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Ítem Acceso Abierto Akap350 recruits Eb1 to the spindle poles, ensuring proper spindle orientation and lumen formation in 3d epithelial cell cultures(Springer Nature, 2017-11-02) Almada, Evangelina; Tonucci, Facundo Mauro; Hidalgo, Florencia; Ferretti, Anabela Cecilia; Pariani, Alejandro Pedro; Favre, Cristian; Larocca, María Cecilia; Ibarra, Solange; Vena, Rodrigo; Girardini, Javier; Kierbel, ArlinetThe organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.Ítem Acceso Abierto Anatomical and biochemical changes induced by Gluconacetobacter diazotrophicus stand up for Arabidopsis thaliana seedlings from Ralstonia solanacearum infection(Frontiers Media, 2019-12-23) Rodriguez, María V.; Tano, Josefina; Ansaldi, Nazarena; Carrau, Analía; Srebot, María S.; Ferreira, Virginia; Martínez, María Laura; Cortadi, Adriana A.; Siri, María I.; Orellano, Elena G.Ítem Acceso Abierto Beyond the binding site: In vivo Identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development(Public Library of Science, 2013-05-07) Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria; Allende, Miguel L.; Calcaterra, Nora B.CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.Ítem Acceso Abierto Brains in metamorphosis: Temporal transcriptome dynamics in hatchery-reared flatfishes(MDPI, 2021-12-02) Guerrero Peña, Laura; Suarez Bregua, Paula; Méndez Martínez, Luis; García Fernández, Pablo; Tur, Ricardo; Rubiolo, Juan Andrés; Tena, Juan J.; Rotllant, JosepÍtem Acceso Abierto Broadening the spectrum of ivermectin: its effect on Trypanosoma cruzi and related trypanosomatids(Frontiers Media, 2022-07-28) Fraccaroli, Laura; Ruiz, María Daniela; Perdomo, Virginia Gabriela; Clausi, Agustina Nicole; Balcazar, Darío Emmanuel; Larocca, Luciana; Carrillo, CarolinaChagas disease is an endemic American parasitosis, caused by Trypanosoma cruzi. The current therapies, benznidazole (BZN) and nifurtimox (NFX), show limited efficacy and multiple side effects. Thus, there is a need to develop new trypanocidal strategies. Ivermectin (IVM) is a broad-spectrum antiparasitic drug with low human and veterinary toxicity with effects against T. brucei and Leishmania spp. Considering this and its relatively low cost, we evaluate IVM as a potential repurposed trypanocidal drug on T. cruzi and other trypanosomatids. We found that IVM affected, in a dose-dependent manner, the proliferation of T. cruzi epimastigotes as well as the amastigotes and trypomastigotes survival. The Selectivity Index for the amastigote stage with respect to Vero cells was 12. The IVM effect was also observed in Phytomonas jma 066 and Leishmania mexicana proliferation but not in Crithidia fasciculata. On the epimastigote stage, the IVM effect was trypanostatic at 50 μM but trypanocidal at 100 μM. The assays of the drug combinations of IVM with BNZ or NFX showed mainly additive effects among combinations. In silico studies showed that classical structures belonging to glutamate-gated Cl channels, the most common IVM target, are absent in kinetoplastids. However, we found in the studied trypanosomatid genomes one copy for putative IMPα and IMPβ, potential targets for IVM. The putative IMPα genes (with 76% similarity) showed conserved Armadillo domains but lacked the canonical IMPβ binding sequence. These results allowed us to propose a novel molecular target in T. cruzi and suggest IVM as a good candidate for drug repurposing in the Chagas disease context.