63 results on '"Nualart F"'
Search Results
2. Superoxide-dependent uptake of vitamin C in human glioma cells: SW04.S17–70
- Author
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Nualart, F., Rodriguez, F., Salazar, K., Jara, N., Garcia-Robles, M., Perez, F., Ferrada, L., and Martinez, F.
- Published
- 2013
3. MCT2 in brain glucose sensing: SW03.S12–38
- Author
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Garcia-Robles, M. A., Cortes-Campos, C., Elizondo, R., Carril, C., Barahona, M. J., and Nualart, F.
- Published
- 2013
4. Relationship between renal distribution of PEPCK and glucocorticoid receptor in diabetic nephropathy: P12-73
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Bertinat, R., Jaramillo, K., Silva, P., Nualart, F., and Yañez, A.
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- 2012
5. Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions
- Author
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Pinzón-Daza M.L., Cuellar-Saenz Y., Nualart F., Ondo-Méndez, Alejandro, Del Riesgo Prendes, Lilia, Castillo-Rivera F., and Garzón R.
- Subjects
Unclassified drug ,Physiology ,Atp binding cassette transporter ,Enzyme linked immunosorbent assay ,Cell hypoxia ,Atp-binding cassette ,Cell survival ,member 2 ,member 1 ,Tumor protein ,Cancer growth ,polycyclic compounds ,Hypoxia ,Priority journal ,Apex1 protein ,Cancer resistance ,Microscopy ,alpha subunit ,sub-family b ,Dna (apurinic or apyrimidinic site) lyase ,Breast cancer resistance protein ,Colon cancer ,confocal ,Neoplasm proteins ,Reverse transcription polymerase chain reaction ,Abcg2 protein ,Human ,Hypoxia inducible factor 1alpha ,tumor ,Colonic neoplasms ,Lyase inhibitor ,Dna-(apurinic or apyrimidinic site) lyase ,P-glycoprotein ,Ht29 cells ,Article ,Enzyme-linked immunosorbent assay ,Multidrug resistance protein 1 ,Genetics ,Ht-29 cell line ,Humans ,sub-family g ,Hypoxia-inducible factor 1 ,Drug effects ,Hif1a protein ,Multidrug resistance protein ,Colon tumor ,Tumor cell line ,Cobalt chloride ,Cancer survival ,Confocal microscopy ,E 3330 ,Metabolism ,Human cell ,Doxorubicin ,Oxidative stress ,Drug resistance ,Protein expression ,Reactive oxygen species ,Cell line ,Reactive oxygen metabolite ,Controlled study - Abstract
P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1? activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1? activity under hypoxic conditions. However, HIF-1? activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 118: 1868–1878, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
- Published
- 2017
6. PO-221 Prostate cancer cells are able to use fructose as a metabolic source
- Author
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Carreño, D., primary, Corro, N., additional, Schmidt, C., additional, Sotomayor, P., additional, Cisternas, P., additional, Inestrosa, N., additional, Nualart, F., additional, Cardenas, J.C., additional, and Godoy, A.S., additional
- Published
- 2018
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7. Sodium vitamin c cotransporter svct2 is expressed in hypothalamic glial cells
- Author
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Garcia, MDL, Salazar, K, Millan, C, Rodriguez, F, Montecinos, H, Caprile, T, Silva, C, Cortes, C, Reinicke, K, Vera, JC, Aguayo, LG, Olate, J, Molina, B, and Nualart, F
- Published
- 2005
8. Vitamin c uptake and recycling among normal and tumor cells from the central nervous system
- Author
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Astuya, A, Caprile, T, Castro, M, Salazar, K, Garcia, MD, Reinicke, K, Rodriguez, F, Vera, JC, Millan, C, Ulloa, V, Low, M, Martinez, F, and Nualart, F
- Published
- 2005
9. Broad expression of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase provide evidence for gluconeogenesis in human tissues other than liver and kidney
- Author
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Yanez, AJ, Nualart, F, Droppelmann, C, Bertinat, R, Brito, M, Concha, II, and Slebe, JC
- Published
- 2003
10. Hypothalamic ependymal-glial cells express the glucose transporter glut2, a protein involved in glucose sensing
- Author
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Garcia, MD, Millan, C, Balmaceda-Aguilera, C, Castro, T, Pastor, P, Montecinos, H, Reinicke, K, Zuniga, F, Vera, JC, Onate, SA, and Nualart, F
- Published
- 2003
11. High-affinity sodium-vitamin c co-transporters (svct) expression in embryonic mouse neurons
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Castro, M, Caprile, T, Astuya, A, Millan, C, Reinicke, K, Vera, JC, Vasquez, O, Aguayo, LG, and Nualart, F
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- 2001
12. Spatial distribution of reissners fiber glycoproteins in the filum terminale of the rat and rabbit
- Author
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Molina, B, Rodriguez, EM, Peruzzo, B, Caprile, T, and Nualart, F
- Published
- 2001
13. Expression and Distribution of Facilitative Glucose (GLUTs) and Monocarboxylate/H+ (MCTs) Transporters in Rat Olfactory Epithelia
- Author
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Nunez-Parra, A., primary, Cortes-Campos, C., additional, Bacigalupo, J., additional, Garcia, M. d. l. A., additional, Nualart, F., additional, and Reyes, J. G., additional
- Published
- 2011
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14. Activation of kinin B1 receptors induces chemotaxis of human neutrophils
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Ehrenfeld, P, primary, Millan, C, additional, Matus, C E, additional, Figueroa, J E, additional, Burgos, R A, additional, Nualart, F, additional, Bhoola, K D, additional, and Figueroa, C D, additional
- Published
- 2006
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15. Expression of the fructose transporter GLUT5 in human breast cancer.
