Institute Biologia Y Medicina Experimental IBYME

Buenos Aires, Argentina

Institute Biologia Y Medicina Experimental IBYME

Buenos Aires, Argentina
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Gambino Y.P.,University of Buenos Aires | Perez Perez A.,University of Seville | Duenas J.L.,Hospital Universitario Virgen Macarena | Calvo J.C.,University of Buenos Aires | And 3 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2012

The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17β-estradiol (E 2) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E 2 conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E 2 also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E 2 induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E 2 genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E 2 regulates leptin expression in placenta and support the importance of leptin in placental physiology. © 2012 Elsevier B.V..

Labovsky V.,Institute Investigaciones en Ingenieria Genetica y Biologia Molecular INGEBI | Labovsky V.,CONICET | Labovsky V.,Institute Biologia y Medicina Experimental IBYME | Hofer E.L.,CONICET | And 11 more authors.
Differentiation | Year: 2010

Bone marrow mesenchymal stromal cells (BM-MSCs) with regenerative potential have been identified in heart. Whether these cells become new cardiac lineage cells by phenomena of transdifferentiation or fusion is also being investigated. Although, these mechanisms give cardiomyocytes, it has to be considered that MSCs transplantation could carry out ossification and calcification processes. An alternative might be the use of myocytes; however, the problem is the arrythmia. For those reasons, is that we investigated how to obtain cardiomyocyte-like cells from human MSCs (hMSCs). The aim of the present work was to evaluate a nuclear reprogramming of the hMSCs by a neonatal rat cardiomyocytes extract (EX) using Streptolysin O (SLO) treatment. hMSCs treated with 57.5 ng/ml SLO presented ball-like, stick-like and myotube-like morphology. In the absence of cardiomyogenic stimuli, hMSCs expressed markers of cardiac phenotype-like sarcomeric α-actinin, connexin-43 and GATA-4. However, when hMSCs were treated with SLO+EX or 10 μM of 5-azacytidine (5-AZA), the expression of these markers were significantly increased and furthermore, expressed SERCA-2, cardiac Troponin I, β-MyHC, desmin, MLC-2a and MLC-2v thus showing the phenotype of mature cardiomyocytes. PCR analysis showed that cardiomyocyte-related genes, such as β1-adrenergic receptor (β1-AR), MLC-2a and cardiac Troponin T, were expressed after SLO+EX treatment like with 5-AZA. We concluded that the extract of neonatal rat cardiomyocytes could promote a nuclear modification of hMSCs to cardiomyogenic-like cells differentiation. Since the 5-AZA treatment appears to be genotoxic and taking into account the obtained results, the nuclear reprogramming by cell extract may be an approach leading to the identification of soluble factors that drives the reprogramming. © 2009 International Society of Differentiation.

Gambino Y.P.,University of Buenos Aires | Maymo J.L.,University of Buenos Aires | Perez-Perez A.,University of Seville | Duenas J.L.,Hospital Universitario Virgen Macarena | And 4 more authors.
Biology of Reproduction | Year: 2010

The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E2) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E 2 effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E2-induced leptin expression. Moreover, E2 treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between - 1951 and -1847 bp is both necessary and sufficient to achieve E2 effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E2 on leptin expression. Moreover, E2 action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E2 could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 μM PD98059 and 0.1 μM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E 2 induction of leptin promoter. On the other hand, E2 treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E2 induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways. © 2010 by the Society for the Study of Reproduction, Inc.

Maymo J.L.,University of Buenos Aires | Perez Perez A.,University of Seville | Maskin B.,Hospital Nacional Profesor Alejandro Posadas | Duenas J.L.,Hospital Universitario Virgen Macarena | And 4 more authors.
PLoS ONE | Year: 2012

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway. © 2012 Maymó et al.

