Centro de Altos Estudios en Ciencias Humanas y de la Salud

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    A functional analysis of the cyclophilin repertoire in the protozoan parasite Trypanosoma cruzi
    (MDPI, 2018-10-31) Fuchs, Alicia Graciela ; Perrone, Alina E ; Milduberger, Natalia A. ; Bustos, Patricia L. ; Bua, Jaqueline
    Trypanosoma cruzi is the etiological agent of Chagas disease. It affects eight million people worldwide and can be spread by several routes, such as vectorborne transmission in endemic areas and congenitally, and is also important in non-endemic regions such as the United States and Europe due to migration from Latin America. Cyclophilins (CyPs) are proteins with enzymatic peptidyl-prolyl isomerase activity (PPIase), essential for protein folding in vivo. Cyclosporin A (CsA) has a high binding affinity for CyPs and inhibits their PPIase activity. CsA has proved to be a parasiticidal drug on some protozoa, including T. cruzi. In this review, we describe the T. cruzi cyclophilin gene family, that comprises 15 paralogues. Among the proteins isolated by CsA-affinity chromatography, we found orthologues of mammalian CyPs. TcCyP19, as the human CyPA, is secreted to the extracellular environment by all parasite stages and could be part of a complex interplay involving the parasite and the host cell. TcCyP22, an orthologue of mitochondrial CyPD, is involved in the regulation of parasite cell death. Our findings on T. cruzi cyclophilins will allow further characterization of these processes, leading to new insights into the biology, the evolution of metabolic pathways, and novel targets for anti-T. cruzi contro
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    A homolog of cyclophilin D is expressed in Trypanosoma cruzi and is involved in the oxidative stress–damage response
    (Cell Death Differentiation Association (ADMC), 2017-2-6) Bustos, Patricia L. ; Volta, Viviana J. ; Perrone, Alina E ; Milduberger, Natalia A. ; Bua, Jaqueline
    Mitochondria have an important role in energy production, homeostasis and cell death. The opening of the mitochondrial permeability transition pore (mPTP) is considered one of the key events in apoptosis and necrosis, modulated by cyclophilin D (CyPD), a crucial component of this protein complex. In Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, we have previously described that mitochondrial permeability transition occurs after oxidative stress induction in a cyclosporin A-dependent manner, a well-known cyclophilin inhibitor. In the present work, a mitochondrial parasite cyclophilin, named TcCyP22, which is homolog to the mammalian CyPD was identified. TcCyP22-overexpressing parasites showed an enhanced loss of mitochondrial membrane potential and loss of cell viability when exposed to a hydrogen peroxide stimulus compared with control parasites. Our results describe for the first time in a protozoan parasite that a mitochondrial cyclophilin is a component of the permeability transition pore and is involved in regulated cell death induced by oxidative stress
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    Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway
    (Elsevier, 2021-5-5) Karadayian, Analía G. ; Bustamante, Juanita ; Lores-Arnaiz, Silvia
    Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD- 95/nNOS pathway.
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    Alcohol hangover: impairments in behavior and bioenergetics in central nervous system
    (Biocell, 2016-4-16) Karadayian, Analía G. ; Bustamante, Juanita ; Lores-Arnaiz, Silvia
    Alcohol hangover (AH) is defined as the temporary state after alcohol binge-like drinking, starting when EtOH is absent in plasma. Results from our laboratory have shown behavioral impairments and mitochondrial dysfunction in an experimental model of AH in mice. Our model consisted in a single i.p. injection of EtOH (3.8 g/kg BW) or saline solution in male and female mice, sacrificing the animals 6 hours after injection. Motor and affective behavior together with mitochondrial function and free radical production were evaluated in brain cortex and cerebellum during AH. Results showed that hangover animals exhibited a significant reduction in neuromuscular coordination, motor strength and locomotion together with a loss of gait stability and walking deficiencies. Moreover, an increment in anxiety-like behavior together with fear-related phenotype and depression signs were observed. In relation to bioenergetics metabolism, AH induced a reduction in oxygen uptake, inhibition of respiratory complexes, changes in mitochondrial membrane permeability, decrease in transmembrane potential, increase in O2•- and H2O2 production and impairment in nitric oxide metabolism. All together our data suggest that the physiopathological state of AH involves behavioral impairments and mitochondrial dysfunction in mouse brain cortex and cerebellum showing the long lasting effects of acute EtOH exposure in CNS.