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Zamora-León, S P, primary, Golde, D W, additional, Concha, I I, additional, Rivas, C I, additional, Delgado-López, F, additional, Baselga, J, additional, Nualart, F, additional, and Vera, J C, additional
- Published
- 1996
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16. Activation of kinin B1receptors induces chemotaxis of human neutrophils
- Author
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Ehrenfeld, P., Millan, C., Matus, C. E., Figueroa, J. E., Burgos, R. A., Nualart, F., Bhoola, K. D., and Figueroa, C. D.
- Abstract
Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B1receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B1receptor agonists, each assay was carried out overnight at 37°C in 5% CO2‐95% air on neutrophils primed with 1 ng/ml interleukin‐1β. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl‐Met‐Leu‐Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys‐des[Arg9]‐bradykinin (LDBK) or des[Arg9]‐bradykinin at 10−10M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 μg/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase‐polymerase chain reaction and in situ hybridization confirmed the expression of the B1receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B1receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B1receptor agonist LDBK. A generation of kinin B1receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.
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- 2006
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17. IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment
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María José Oviedo, Eder Ramírez, Isabelle De Lima, Natalia Saldivia, Ninoschka Troncoso, Katterine Salazar, Luciano Ferrada, Francisco Nualart, Fernando Martínez, Manuel Cifuentes, Victor Baeza, [Baeza,V, Martínez,F, Ramírez,E, Nualart,F, Oviedo,MJ, De Lima,I, Troncoso,N, Saldivia,N, Salazar,K] Laboratory of Neurobiology and Stem Cells, NeuroCellT, Department of Cellular Biology, Faculty of Biological Sciences, University of Concepcion, Concepcion, Chile. [Nualart,F, Ferrada,L, Salazar,K] Faculty of Biological Sciences, Center for Advanced Microscopy CMA BIOBIO, University of Concepcion, Concepcion, Chile. [Cifuentes,M] Department of Cell Biology, Genetics and Physiology, University of Malaga, IBIMA, Malaga, Spain. [Cifuentes,M] Andalusian Center for Nanomedicine and Biotechnology, BIONAND, Malaga, Spain. [Cifuentes,M] Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, Malaga, Spain., and This work was supported by a Fondecyt Regular Grant Number: 1190848 (to Katterine Salazar) and a PIA-CONICYT, Grant Number: ECM‐12 (to Francisco Nualart).
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Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Differentiation [Medical Subject Headings] ,Diseases::Nervous System Diseases::Central Nervous System Diseases::Brain Diseases::Hydrocephalus [Medical Subject Headings] ,Organisms::Viruses::DNA Viruses::Adenoviridae [Medical Subject Headings] ,Anatomy::Cells [Medical Subject Headings] ,Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Rodentia::Muridae::Murinae::Rats::Rats, Sprague-Dawley [Medical Subject Headings] ,Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Peptide Hydrolases::Cysteine Proteases::Cysteine Endopeptidases::Caspases::Caspases, Effector::Caspase 3 [Medical Subject Headings] ,Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm [Medical Subject Headings] ,Rats, Sprague-Dawley ,Loss of Function Mutation ,Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Nerve Tissue Proteins [Medical Subject Headings] ,Organisms::Eukaryota::Animals [Medical Subject Headings] ,Adherens junctions ,Adenovirus ,Gliosis ,Cells, Cultured ,Multidisciplinary ,Ependimoma ,Chemistry ,Cilium ,Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Adhesion [Medical Subject Headings] ,Adherens Junctions ,Cadherins ,Astrogliosis ,Cell biology ,Caspase-3 ,Ependymoma ,Medicine ,Anatomy::Nervous System::Central Nervous System::Spinal Cord [Medical Subject Headings] ,Anatomy::Cells::Cellular Structures::Cell Membrane::Cell Membrane Structures::Intercellular Junctions::Adherens Junctions [Medical Subject Headings] ,Programmed cell death ,Ependymal Cell ,Science ,Cells ,Nerve Tissue Proteins ,Article ,Adenoviridae ,Adherens junction ,Células ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Microscopy::Microscopy, Electron::Microscopy, Electron, Transmission [Medical Subject Headings] ,Ependyma ,Phenomena and Processes::Cell Physiological Phenomena::Cell Polarity [Medical Subject Headings] ,medicine ,Cell Adhesion ,Animals ,Caspasa 3 ,Uniones adherentes ,Diseases::Neoplasms::Neoplasms by Histologic Type::Neoplasms, Germ Cell and Embryonal::Neuroectodermal Tumors::Neoplasms, Neuroepithelial::Glioma::Ependymoma [Medical Subject Headings] ,Neoplastic transformation ,Persons::Persons::Age Groups::Child [Medical Subject Headings] ,Apical cytoplasm ,Persons::Persons::Age Groups::Adult [Medical Subject Headings] ,Cadherin ,Glial biology ,Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::Cell Adhesion Molecules::Cadherins [Medical Subject Headings] ,medicine.disease ,Diseases::Pathological Conditions, Signs and Symptoms::Pathologic Processes::Gliosis [Medical Subject Headings] ,Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Death [Medical Subject Headings] ,Cellular neuroscience ,Anatomy::Nervous System::Central Nervous System::Brain::Cerebral Ventricles::Ependyma [Medical Subject Headings] ,Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Cytoskeletal Proteins::Catenins [Medical Subject Headings] ,Anatomy::Cells::Cells, Cultured [Medical Subject Headings] ,Cadherinas - Abstract
Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/β-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a “balloon-like” morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with “cilia rigidity” morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.
- Published
- 2021
18. Dehydroascorbic acid sensitizes cancer cells to system x c - inhibition-induced ferroptosis by promoting lipid droplet peroxidation.
