miR-210 y miR-486, en el cuerpo cavernoso de ratas sometidas a un modelo de alcoholismo semivoluntario. Se dividieron 24 ratas Wistar en dos grupos: control (C) grupo de ratas tratadas con etanol al 20 % (A) durante siete semanas. Las muestras del cuerpo cavernoso se prepararon para el análisis inmunohistoquímico de la expresión de la proteína AIF y PARP, y la expresión del gen microRNAs miR-145, miR-210, miR-486 en tejido cavernoso se realizó por PCR en tiempo real. El análisis inmunohistoquímico mostró escaso etiquetado nuclear positivo para la proteína PARP y AIF en el cuerpo cavernoso de los animales de control y tratados con etanol. Después del análisis de la expresión de microARN miR-145, -210 y -486 no se encontraron resultados con diferencias estadísticas significativas entre los grupos control y alcoholizados. La expresión de AIF y PARP y sus microARN reguladores involucrados en el proceso apoptótico (miR-145, miR-210 y miR-486) no se alteraron en el cuerpo cavernoso de las ratas sometidas a alcoholismo semivoluntario.

Animals , Rats , Apoptosis , Alcoholism/metabolism , Erectile Dysfunction/metabolism , Penis/physiopathology , Penis/chemistry , Immunohistochemistry , Rats, Wistar , MicroRNAs/analysis , MicroRNAs/genetics , MicroRNAs/metabolism , Disease Models, Animal , Alcoholism/physiopathology , Apoptosis Inducing Factor/analysis , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Real-Time Polymerase Chain Reaction , Erectile Dysfunction/physiopathology
Rev. méd. Chile ; 143(2): 175-182, feb. 2015. tab
Article in Spanish | LILACS | ID: lil-742568


Background: In 2007, a Clinical-Case-Portfolio (CCP) was introduced as a new assessment instrument for fourth grade undergraduate medical students. Since then, several changes have been implemented such as reduction on the number of clinical cases, peer review and the introduction of virtual patient to the portfolio. Aim: To describe the virtual patient model incorporated to the CCP and assess the perception of this change and its effects on the performance of undergraduate students. Material and Methods: Virtual patients were implemented based on prototype clinical cases with specific syndromes. Students’ perceptions about CCP before and after the introduction of virtual patients were evaluated using a validated questionnaire that was answered voluntarily and anonymously. Results: Overall perception of CCP significantly improved after the incorporation of virtual patients (97.1 ± 24.9 and 111.3 ± 25.7 points; 57.8 and 66.2% respectively). The same improvements were observed for the domains “Student Learning”, “Organization and Evaluation”, “Teaching Methodology” and “Integration”. In both years, students obtained high grades in CCP evaluations. However CCP grades were not significantly correlated with integrated final grades. Conclusions: The incorporation of virtual patients improved undergraduate students’ perception of CCP.

Animals , Mice , Apoptosis , Axin Protein/metabolism , Enzyme Activation , Poly(ADP-ribose) Polymerases/metabolism , Protein-Serine-Threonine Kinases/antagonists & inhibitors , Aurora Kinases , Adenosine Triphosphate/metabolism , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Cell Line , Cell Membrane/metabolism , Cell Membrane/physiology , Mitochondria/metabolism , Protein Kinase Inhibitors/pharmacology , RNA Interference , Time-Lapse Imaging
Biocell ; 36(3): 121-126, Dec. 2012. ilus, graf
Article in English | LILACS | ID: lil-694712


Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluorescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide production in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.

Humans , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/physiology , Electron Transport Complex I/metabolism , Cell Line , Cell Line, Tumor , Fluorescent Dyes/pharmacology , Gene Silencing , HeLa Cells , Image Processing, Computer-Assisted , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Phenanthridines/pharmacology , Superoxides/metabolism , Telomerase/metabolism , Telomere/ultrastructure
Biol. Res ; 42(2): 249-260, 2009. ilus, tab
Article in English | LILACS | ID: lil-524896


Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

Humans , Alcohol Oxidoreductases/drug effects , Apoptosis Inducing Factor/pharmacology , Apoptosis/drug effects , DNA, Complementary/drug effects , DNA-Binding Proteins/drug effects , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Apoptosis/genetics , Blotting, Western , DNA, Complementary/genetics , DNA, Complementary/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique, Indirect , Jurkat Cells , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , Transfection