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    Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging
    (Neurochemical research, 2016-1-28) Lores-Arnaiz, Silvia ; Lombardi, Paulina ; Karadayian, Analía G. ; Orgambide, Federico ; Cicerchia, Daniela ; Bustamante, Juanita
    Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decresed by 26 and 33% in sunaptosomes from 17-months old mice, but spare respiratory rate was decreased by 45% in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animales, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non synaptic mitochondrian would not be affectred by calcium overload. UCP2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.
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    Circulating cytokine and chemokine profiles of trypanosoma cruzi-infected women during pregnancy and its association with congenital transmission
    (Infectious Diseases Society of America (IDSA), 2021-9-15) Volta, Viviana J. ; Bustos, Patricia L. ; González, Carolina ; Natale, María Ailen ; Perrone, Alina E ; Milduberger, Natalia A. ; Laucella, Susana A. ; Bua, Jacqueline
    Background. Trypanosoma cruzi, the causative agent of Chagas disease, can be transmitted to the offspring of infected women, which constitutes an epidemiologically significant parasite transmission route in nonendemic areas. It is relevant to evaluate differ entially expressed factors in T. cruzi-infected pregnant women as potential markers of Chagas congenital transmission. Methods. Circulating levels of 12 cytokines and chemokines were measured by enzyme-linked immunosorbent assay or cytometric bead array in T. cruzi-infected and uninfected pregnant women in their second trimester of pregnancy and control groups of T. cruzi-infected and uninfected nonpregnant women. Results. Trypanosoma cruzi-infected women showed a proinflammatory Th1-biased profile, with increased levels of tumor ne crosis factor (TNF)-a, interleukin (IL)-12p70, IL-15, and monokine induced by interferon-gamma (MIG). Uninfected pregnant women presented a biased response towards Th2/Th17/Treg profiles, with increased plasma levels of IL-5, IL-6, IL-1ß, IL-17A, and IL-10. Finally, we identified that high parasitemia together with low levels of TNF-a, IL-15, and IL-17, low TNF-a/IL-10 ratio, and high IL-12p70 levels are factors associated with an increased probability of Chagas congenital transmission. Conclusions. Trypanosoma cruzi-infected pregnant women who did not transmit the infection to their babies exhibited a dis tinct proinflammatory cytokine profile that might serve as a potential predictive marker of congenital transmission.
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    Descripción de las características clínicas y epidemiológicas de las personas mayores de edad diagnosticadas de COVID-19 durante el año 2021 en el Departamento Rosario, Santa Fe, Argentina
    (Círculo Médico de Rosario, 2023) Weisburd, Guillermo Jaime ; Fernández, Florencia ; Gabini, Sebastián Manuel
    Objetivo: describir las características clínicas y epidemiológicas de las personas mayores de edad diagnosticadas de COVID-19 durante el año 2021 en el departamento Rosario, Santa Fe, Argentina. Estudio descriptivo, cuantitativo, transversal y retrospectivo. Los datos se recolectaron a partir de los registros oficiales del Ministerio de Salud de la Provincia de Santa Fe cargados en Sistema Integrado de Información Sanitaria Argentino. Se registraron un total de 111.095 casos confirmados. 69,4% se presentaron entre los meses de abril a julio. 51,4% eran mujeres y 48,6% varones. El promedio de edad fue de 42,82 años (DE = 15,94). Fueron internados 2.810 pacientes, 53,8% varones y 46,2% mujeres. 63,8% tenían más de 60 años. De los pacientes internados el 23,27% fueron derivados a la Unidad de Cuidados Intensivos (UCI) y 56% eran varones. De los pacientes internados el 93,1% presentó al menos una comorbilidad y ésta se comoprobó en 91,4% de los ingresados a la UCI. Hubo 1.962 decesos, el 56,8% fueron varones y el 74,6% en mayores de 60 años. La mayor cantidad de confirmados se presentó en la temporada otoño-invierno. Predominaron las mujeres y menores de 60 años. En los casos de gravedad y defunción preponderaron los varones mayores de 60 años y asociados a comorbilidad.