- Author
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Ferrada L, Barahona MJ, Vera M, Stockwell BR, and Nualart F
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- Humans, Dehydroascorbic Acid pharmacology, Lipid Droplets, Cell Death, Lipid Peroxidation, Ferroptosis, Neoplasms
- Abstract
Since the discovery of ferroptosis, it has been postulated that this type of cell death could be utilized in treatments for cancer. Unfortunately, several highly aggressive tumor models are resistant to the pharmacological induction of ferroptosis. However, with the use of combined therapies, it is possible to recover sensitivity to ferroptosis in certain cellular models. Here, we discovered that co-treatment with the metabolically stable ferroptosis inducer imidazole ketone erastin (IKE) and the oxidized form of vitamin C, dehydroascorbic acid (DHAA), is a powerful therapy that induces ferroptosis in tumor cells previously resistant to IKE-induced ferroptosis. We determined that DHAA and IKE + DHAA delocalize and deplete GPX4 in tumor cells, specifically inducing lipid droplet peroxidation, which leads to ferroptosis. Moreover, in vivo, IKE + DHAA has high efficacy with regard to the eradication of highly aggressive tumors such as glioblastomas. Thus, the use of IKE + DHAA could be an effective and safe therapy for the eradication of difficult-to-treat cancers., (© 2023. The Author(s).)
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- 2023
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19. Enhanced Astrocyte Activity and Excitatory Synaptic Function in the Hippocampus of Pentylenetetrazole Kindling Model of Epilepsy.
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Díaz F, Aguilar F, Wellmann M, Martorell A, González-Arancibia C, Chacana-Véliz L, Negrón-Oyarzo I, Chávez AE, Fuenzalida M, Nualart F, Sotomayor-Zárate R, and Bonansco C
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- Mice, Animals, Pentylenetetrazole adverse effects, Astrocytes metabolism, Seizures metabolism, Hippocampus metabolism, Epilepsy metabolism, Kindling, Neurologic metabolism
- Abstract
Epilepsy is a chronic condition characterized by recurrent spontaneous seizures. The interaction between astrocytes and neurons has been suggested to play a role in the abnormal neuronal activity observed in epilepsy. However, the exact way astrocytes influence neuronal activity in the epileptogenic brain remains unclear. Here, using the PTZ-induced kindling mouse model, we evaluated the interaction between astrocyte and synaptic function by measuring astrocytic Ca
2+ activity, neuronal excitability, and the excitatory/inhibitory balance in the hippocampus. Compared to control mice, hippocampal slices from PTZ-kindled mice displayed an increase in glial fibrillary acidic protein (GFAP) levels and an abnormal pattern of intracellular Ca2+ -oscillations, characterized by an increased frequency of prolonged spontaneous transients. PTZ-kindled hippocampal slices also showed an increase in the E/I ratio towards excitation, likely resulting from an augmented release probability of excitatory inputs without affecting inhibitory synapses. Notably, the alterations in the release probability seen in PTZ-kindled slices can be recovered by reducing astrocyte hyperactivity with the reversible toxin fluorocitrate. This suggests that astroglial hyper-reactivity enhances excitatory synaptic transmission, thereby impacting the E/I balance in the hippocampus. Altogether, our findings support the notion that abnormal astrocyte-neuron interactions are pivotal mechanisms in epileptogenesis.- Published
- 2023
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20. Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing.
- Author
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Nualart F, Cifuentes M, Ramírez E, Martínez F, Barahona MJ, Ferrada L, Saldivia N, Bongarzone ER, Thorens B, and Salazar K
- Subjects
- Animals, Mice, Rats, Neuroglia, Glucose, Wnt-5a Protein genetics, Connexin 43, Hyperglycemia
- Abstract
Hyperglycemia increases glucose concentrations in the cerebrospinal fluid (CSF), activating glucose-sensing mechanisms and feeding behavior in the hypothalamus. Here, we discuss how hyperglycemia temporarily modifies ependymal cell ciliary beating to increase hypothalamic glucose sensing. A high level of glucose in the rat CSF stimulates glucose transporter 2 (GLUT2)-positive subcommissural organ (SCO) cells to release SCO-spondin into the dorsal third ventricle. Genetic inactivation of mice GLUT2 decreases hyperglycemia-induced SCO-spondin secretion. In addition, SCO cells secrete Wnt5a-positive vesicles; thus, Wnt5a and SCO-spondin are found at the apex of dorsal ependymal cilia to regulate ciliary beating. Frizzled-2 and ROR2 receptors, as well as specific proteoglycans, such as glypican/testican (essential for the interaction of Wnt5a with its receptors) and Cx43 coupling, were also analyzed in ependymal cells. Finally, we propose that the SCO-spondin/Wnt5a/Frizzled-2/Cx43 axis in ependymal cells regulates ciliary beating, a cyclic and adaptive signaling mechanism to control glucose sensing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nualart et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2023
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21. Role of vitamin C and SVCT2 in neurogenesis.
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Salazar K, Jara N, Ramírez E, de Lima I, Smith-Ghigliotto J, Muñoz V, Ferrada L, and Nualart F
- Abstract
Different studies have established the fundamental role of vitamin C in proliferation, differentiation, and neurogenesis in embryonic and adult brains, as well as in in vitro cell models. To fulfill these functions, the cells of the nervous system regulate the expression and sorting of sodium-dependent vitamin C transporter 2 (SVCT2), as well as the recycling of vitamin C between ascorbic acid (AA) and dehydroascorbic acid (DHA) via a bystander effect. SVCT2 is a transporter preferentially expressed in neurons and in neural precursor cells. In developmental stages, it is concentrated in the apical region of the radial glia, and in adult life, it is expressed preferentially in motor neurons of the cerebral cortex, starting on postnatal day 1. In neurogenic niches, SVCT2 is preferentially expressed in precursors with intermediate proliferation, where a scorbutic condition reduces neuronal differentiation. Vitamin C is a potent epigenetic regulator in stem cells; thus, it can induce the demethylation of DNA and histone H3K27m3 in the promoter region of genes involved in neurogenesis and differentiation, an effect mediated by Tet1 and Jmjd3 demethylases, respectively. In parallel, it has been shown that vitamin C induces the expression of stem cell-specific microRNA, including the Dlk1-Dio3 imprinting region and miR-143, which promotes stem cell self-renewal and suppresses de novo expression of the methyltransferase gene Dnmt3a. The epigenetic action of vitamin C has also been evaluated during gene reprogramming of human fibroblasts to induced pluripotent cells, where it has been shown that vitamin C substantially improves the efficiency and quality of reprogrammed cells. Thus, for a proper effect of vitamin C on neurogenesis and differentiation, its function as an enzymatic cofactor, modulator of gene expression and antioxidant is essential, as is proper recycling from DHA to AA by various supporting cells in the CNS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Salazar, Jara, Ramírez, de Lima, Smith-Ghigliotto, Muñoz, Ferrada and Nualart.)