Ítem Acceso Abierto Candida infections, causes, targets, and resistance mechanisms: traditional and alternative antifungal agents(Hindawi, 2013) Spampinato, Claudia P.; Leonardi, Darío; https://orcid.org/0000-0003-2292-3570The genus Candida includes about 200 different species, but only a few species are human opportunistic pathogens and cause infections when the host becomes debilitated or immunocompromised.Candida infections can be superficial or invasive. Superficial infections often affect the skin or mucous membranes and can be treated successfully with topical antifungal drugs. However, invasive fungal infections are often life-threatening, probably due to inefficient diagnostic methods and inappropriate initial antifungal therapies. Here, we briefly review our current knowledge of pathogenic species of the genus Candida and yeast infection causes and then focus on current antifungal drugs and resistance mechanisms. An overview of new therapeutic alternatives for the treatment of Candida infections is also provided.Ítem Acceso Abierto Centrosomal AKAP350 modulates the G1/S transition(Landes Bioscience, 2013-10-10) Mattaloni, Stella M.; Ferretti, Anabela Cecilia; Tonucci, Facundo Mauro; Favre, Cristian; Goldenring, James R.; Larocca, María CeciliaAKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participatein the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1 /S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.Ítem Acceso Abierto Cloning and characterization of the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa(Springer, 2017-04-27) Ceccoli, Romina Denis; Bianchi, Dario A.; Fink, Michael J.; Mihovilovic, Marko D.; Rial, Daniela V.Ítem Acceso Abierto CNBP controls transcription by unfolding DNA G-quadruplex structures(Oxford University Press, 2019-06-20) David, Aldana P.; Pipier, Angélique; Pascutti, Federico; Binolfi, Andrés; Weiner, Andrea María Julia; Challier, Emilse; Heckel, Sofía; Calsou, Patrick; Gomez, Dennis; Calcaterra, Nora B.; Armas, PabloÍtem Acceso Abierto Conservation of zebrafish microrna-145 and its role during neural crest cell development(MDPI, 2021) Steeman, Tomás José; Rubiolo, Juan Andrés; Sánchez, Laura E.; Calcaterra, Nora B.; Weiner, Andrea María JuliaÍtem Acceso Abierto Core clock genes adjust growth cessation time to day-night switches in poplar(Springer Nature, 2024-02-27) Alique García, Daniel; Redondo López, Arturo; González Schain, Nahuel; Allona, Isabel; Wabnik, Krzysztof; Perales, Mariano; https://orcid.org/0000-0001-7869-9411; https://orcid.org/0000-0002-7012-2850; https://orcid.org/0000-0001-7263-0560; https://orcid.org/0000-0002-7351-8439Poplar trees use photoperiod as a precise seasonal indicator, synchronizing plant phenology with the environment. Daylength cue determines FLOWERING LOCUS T 2 (FT2) daily expression, crucial for shoot apex development and establishment of the annual growing period. However, limited evidence exists for the molecular factors controlling FT2 transcription and the conservation with the photoperiodic control of Arabidopsis flowering. We demonstrate that FT2 expression mediates growth cessation response quantitatively, and we provide a minimal data-driven model linking core clock genes to FT2 daily levels. GIGANTEA (GI) emerges as a critical inducer of the FT2 activation window, time-bound by TIMING OF CAB EXPRESSION (TOC1) and LATE ELONGATED HYPOCOTYL (LHY2) repressions. CRISPR/Cas9 loss-of-function lines validate these roles, identifying TOC1 as a long-sought FT2 repressor. Additionally, model simulations predict that FT2 downregulation upon daylength shortening results from a progressive narrowing of this activation window, driven by the phase shift observed in the preceding clock genes. This circadian-mediated mechanism enables poplar to exploit FT2 levels as an accurate daylength-meter.Ítem Acceso Abierto Crambescin C1 acts as a possible substrate of iNOS and eNOS increasing nitric oxide production and inducing in vivo hypotensive effect(Frontiers Media, 2021-07-07) Rubiolo, Juan Andrés; Lence, Emilio; González Bello, Concepción; Roel, María; Gil Longo, José; Campos Toimil, Manuel; Ternon, Eva; Thomas, Olivier P.; González Cantalapiedra, Antonio; López Alonso, Henar; Vieytes, Mercedes R.; Botana, Luis M.Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.Ítem Acceso Abierto Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri(Hindawi, 2013-08-01) Tondo, María Laura; Hurtado-Guerrero, Ramón; Ceccarelli, Eduardo Augusto; Medina, Milagros; Orellano, Elena G.; Martínez-Júlvez, Marta; https://orcid.org/0000-0002-3122-9401We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5 Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen.Ítem Acceso Abierto Deciphering the number and location of active sites in the monomeric glyoxalase I of Zea mays(Wiley, 2019-04-16) Gonzalez, Javier M.; Agostini, Romina B.; Alvarez, Clarisa Ester; Klinke, Sebastián; Andreo, Carlos S.; Campos Bermudez, Valeria A.Ítem Acceso Abierto Elucidating the impact of low doses of nanoformulated benznidazole in acute experimental Chagas disease(Public Library of Science (PLOS), 2017-12-21) Rial, Marcela S.; Scalise, María L.; Arrúa, Eva Carolina; Esteva, Mónica I.; Salomon, Claudio; Fichera, Laura E.Ítem Acceso Abierto Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis(Royal Society of Chemistry, 2018-06-28) Espinoza Cara, Andrés; Zitare, Ulises A.; Alvarez Paggi , Damián; Klinke, Sebastián; Otero, Lisandro H.; Murgida, Daniel H.; Vila, Alejandro J.; https://orcid.org/0000-0003-0500-4513; https://orcid.org/0000-0003-0248-6916; https://orcid.org/0000-0002-1507-9685; https://orcid.org/0000-0002-8777-0870; https://orcid.org/0000-0002-5448-5483; https://orcid.org/0000-0001-5173-0183; https://orcid.org/0000-0002-7978-3233Copper sites in proteins are designed to perform either electron transfer or redox catalysis. Type 1 and CuA sites are electron transfer hubs bound to a rigid protein fold that prevents binding of exogenous ligands and side reactions. Here we report the engineering of two Type 1 sites by loop-directed mutagenesis within a CuA scaffold with unique electronic structures and functional features. A copper–thioether axial bond shorter than the copper–thiolate bond is responsible for the electronic structure features, in contrast to all other natural or chimeric sites where the copper thiolate bond is short. These sites display highly unusual features, such as: (1) a high reduction potential despite a strong interaction with the axial ligand, which we attribute to changes in the hydrogen bond network and (2) the ability to bind exogenous ligands such as imidazole and azide. This strategy widens the possibility of using natural protein scaffolds with functional features not present in nature.Ítem Acceso Abierto Expression of a Chloroplast-Targeted Cyanobacterial Flavodoxin in tomato plants increases harvest index by altering plant size and productivity(Frontiers Media, 2019-11-08) Mayta, Martín L.; Arce, Rocío C.; Zurbriggen, Matias D.; Valle, Estela M.; Hajirezaei, Mohammad-Reza; Zanor, María Inés; Carrillo, NéstorÍtem Acceso Abierto Expression of a plastid-targeted flavodoxin decreases chloroplast reactive oxygen species accumulation and delays senescence in aging tobacco leaves(Frontiers Media, 2018-07-17) Mayta, Martín L.; Lodeyro, Anabella F.; Guiamet, Juan J.; Tognetti, Vanesa B.; Melzer, Michael; Hajirezaei, Mohammad-Reza; Carrillo, NéstorÍtem Acceso Abierto FITNESS acts as a negative regulator of immunity and influences the plant reproductive output after Pseudomonas syringae infection(Frontiers Media, 2021-02-04) Mengarelli, Diego Alberto; Tewes, Lara Roldán; Balazadeh, Salma; Zanor, María InésÍtem Acceso Abierto Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications(Frontiers Media, 2014-02-06) Ceccoli, Romina Denis; Bianchi, Dario A.; Rial, Daniela V.External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.