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    Descripción de las características clínicas y epidemiológicas de las personas mayores de edad diagnosticadas de COVID-19 durante el año 2021 en el Departamento Rosario, Santa Fe, Argentina
    (Círculo Médico de Rosario, 2023) Weisburd, Guillermo Jaime ; Fernández, Florencia ; Gabini, Sebastian Manuel
    Objetivo: describir las características clínicas y epidemiológicas de las personas mayores de edad diagnosticadas de COVID-19 durante el año 2021 en el departamento Rosario, Santa Fe, Argentina. Estudio descriptivo, cuantitativo, transversal y retrospectivo. Los datos se recolectaron a partir de los registros oficiales del Ministerio de Salud de la Provincia de Santa Fe cargados en Sistema Integrado de Información Sanitaria Argentino. Se registraron un total de 111.095 casos confirmados. 69,4% se presentaron entre los meses de abril a julio. 51,4% eran mujeres y 48,6% varones. El promedio de edad fue de 42,82 años (DE = 15,94). Fueron internados 2.810 pacientes, 53,8% varones y 46,2% mujeres. 63,8% tenían más de 60 años. De los pacientes internados el 23,27% fueron derivados a la Unidad de Cuidados Intensivos (UCI) y 56% eran varones. De los pacientes internados el 93,1% presentó al menos una comorbilidad y ésta se comoprobó en 91,4% de los ingresados a la UCI. Hubo 1.962 decesos, el 56,8% fueron varones y el 74,6% en mayores de 60 años. La mayor cantidad de confirmados se presentó en la temporada otoño-invierno. Predominaron las mujeres y menores de 60 años. En los casos de gravedad y defunción preponderaron los varones mayores de 60 años y asociados a comorbilidad.
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    Descripción de las características clínicas y epidemiológicas de las personas mayores diagnosticadas de COVID-19 durante el año 2021 en el departamento de Rosario, Santa Fe, Argentina
    (Revista Médica de Rosario, 2023) Weisburd, Guillermo Jaime ; Fernández, Florencia ; Gabini, Sebastián Manuel
    Objetivo: describir las características clínicas y epidemiológicas de las personas mayores de edad diagnosticadas de COVID-19 durante el año 2021 en el departamento Rosario, Santa Fe, Argentina. Estudio descriptivo, cuantitativo, transversal y retrospectivo. Los datos se recolectaron a partir de los registros oficiales del Ministerio de Salud de la Provincia de Santa Fe cargados en Sistema Integrado de Información Sanitaria Argentino. Se registraron un total de 111.095 casos confirmados. 69,4% se presentaron entre los meses de abril a julio. 51,4% eran mujeres y 48,6% varones. El promedio de edad fue de 42,82 años (DE = 15,94). Fueron internados 2.810 pacientes, 53,8% varones y 46,2% mujeres. 63,8% tenían más de 60 años. De los pacientes internados el 23,27% fueron derivados a la Unidad de Cuidados Intensivos (UCI) y 56% eran varones. De los pacientes internados el 93,1% presentó al menos una comorbilidad y ésta se comprobó en 91,4% de los ingresados a la UCI. Hubo 1.962 decesos, el 56,8% fueron varones y el 74,6% en mayores de 60 años. La mayor cantidad de confirmados se presentó en la temporada otoño-invierno. Predominaron las mujeres y menores de 60 años. En los casos de gravedad y defunción preponderaron los varones mayores de 60 años y asociados a comorbilidad.
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    Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset
    (Elsevier, 2017-7) Karadayian, Analía G. ; Malanga, Gabriela ; Czerniczyniec, Analía G. ; Lombardi, Paulina ; Bustamante, Juanita ; Lores-Arnaiz, Silvia
    Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals.