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- 2023
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22. Is IIIG9 a New Protein with Exclusive Ciliary Function? Analysis of Its Potential Role in Cancer and Other Pathologies.
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Oviedo MJ, Ramírez E, Cifuentes M, Farkas C, Mella A, Bertinat R, Gajardo R, Ferrada L, Jara N, De Lima I, Martínez F, Nualart F, and Salazar K
- Subjects
- Child, Humans, Brain metabolism, Proteins metabolism, Neoplasms, Protein Phosphatase 1 metabolism
- Abstract
The identification of new proteins that regulate the function of one of the main cellular phosphatases, protein phosphatase 1 (PP1), is essential to find possible pharmacological targets to alter phosphatase function in various cellular processes, including the initiation and development of multiple diseases. IIIG9 is a regulatory subunit of PP1 initially identified in highly polarized ciliated cells. In addition to its ciliary location in ependymal cells, we recently showed that IIIG9 has extraciliary functions that regulate the integrity of adherens junctions. In this review, we perform a detailed analysis of the expression, localization, and function of IIIG9 in adult and developing normal brains. In addition, we provide a 3D model of IIIG9 protein structure for the first time, verifying that the classic structural and conformational characteristics of the PP1 regulatory subunits are maintained. Our review is especially focused on finding evidence linking IIIG9 dysfunction with the course of some pathologies, such as ciliopathies, drug dependence, diseases based on neurological development, and the development of specific high-malignancy and -frequency brain tumors in the pediatric population. Finally, we propose that IIIG9 is a relevant regulator of PP1 function in physiological and pathological processes in the CNS.
- Published
- 2022
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23. Vitamin C Deficiency Reduces Neurogenesis and Proliferation in the SVZ and Lateral Ventricle Extensions of the Young Guinea Pig Brain.
- Author
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Jara N, Cifuentes M, Martínez F, González-Chavarría I, Salazar K, Ferrada L, and Nualart F
- Abstract
Although scurvy, the severe form of vitamin C deficiency, has been almost eradicated, the prevalence of subclinical vitamin C deficiency is much higher than previously estimated and its impact on human health might not be fully understood. Vitamin C is an essential molecule, especially in the central nervous system where it performs numerous, varied and critical functions, including modulation of neurogenesis and neuronal differentiation. Although it was originally considered to occur only in the embryonic brain, it is now widely accepted that neurogenesis also takes place in the adult brain. The subventricular zone (SVZ) is the neurogenic niche where the largest number of new neurons are born; however, the effect of vitamin C deficiency on neurogenesis in this key region of the adult brain is unknown. Therefore, through BrdU labeling, immunohistochemistry, confocal microscopy and transmission electron microscopy, we analyzed the proliferation and cellular composition of the SVZ and the lateral ventricle (LVE) of adult guinea pigs exposed to a vitamin-C-deficient diet for 14 and 21 days. We found that neuroblasts in the SVZ and LVE were progressively and significantly decreased as the days under vitamin C deficiency elapsed. The neuroblasts in the SVZ and LVE decreased by about 50% in animals with 21 days of deficiency; this was correlated with a reduction in BrdU positive cells in the SVZ and LVE. In addition, the reduction in neuroblasts was not restricted to a particular rostro-caudal area, but was observed throughout the LVE. We also found that vitamin C deficiency altered cellular morphology at the ultrastructural level, especially the cellular and nuclear morphology of ependymal cells of the LVE. Therefore, vitamin C is essential for the maintenance of the SVZ cell populations required for normal activity of the SVZ neurogenic niche in the adult guinea pig brain. Based on our results from the guinea pig brain, we postulate that vitamin C deficiency could also affect neurogenesis in the human brain.
- Published
- 2022
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24. The Differential Paracrine Role of the Endothelium in Prostate Cancer Cells.
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Torres-Estay V, Mastri M, Rosario S, Fuenzalida P, Echeverría CE, Flores E, Watts A, Cerda-Infante J, Montecinos VP, Sotomayor PC, Amigo J, Escudero C, Nualart F, Ebos JML, Smiraglia DJ, and Godoy AS
- Abstract
The survival of patients with solid tumors, such as prostate cancer (PCa), has been limited and fleeting with anti-angiogenic therapies. It was previously thought that the mechanism by which the vasculature regulates tumor growth was driven by a passive movement of oxygen and nutrients to the tumor tissue. However, previous evidence suggests that endothelial cells have an alternative role in changing the behavior of tumor cells and contributing to cancer progression. Determining the impact of molecular signals/growth factors released by endothelial cells (ECs) on established PCa cell lines in vitro and in vivo could help to explain the mechanism by which ECs regulate tumor growth. Using cell-conditioned media collected from HUVEC (HUVEC-CM), our data show the stimulated proliferation of all the PCa cell lines tested. However, in more aggressive PCa cell lines, HUVEC-CM selectively promoted migration and invasion in vitro and in vivo. Using a PCa-cell-line-derived xenograft model co-injected with HUVEC or preincubated with HUVEC-CM, our results are consistent with the in vitro data, showing enhanced tumor growth, increased tumor microvasculature and promoted metastasis. Gene set enrichment analyses from RNA-Seq gene expression profiles showed that HUVEC-CM induced a differential effect on gene expression when comparing low versus highly aggressive PCa cell lines, demonstrating epigenetic and migratory pathway enrichments in highly aggressive PCa cells. In summary, paracrine stimulation by HUVEC increased PCa cell proliferation and tumor growth and selectively promoted migration and metastatic potential in more aggressive PCa cell lines.