Bilotas M.,Institute Biologia y Medicina Experimental IBYME | Meresman G.,Institute Biologia y Medicina Experimental IBYME | Buquet R.,Hospital Of Clinicas Jose Of San Martin | Baranao R.I.,Institute Biologia y Medicina Experimental IBYME
Journal of Reproductive Immunology | Year: 2010

The aim of this study was to evaluate the effect of vascular endothelial growth factor (VEGF) and interleukin-1β (IL-1β) on apoptosis induced by leuprolide acetate (LA) in endometrial epithelial cell cultures from patients with endometriosis. Primary endometrial epithelial cell cultures were obtained from uterine endometrial biopsies of patients with endometriosis and control women. Endometrial epithelial cells were incubated with LA; a combination of LA and VEGF; a combination of LA and IL-1β; or in basal conditions. LA was added 3 h prior to addition of VEGF and IL-1β. After stimulation, the percentage of apoptotic cells was evaluated by the acridine orange-ethidium bromide technique and Bax expression was assessed by western blot. Treatment with LA enhanced the percentage of apoptotic cells in endometrial epithelial cells from subjects with endometriosis and control subjects. Addition of either VEGF or IL-1β after exposure to LA restored the percentage of apoptotic cells to basal levels. Moreover, treatment with LA increased Bax expression in endometrial epithelial cells from patients with endometriosis. This effect was reverted by the addition of either VEGF or IL-1β. Our results show that VEGF and IL-1β reduce apoptosis and decrease Bax expression in endometrial epithelial cells from patients with endometriosis. This study suggests that VEGF and IL-1β may protect endometriotic cells from undergoing apoptosis in addition to exerting their pro-angiogenic role. © 2010 Elsevier Ireland Ltd. All rights reserved.

Schillaci R.,CONICET | Guzman P.,University of the Frontier | Cayrol F.,CONICET | Beguelin W.,CONICET | And 10 more authors.
BMC Cancer | Year: 2012

Background: The biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2) presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK) guidelines were used as reference.Methods: Tissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2) and NuclErbB-2 expression by an immunofluorescence (IF) protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC) with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS.Results: The IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors.Conclusions: We revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration. © 2012 Schillaci et al; BioMed Central Ltd.

Albrecht S.,BlackRock | Lane J.A.,Teagasc | Marino K.,BlackRock | Marino K.,Institute Biologia y Medicina Experimental IByME | And 4 more authors.
British Journal of Nutrition | Year: 2014

The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultra-performance liquid chromatography-hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencing with linkage- and sugar-specific exoglycosidases. Relative peak quantification of the 2-aminobenzamide-labelled oligosaccharides provided additional information. Milk from domestic animals contained a much larger variety of complex oligosaccharides than was previously assumed, and thirteen of these structures have been identified previously in human milk. The direct comparison of the oligosaccharide mixtures reflects their role in the postnatal maturation of different types of gastrointestinal systems, which, in this way, are prepared for certain post-weaning diets. The potential value of animal milk for the commercial extraction of oligosaccharides to be used in human and animal health is highlighted. © The Authors 2013.

Labovsky V.,Institute Biologia y Medicina Experimental IBYME | Labovsky V.,CONICET | Vallone V.B.F.,Institute Biologia y Medicina Experimental IBYME | Vallone V.B.F.,CONICET | And 4 more authors.
Cancer Cell International | Year: 2012

Background: While breast cancer (BC) is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), stromal cell-derived factor-1 (SDF-1), and their receptors (R) in 2 human BC cell lines, MDA-MB-231 and MCF-7.Methods: OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses.Results: MCF-7 cells released higher levels of OPG in conditioned media (CM) than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3.Conclusions: MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention. © 2012 Labovsky et al.; licensee BioMed Central Ltd.

Sanz S.,University of Barcelona | Bandini G.,University of Dundee | Bandini G.,Boston University | Ospina D.,University of Barcelona | And 5 more authors.
Journal of Biological Chemistry | Year: 2013

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Veron G.L.,Institute Biologia y Medicina Experimental IBYME | Molina R.I.,Laboratorio Of Andrologia Y Reproduccion | Tissera A.D.,Laboratorio Of Andrologia Y Reproduccion | Estofan G.M.,Centro Integral Of Ginecologia | And 2 more authors.
American Journal of Reproductive Immunology | Year: 2016

Problem: Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce. Method of study: A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR™ (IgG) test), and computer-assisted sperm analysis (CASA). Results: A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results. Conclusions: ASA evaluation is highly relevant in full semen assessment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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