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    Hemocytes of the ribbed mussel Aulacomya atra atra from Nuevo Gulf (Chubut) as biomarkers of oxidative stress
    (Nova Science Publishers, Inc, 2018) Malanga, Gabriela ; Giarratano, Erica ; Lores-Arnaiz, Silvia ; Puntarulo, Susana Ángela ; Bustamante, Juanita
    Bivalve mollusks exposed to a wide variety of natural and anthropogenic environmental changes are widely used as sentinels. These factors can cause an imbalance between the generation and elimination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to an oxidative stress that is manifested by alterations of the antioxidant defense system and/or oxidative damage. The hemocytes constitute the immune system in bivalves, and cell death processes have been recently described as part of the mechanism of defense against various pathogens and contaminants. Previous studies showed a higher content of trace metals Fe and Cd in the gills and digestive gland of the Aulacomya atra atra from Folías Wreck (impacted area) than from Punta Cuevas (control area). In order to compare oxidative stress conditions in bivalves obtained from both sites, we evaluated the production of ROS and oxidative stress biomarkers in hemocytes from the A. atra atra during the month of September of 2015. The results obtained by flow cytometry, using MitoSox as probe, showed that superoxide anion was 58% higher in bivalve’s hemocytes from Folías Wreck than in those from the reference place Punta Cuevas. The oxidation of the dye 2´ 7´ dichlorofluorescein diacetate (DCFH-DA), as a general indicator of oxidative stress, showed a 14% increase in bivalve’s hemocytes from Folías Wreck, as compared to hemocytes from Punta Cuevas. Thiobarbituric acid reactive substances (TBARS) content showed no differences between hemocytes from animals isolated from both locations. In addition, the content of lipid radical measured by Electron Spin Resonance increased 2.1 fold in the hemocytes from Folías Wreck samples as compared to the level obtained in hemocytes from bivalves collected from Punta Cuevas. The oxidized/depleted cardiolipin was 16% higher in samples obtained from Folías Wreck than in Punta Cuevas. Based on these results, it can be concluded that hemocytes from the ribbed mussel A. atra atra could be used as a model to evaluate oxidative stress induced by pollutants or other environmental stressors.
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    Improved immuno-detection of a low-abundance cyclophilin allows the confirmation of its expression in a protozoan parasite
    (Hilaris, 2015-10-6) Bustos, Patricia L. ; Perrone, Alina E ; Milduberger, Natalia A. ; Bua, Jaqueline
    Protein samples can be challenging to analyze due to the presence of high-abundance proteins masking low abundance proteins of interest, such as biomarkers and novel physiological mediators. Cyclophilins are chaperones involved in the cis/trans isomerization of peptidyl-prolyl bonds in peptides or proteins and have been found in every organism sequenced to date. Although considerable progress has been made in the characterization of some cyclophilins expressed in diverse parasites invading humans, the main aspects of low-abundance members of this family remain unknown. In the present work, we present that the combined strategy of using more specific antibodies and increasing the presence of subcellular proteins in the sample, allowed us to confirm the expression of a 21.1 kDa cyclophilin for the first time in Trypanosoma cruzi.
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    Improved Immuno-Detection of a Low-Abundance Cyclophilin Allows the Confirmation of its Expression in a Protozoan Parasite
    (Immunochemistry & Inmunopathology, 2015) Bustos, Patricia Laura ; Perrone, Alina E ; Milduberger, Natalia A. ; Bua, Jaqueline
    Protein samples can be challenging to analyze due to the presence of high-abundance proteins masking low-abundance proteins of interest, such as biomarkers and novel physiological mediators. Cyclophilins are chaperones involved in the cis/trans isomerization of peptidyl-prolyl bonds in peptides or proteins and have been found in every organism sequenced to date. Although considerable progress has been made in the characterization of some cyclophilins expressed in diverse parasites invading humans, the main aspects of low-abundance members of this family remain unknown. In the present work, we present that the combined strategy of using more specific antibodies and increasing the presence of subcellular proteins in the sample, allowed us to confirm the expression of a 21.1 kDa cyclophilin for the first time in Trypanosoma cruzi.
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    Ketamine effect on intracellular and mitochondrial calcium mobilization
    (Biocell, 2016) Bustamante, Juanita ; Czerniczyniec, Analía G. ; Lores-Arnaiz, Silvia
    The suppressive effects of ketamine on intracellular calcium has been reported in a variety of cells although the mechanisms involved are not well understood. The aim of this work was to evaluate the ketamine effect on the mitochondrial Ca2+ accumulation and the cellular Ca2+ mobilization using FLUO4-AM and flow cytometry. The results showed that mitochondria from ketamine injected animals presented a lower ability to retain calcium at concentrations higher than 20 µM, as compared with controls (saline injected animals). In addition, ketamine showed a significant decreased KCl-induced intracellular calcium concentration. KCl increased calcium influx through cellular depolarization. According to the data presented herein, ketamine presents a clear inhibitory effect on cytosolic Ca2+ transport mechanisms, independently from their action on the calcium channel associated NMDA receptor.