- Published
- 2022
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25. Biofilms in hoses utilized to divert colostrum and milk on dairy farms: A report exploring their potential role in herd health, milk quality, and public health.
- Author
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Latorre AA, Oliva R, Pugin J, Estay A, Nualart F, Salazar K, Garrido N, and Muñoz MA
- Abstract
Biofilms in milking equipment on dairy farms have been associated with failures in cleaning and sanitizing protocols. These biofilms on milking equipment can be a source of contamination for bulk tank milk and a concern for animal and public health, as biofilms can become on-farm reservoirs for pathogenic bacteria that cause disease in cows and humans. This report describes a cross-sectional study on 3 dairy farms, where hoses used to divert waste milk, transition milk, and colostrum were analyzed by culture methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to assess the presence of pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa , and Klebsiella spp. In addition, the presence of biofilms was analyzed using scanning electron microscopy and confocal spectral microscopy. Biofilms composed of multispecies microbial communities were observed on the surfaces of all milk hoses. In two dairy farms, S. aureus, P. aeruginosa, Klebsiella pneumoniae , and Klebsiella oxytoca were isolated from the milk hose samples collected. Cleaning and sanitation protocols of all surfaces in contact with milk or colostrum are crucial. Hoses used to collect waste milk, colostrum, and transition milk can be a source of biofilms and hence pathogenic bacteria. Waste milk used to feed calves can constitute a biosecurity issue and a source of pathogens, therefore an increased exposure and threat for the whole herd health and, potentially, for human health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Latorre, Oliva, Pugin, Estay, Nualart, Salazar, Garrido and Muñoz.)
- Published
- 2022
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26. Pharmacological targets for the induction of ferroptosis: Focus on Neuroblastoma and Glioblastoma.
- Author
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Ferrada L, Barahona MJ, Salazar K, Godoy AS, Vera M, and Nualart F
- Abstract
Neuroblastomas are the main extracranial tumors that affect children, while glioblastomas are the most lethal brain tumors, with a median survival time of less than 12 months, and the prognosis of these tumors is poor due to multidrug resistance. Thus, the development of new therapies for the treatment of these types of tumors is urgently needed. In this context, a new type of cell death with strong antitumor potential, called ferroptosis, has recently been described. Ferroptosis is molecularly, morphologically and biochemically different from the other types of cell death described to date because it continues in the absence of classical effectors of apoptosis and does not require the necroptotic machinery. In contrast, ferroptosis has been defined as an iron-dependent form of cell death that is inhibited by glutathione peroxidase 4 (GPX4) activity. Interestingly, ferroptosis can be induced pharmacologically, with potential antitumor activity in vivo and eventual application prospects in translational medicine. Here, we summarize the main pathways of pharmacological ferroptosis induction in tumor cells known to date, along with the limitations of, perspectives on and possible applications of this in the treatment of these tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ferrada, Barahona, Salazar, Godoy, Vera and Nualart.)
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- 2022
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27. IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment.
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Baeza V, Cifuentes M, Martínez F, Ramírez E, Nualart F, Ferrada L, Oviedo MJ, De Lima I, Troncoso N, Saldivia N, and Salazar K
- Subjects
- Adherens Junctions ultrastructure, Animals, Cell Adhesion, Cells, Cultured, Ependyma metabolism, Ependyma ultrastructure, Loss of Function Mutation, Nerve Tissue Proteins genetics, Rats, Sprague-Dawley, Rats, Adherens Junctions metabolism, Ependyma cytology, Nerve Tissue Proteins metabolism
- Abstract
Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/β-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation., (© 2021. The Author(s).)
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- 2021
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28. SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, Which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes.
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Salazar K, Espinoza F, Cerda-Gallardo G, Ferrada L, Magdalena R, Ramírez E, Ulloa V, Saldivia N, Troncoso N, Oviedo MJ, Barahona MJ, Martínez F, and Nualart F
- Abstract
During brain development, sodium-vitamin C transporter (SVCT2) has been detected primarily in radial glial cells in situ, with low-to-absent expression in cerebral cortex neuroblasts. However, strong SVCT2 expression is observed during the first postnatal days, resulting in increased intracellular concentration of vitamin C. Hippocampal neurons isolated from SVCT2 knockout mice showed shorter neurites and low clustering of glutamate receptors. Other studies have shown that vitamin C-deprived guinea pigs have reduced spatial memory, suggesting that ascorbic acid (AA) and SVCT2 have important roles in postnatal neuronal differentiation and neurite formation. In this study, SVCT2 lentiviral overexpression induced branching and increased synaptic proteins expression in primary cultures of cortical neurons. Analysis in neuroblastoma 2a (Neuro2a) and human subventricular tumor C3 (HSVT-C3) cells showed similar branching results. SVCT2 was mainly observed in the cell membrane and endoplasmic reticulum; however, it was not detected in the mitochondria. Cellular branching in neuronal cells and in a previously standardized neurosphere assay is dependent on the recycling of vitamin C or reduction in dehydroascorbic acid (DHA, produced by neurons) by glial cells. The effect of WZB117, a selective glucose/DHA transporter 1 (GLUT1) inhibitor expressed in glial cells, was also studied. By inhibiting GLUT1 glial cells, a loss of branching is observed in vitro, which is reproduced in the cerebral cortex in situ. We concluded that vitamin C recycling between neurons and astrocyte-like cells is fundamental to maintain neuronal differentiation in vitro and in vivo. The recycling activity begins at the cerebral postnatal cortex when neurons increase SVCT2 expression and concomitantly, GLUT1 is expressed in glial cells.
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- 2021
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29. Two Distinct Faces of Vitamin C: AA vs. DHA.