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    Lactic acid transport mediated by aquaporin-9 : implications on the pathophysiology of preeclampsia
    (Frontiers, 2021) Acosta, Lucas Hernán ; Medina, Yollyseth ; Reppetti, Julieta ; Corominas, Ana ; Bustamante, Juanita ; Szpilbarg, Natalia ; Damiano, Alicia E.
    Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.
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    Mitochondrial permeability transition in protozoan parasites: what we learned from Trypanosoma cruzi
    (Official journal of the Cell Death Differentiation Association, 2017-9-21) Bustos, Patricia Laura ; Perrone, Alina E ; Milduberger, Natalia A. ; Bua, Jaqueline
    Regulated cell death (RCD) involves a genetically encoded molecular machinery, which can be altered by means of pharmacologic and/or genetics interventions targeting the key components of such machinery. A variant of RCD that often manifests with necrotic morphotype critically relies on Cyclophilin D (CyPD), a mitochondrial matrix peptidyl-prolyl isomerase, which is encoded by the Ppif gene. A lot a research has been done in mammals, but still very little is known for protozoan parasites, one of the most ancient phylogenic branches of unicellular eukaryotes. In the present work, we revised the knowledge about mitochondrial permeability transition and regulated cell death in the parasite Trypanosoma cruzi, the causative agent of Chagas disease, that affects 7?8 million people only in South America as well as in other parts of the world through migrations from endemic areas. We also included other protozoan parasites of medical importance to briefly summarize and compare what is known so far about this exciting field of parasitology. We finalized the present article explaining the finding that a homologue of Cyclophilin D, which is the unique genetically confirmed regulator of the mitochondrial permeability transition in mammalian cells, is also expressed in T. cruzi and may be involved in regulated cell death in the parasite. These results were published earlier this year in Cell Death Discovery (Cell Death Discov 3, 16092. 2017 Feb 06). To our knowledge, this is the only Cyclophilin D homologue that has been described in a protozoan parasite. We consider that this parasite mitochondrial cyclophilin could be a valuable drug target for the therapeutic of Chagas disease.
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    Oxidative stress damage in the protozoan parasite Trypanosoma cruzi is inhibited by cyclosporin A
    (Parasitology, Cambridge University Press, 2015-2-6) Bustos, Patricia L. ; Perrone, Alina E ; Milduberger, Natalia A. ; Postan, Miriam ; Bua, Jaqueline
    Cyclosporin A (CsA) specifically inhibits the mitochondrial permeability transition pore (mPTP). Opening of the mPTP, which is triggered by high levels of matrix [Ca2+] and/or oxidative stress, leads to mitochondrial dysfunction and thus to cell death by either apoptosis or necrosis. In the present study, we analysed the response of Trypanosoma cruzi epimastigote parasites to oxidative stress with 5 mM H2O2, by studying several features related to programmed cell death and the effects of pre-incubation with 1 µM of CsA. We evaluated TcPARP cleavage, DNA integrity, cytochrome c translocation, Annexin V/propidium iodide staining, reactive oxygen species production. CsA prevented parasite oxidative stress damage as it significantly inhibited DNA degradation, cytochrome c translocation to cytosol and TcPARP cleavage. The calcein-AM/CoCl2 assay, used as a selective indicator of mPTP opening in mammals, was also performed in T. cruzi parasites. H2O2 treatment decreased calcein fluorescence, but this decline was partially inhibited by pre-incubation with CsA. Our results encourage further studies to investigate if there is a mPTP-like pore and a mitochondrial cyclophilin involved in this protozoan parasite.