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Ferrada L, Magdalena R, Barahona MJ, Ramírez E, Sanzana C, Gutiérrez J, and Nualart F
- Abstract
Historically, vitamin C has been associated with many regulatory processes that involve specific signaling pathways. Among the most studied signaling pathways are those involved in the regulation of aging, differentiation, neurotransmission, proliferation, and cell death processes in cancer. This wide variety of regulatory effects is due to the fact that vitamin C has a dual mechanism of action. On the one hand, it regulates the expression of genes associated with proliferation (Ccnf and Ccnb1), differentiation (Sox-2 and Oct-4), and cell death (RIPK1 and Bcl-2). At the same time, vitamin C can act as a regulator of kinases, such as MAPK and p38, or by controlling the activation of the NF-kB pathway, generating chronic responses related to changes in gene expression or acute responses associated with the regulation of signal transduction processes. To date, data from the literature show a permanent increase in processes regulated by vitamin C. In this review, we critically examine how vitamin C regulates these different cellular programs in normal and tumor cells.
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- 2021
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30. Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro.
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Espinoza F, Magdalena R, Saldivia N, Jara N, Martínez F, Ferrada L, Salazar K, Ávila F, and Nualart F
- Abstract
The reduced form of vitamin C, ascorbic acid (AA), has been related with gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron-astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown. To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells. AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells that efficiently capture DHA. In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. We demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.
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- 2020
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31. The constitutive androstane receptor and pregnane X receptor in the brain.
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Torres-Vergara P, Ho YS, Espinoza F, Nualart F, Escudero C, and Penny J
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- Brain metabolism, Constitutive Androstane Receptor, Pregnane X Receptor, Receptors, Cytoplasmic and Nuclear, Receptors, Steroid metabolism
- Abstract
Since their discovery, the orphan nuclear receptors constitutive androstane receptor (CAR;NR1I3) and pregnane X receptor (PXR;NR1I2) have been regarded as master regulators of drug disposition and detoxification mechanisms. They regulate the metabolism and transport of endogenous mediators and xenobiotics in organs including the liver, intestine and brain. However, with proposals of new physiological functions for NR1I3 and NR1I2, there is increasing interest in the role of these receptors in influencing brain function. This review will summarise key findings regarding the expression and function of NR1I3 and NR1I2 in the brain, hereby highlighting the need for further research in this field., (© 2020 The British Pharmacological Society.)
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- 2020
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32. Vitamin C controls neuronal necroptosis under oxidative stress.
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Ferrada L, Barahona MJ, Salazar K, Vandenabeele P, and Nualart F
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- Dehydroascorbic Acid, Neurons, Oxidation-Reduction, Oxidative Stress, Ascorbic Acid pharmacology, Necroptosis
- Abstract
Under physiological conditions, vitamin C is the main antioxidant found in the central nervous system and is found in two states: reduced as ascorbic acid (AA) and oxidized as dehydroascorbic acid (DHA). However, under pathophysiological conditions, AA is oxidized to DHA. The oxidation of AA and subsequent production of DHA in neurons are associated with a decrease in GSH concentrations, alterations in glucose metabolism and neuronal death. To date, the endogenous molecules that act as intrinsic regulators of neuronal necroptosis under conditions of oxidative stress are unknown. Here, we show that treatment with AA regulates the expression of pro- and antiapoptotic genes. Vitamin C also regulates the expression of RIPK1/MLKL, whereas the oxidation of AA in neurons induces morphological alterations consistent with necroptosis and MLKL activation. The activation of necroptosis by AA oxidation in neurons results in bubble formation, loss of membrane integrity, and ultimately, cellular explosion. These data suggest that necroptosis is a target for cell death induced by vitamin C., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)
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- 2020
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33. Basal Sodium-Dependent Vitamin C Transporter 2 polarization in choroid plexus explant cells in normal or scorbutic conditions.
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Ulloa V, Saldivia N, Ferrada L, Salazar K, Martínez F, Silva-Alvarez C, Magdalena R, Oviedo MJ, Montecinos H, Torres-Vergara P, Cifuentes M, and Nualart F
- Subjects
- Animals, Blood-Brain Barrier growth & development, Blood-Brain Barrier metabolism, Brain growth & development, Cell Membrane metabolism, Cells, Cultured, Choroid Plexus metabolism, Embryonic Development genetics, Epithelial Cells metabolism, Epithelial Cells pathology, Gene Expression Regulation, Developmental genetics, Guinea Pigs, Mice, Monocarboxylic Acid Transporters genetics, Neurons metabolism, Sodium-Coupled Vitamin C Transporters cerebrospinal fluid, Swine, Symporters genetics, Ascorbic Acid metabolism, Brain metabolism, Glucose Transporter Type 1 blood, Sodium-Coupled Vitamin C Transporters blood
- Abstract
Vitamin C is incorporated into the cerebrospinal fluid (CSF) through choroid plexus cells. While the transfer of vitamin C from the blood to the brain has been studied functionally, the vitamin C transporter, SVCT2, has not been detected in the basolateral membrane of choroid plexus cells. Furthermore, it is unknown how its expression is induced in the developing brain and modulated in scurvy conditions. We concluded that SVCT2 is intensely expressed in the second half of embryonic brain development and postnatal stages. In postnatal and adult brain, SVCT2 is highly expressed in all choroidal plexus epithelial cells, shown by colocalization with GLUT1 in the basolateral membranes and without MCT1 colocalization, which is expressed in the apical membrane. We confirmed that choroid plexus explant cells (in vitro) form a sealed epithelial structure, which polarized basolaterally, endogenous or overexpressed SVCT2. These results are reproduced in vivo by injecting hSVCT2wt-EYFP lentivirus into the CSF. Overexpressed SVCT2 incorporates AA (intraperitoneally injected) from the blood to the CSF. Finally, we observed in Guinea pig brain under scorbutic condition, that normal distribution of SVCT2 in choroid plexus may be regulated by peripheral concentrations of vitamin C. Additionally, we observed that SVCT2 polarization also depends on the metabolic stage of the choroid plexus cells.
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- 2019
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34. Fact or Fiction, It Is Time for a Verdict on Vasculogenic Mimicry?
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Valdivia A, Mingo G, Aldana V, Pinto MP, Ramirez M, Retamal C, Gonzalez A, Nualart F, Corvalan AH, and Owen GI
- Abstract
The term vasculogenic mimicry (VM) refers to the capacity of certain cancer cells to form fluid-conducting structures within a tumor in an endothelial cell (EC)-free manner. Ever since its first report by Maniotis in 1999, the existence of VM has been an extremely contentious issue. The overwhelming consensus of the literature suggests that VM is frequently observed in highly aggressive tumors and correlates to lower patient survival. While the presence of VM in vivo in animal and patient tumors are claimed upon the strong positive staining for glycoproteins (Periodic Acid Schiff, PAS), it is by no means universally accepted. More controversial still is the existence of an in vitro model of VM that principally divides the scientific community. Original reports demonstrated that channels or tubes occur in cancer cell monolayers in vitro when cultured in matrigel and that these structures may support fluid movement. However, several years later many papers emerged stating that connections formed between cancer cells grown on matrigel represented VM. We speculate that this became accepted by the cancer research community and now the vast majority of the scientific literature reports both presence and mechanisms of VM based on intercellular connections, not the presence of fluid conducting tubes. In this opinion paper, we call upon evidence from an exhaustive review of the literature and original data to argue that the majority of in vitro studies presented as VM do not correspond to this phenomenon. Furthermore, we raise doubts on the validity of concluding the presence of VM in patient samples and animal models based solely on the presence of PAS+ staining. We outline the requirement for new biomarkers of VM and present criteria by which VM should be defined in vitro and in vivo .
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- 2019
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35. The median eminence as the hypothalamic area involved in rapid transfer of glucose to the brain: functional and cellular mechanisms.
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Martínez F, Cifuentes M, Tapia JC, and Nualart F
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- Animals, Biological Transport, Active physiology, Gene Expression Regulation physiology, Male, Rats, Rats, Sprague-Dawley, Blood-Brain Barrier metabolism, Glucose metabolism, Hypothalamus metabolism, Mitochondria metabolism
- Abstract
Our data proposes that glucose is transferred directly to the cerebrospinal fluid (CSF) of the hypothalamic ventricular cavity through a rapid "fast-track-type mechanism" that would efficiently stimulate the glucosensing areas. This mechanism would occur at the level of the median eminence (ME), a periventricular hypothalamic zone with no blood-brain barrier. This "fast-track" mechanism would involve specific glial cells of the ME known as β2 tanycytes that could function as "inverted enterocytes," expressing low-affinity glucose transporters GLUT2 and GLUT6 in order to rapidly transfer glucose to the CSF. Due to the large size of tanycytes, the presence of a high concentration of mitochondria and the expression of low-affinity glucose transporters, it would be expected that these cells accumulate glucose in the endoplasmic reticulum (ER) by sequestering glucose-6-phosphate (G-6-P), in a similar way to that recently demonstrated in astrocytes. Glucose could diffuse through the cells by micrometric distances to be released in the apical region of β2 tanycytes, towards the CSF. Through this mechanism, levels of glucose would increase inside the hypothalamus, stimulating glucosensing mechanisms quickly and efficiently. KEY MESSAGES: • Glucose diffuses through the median eminence cells (β2 tanycytes), towards the hypothalamic CSF. • Glucose is transferred through a rapid "fast-track-type mechanism" via GLUT2 and GLUT6. • Through this mechanism, hypothalamic glucose levels increase, stimulating glucosensing.
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- 2019
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36. The Antidiabetic Agent Sodium Tungstate Induces Abnormal Glycogen Accumulation in Renal Proximal Tubules from Diabetic IRS2-Knockout Mice.
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Bertinat R, Westermeier F, Silva P, Gatica R, Oliveira JM, Nualart F, Gomis R, and Yáñez AJ
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- Animals, Diabetes Mellitus, Experimental metabolism, Diabetic Nephropathies metabolism, Insulin metabolism, Insulin Receptor Substrate Proteins genetics, Insulin Resistance, Kidney metabolism, Kidney Tubules, Proximal drug effects, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphoenolpyruvate Carboxykinase (ATP) metabolism, Signal Transduction drug effects, Diabetes Mellitus, Experimental pathology, Glycogen metabolism, Hypoglycemic Agents pharmacology, Kidney Tubules, Proximal metabolism, Tungsten Compounds pharmacology
- Abstract
The kidney is an insulin-sensitive organ involved in glucose homeostasis. One major effect of insulin is to induce glycogen storage in the liver and muscle. However, no significant glycogen stores are detected in normal kidneys, but diabetic subjects present a characteristic renal histopathological feature resulting from extensive glycogen deposition mostly in nonproximal tubules. The mechanism of renal glycogen accumulation is yet poorly understood. Here, we studied in situ glycogen accumulation in the kidney from diabetic IRS2-knockout mice and the effect of the insulin-mimetic agent sodium tungstate (NaW). IRS2-knockout mice displayed hyperglycemia and hyperinsulinemia. NaW only normalized glycemia. There was no evident morphological difference between kidneys from untreated wild-type (WT), NaW-treated WT, and untreated IRS2-knockout mice. However, NaW-treated IRS2-knockout mice showed tubular alterations resembling clear cells in the cortex, but not in the outer medulla, that were correlated with glycogen accumulation. Immunohistochemical detection of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, mostly expressed by renal proximal tubules, showed that altered tubules were of proximal origin. Our preliminary study suggests that IRS2 differentially regulates glycogen accumulation in renal tubules and that NaW treatment in the context of IRS2 ablation induces abnormal glycogen accumulation in cortical proximal tubules.
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- 2018
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37. Structural and functional identification of vasculogenic mimicry in vitro.