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    Paraoxonase 1 gene polymorphisms and enzyme activities in coronary artery disease and its relationship to serum lipids and glycemia
    (Elsevier, 2016-8-1) Fridman, Osvaldo ; Riviere, Stephanie ; Fuchs, Alicia Graciela ; Potenzoni, Miguel Ángel ; Porcile, Rafael ; Gariglio, Luis Osvaldo
    Objectives: Oxidative stress and inflammation are important processes in development of atherosclerosis. Paraoxonase 1 (PON1) is a bioscavenger enzyme associated with inflammation and oxidative stress. We evaluate the association of two single nucleotide polymorphisms in PON1 gene, and enzyme activities with lipid profile and glycemia. Methods: This case-control study consisted of 126 patients with coronary artery disease (CAD) and 203 healthy controls. PON Q192R and L55M polymorphisms were detected by real-time PCR. Paraoxonase and arylesterase activities were determined spectrophotometrically. Blood glucose, cholesterol, triglycerides, HDL, and LDL were measured. Results: PON1 QR192 polymorphism had a major effect on paraoxonase but no effect on arylesterase serum activities. Paraoxonase activity was higher in RR genotype and lowest in QQ genotype. Paraoxonase and arylesterase activities were higher in LL and lower in MM genotypes of PON1 LM55 polymorphism. RQ and LM variants showed intermediate activities between respective homozygous. Elevated concentrations of triglycerides in cases correlate with QQ variant or the presence of M allele. Glucose levels were elevated in cases with QQ variant or with the presence of M allele. Cholesterol and LDL did not show variations in control and cases with any variant of both polymorphisms. HDL is lower in cases with respect to controls independently of genotypes. All differences were significant with p < 0.05.
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    Phospholipid changes in Rhinella arenarum embryos under different acclimation conditions to copper
    (Elsevier, 2016-10) Herkovits, Jorge ; Fridman, Osvaldo ; Fonovich, Teresa M. ; Pérez-Coll, Cristina S. ; José L., D'eramo
    We report phospholipid changes in Rhinella arenarum embryos after applying three acclimation protocols to copper between 40 and 420 ng L- 1. The lower and higher acclimation treatments resulted in embryos' enhanced resistance to this metal. Phospholipid remodeling activity, evident through arachidonic acid radioactivity incorporation increase in phosphatidylcholine (PC) and sphingomyelin (SPH) fractions, was registered in embryos acclimated to the intermediate exposure condition. Concomitantly, a decrease in phosphatidic acid fraction (PA) was registered in the higher acclimation condition. PC/PE radioactivity ratio increased both for medium and high acclimation conditions from 0.493 in control embryos to 1.378 and 1.032 respectively. Phospholipid changes could be relevant for changes in membrane features associated with low level exposures to copper, preparing the embryo for a higher resistance to this metal. The increased resistance to copper could also be associated with both an increase in metallothioneins concentration, as registered with HPLC in all the acclimation conditions, and an increase in the copper bound to the third fraction of metallothioneins separated by this method. Our results point out that even very low level exposure to copper results in phospholipid metabolism changes that could be relevant for the acclimation phenomena.
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    Response to the letter to the editor : mitochondria isolated from the striatum of the brain exhibit a higher degree of oxidative phosphorylation coupling, which shows that they are not subject to energetic dysfunction upon acute paraquat administration
    (Springer, 2016-9-29) Czerniczyniec, Analía ; Karadayian, Analía G. ; Bustamante, Juanita ; Lores-Arnaiz, Silvia
    In response to criticisms raised by Professor Rendon regarding our original study “Impairment of striatal mitochondrial function by acute paraquat poisoning” (J Bioenerg Biomembr 47:395–408, 2015), we re-evaluated key methodological aspects and data interpretation. Oxygen consumption rates were measured in the absence and presence of KCN (1.3 mM) and diphenyleneiodonium (DPI, 1 µM) to discriminate KCN-sensitive respiration from paraquat redox cycling. Paraquat inhibited state 4 and state 3 KCN-sensitive respiration by 80% and 62%, respectively, while DPI-sensitive oxygen uptake increased 2.2- to 2.3-fold, confirming both respiratory-chain inhibition and redox-cycling contributions. Respiratory control ratios were deliberately omitted for KCN-sensitive data, as they do not accurately reflect mitochondrial viability under these conditions; instead, direct analysis of metabolic states 4 and 3 revealed clear bioenergetic impairment. Submitochondrial membrane preparations were tested for vesicle formation using FCCP (4 µM); no stimulation of NADH-cytochrome c reductase, succinate-cytochrome c reductase, or cytochrome oxidase activities occurred, and paraquat-induced inhibition (27% in complex I–III and 19% in complex IV) persisted unchanged. Rotenone (3 µM) inhibited NADH-cytochrome c reductase by ~80% in both control and paraquat samples, confirming that measured activity was predominantly rotenone-sensitive complex I–III (32% inhibition by paraquat). These additional controls validate our original methodology and support the conclusion that acute paraquat poisoning impairs striatal mitochondrial bioenergetics through direct respiratory-chain inhibition and increased free-radical production.