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Racordon D, Valdivia A, Mingo G, Erices R, Aravena R, Santoro F, Bravo ML, Ramirez C, Gonzalez P, Sandoval A, González A, Retamal C, Kogan MJ, Kato S, Cuello MA, Osorio G, Nualart F, Alvares P, Gago-Arias A, Fabri D, Espinoza I, Sanchez B, Corvalán AH, Pinto MP, and Owen GI
- Subjects
- Female, Humans, Imaging, Three-Dimensional, Microscopy, Confocal, Microscopy, Fluorescence, Organ Culture Techniques, Staining and Labeling, Tumor Cells, Cultured, X-Ray Microtomography, Models, Biological, Ovarian Neoplasms pathology
- Abstract
Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.
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- 2017
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38. Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.
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Caviedes A, Varas-Godoy M, Lafourcade C, Sandoval S, Bravo-Alegria J, Kaehne T, Massmann A, Figueroa JP, Nualart F, and Wyneken U
- Abstract
Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N -methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.
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- 2017
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39. Arachidonic acid triggers [Ca2+]i increases in rat round spermatids by a likely GPR activation, ERK signalling and ER/acidic compartments Ca2+ release.
- Author
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Paillamanque J, Sanchez-Tusie A, Carmona EM, Treviño CL, Sandoval C, Nualart F, Osses N, and Reyes JG
- Subjects
- Animals, Cells, Cultured, Dose-Response Relationship, Drug, Endoplasmic Reticulum metabolism, Endosomes drug effects, Endosomes metabolism, Kinetics, Male, Microscopy, Confocal, NADP analogs & derivatives, NADP metabolism, Phospholipases A2 metabolism, Rats, Sprague-Dawley, Receptors, G-Protein-Coupled metabolism, Salicylates pharmacology, Sesterterpenes pharmacology, Spermatids cytology, Spermatids metabolism, Testis cytology, Testis drug effects, Testis metabolism, Arachidonic Acid pharmacology, Calcium metabolism, Endoplasmic Reticulum drug effects, MAP Kinase Signaling System drug effects, Receptors, G-Protein-Coupled agonists, Spermatids drug effects
- Abstract
Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
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40. Cytoarchitecture, Proliferative Activity and Neuroblast Migration in the Subventricular Zone and Lateral Ventricle Extension of the Adult Guinea Pig Brain.
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Jara N, Cifuentes M, Martínez F, Salazar K, and Nualart F
- Subjects
- Animals, Cell Proliferation, Cell Shape, Guinea Pigs, Lateral Ventricles ultrastructure, Male, Aging physiology, Cell Movement, Lateral Ventricles cytology, Neurons cytology
- Abstract
In the mouse brain, neuroblasts generated in the subventricular zone (SVZ) migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS). Although the RMS is not present in the human brain, a migratory pathway that is organized around a ventricular cavity that reaches the OB has been reported. A similar cavity, the lateral ventricle extension (LVE), is found in the adult guinea pig brain. Therefore, we analyzed cytoarchitecture, proliferative activity and precursor cell migration in the SVZ and LVE of 1-, 6- and 12-month-old guinea pigs. In young animals, we used confocal spectral and transmission electron microscopy to identify neuroblasts, astrocytes, and progenitor cells in the SVZ and LVE. Analysis of peroxidase diffusion demonstrated that the LVE was a continuous cavity lined by ependymal cells and surrounded by neuroblasts. Precursor cells were mostly located in the SVZ and migrated from the SVZ to the OB through the LVE. Finally, analysis of 6- and 12-month-old guinea pigs revealed that the LVE was preserved in older animals; however, the number of neurogenic cells was significantly reduced. Consequently, we propose that the guinea pig brain may be used as a new neurogenic model with increased similarity to humans, given that the LVE connects the LV with the OB, as has been described in humans, and that the LVE works a migratory pathway. Stem Cells 2016;34:2574-2586., (© 2016 AlphaMed Press.)
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- 2016
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41. Dynamic localization of glucokinase and its regulatory protein in hypothalamic tanycytes.
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Salgado M, Tarifeño-Saldivia E, Ordenes P, Millán C, Yañez MJ, Llanos P, Villagra M, Elizondo-Vega R, Martínez F, Nualart F, Uribe E, and de Los Angeles García-Robles M
- Subjects
- Animals, Blotting, Western, Cytoplasm metabolism, Escherichia coli genetics, Gene Expression Regulation, Glucose pharmacology, Male, Rats, Rats, Sprague-Dawley, Saccharomyces cerevisiae genetics, Ependymoglial Cells metabolism, Glucokinase analysis
- Abstract
Glucokinase (GK), the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose.
- Published
- 2014
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42. SVCT2 vitamin C transporter expression in progenitor cells of the postnatal neurogenic niche.
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Pastor P, Cisternas P, Salazar K, Silva-Alvarez C, Oyarce K, Jara N, Espinoza F, Martínez AD, and Nualart F
- Abstract
Known as a critical antioxidant, recent studies suggest that vitamin C plays an important role in stem cell generation, proliferation and differentiation. Vitamin C also enhances neural differentiation during cerebral development, a function that has not been studied in brain precursor cells. We observed that the rat neurogenic niche is structurally organized at day 15 of postnatal development, and proliferation and neural differentiation increase at day 21. In the human brain, a similar subventricular niche was observed at 1-month of postnatal development. Using immunohistochemistry, sodium-vitamin C cotransporter 2 (SVCT2) expression was detected in the subventricular zone (SVZ) and rostral migratory stream (RMS). Low co-distribution of SVCT2 and βIII-tubulin in neuroblasts or type-A cells was detected, and minimal co-localization of SVCT2 and GFAP in type-B or precursor cells was observed. Similar results were obtained in the human neurogenic niche. However, BrdU-positive cells also expressed SVCT2, suggesting a role of vitamin C in neural progenitor proliferation. Primary neurospheres prepared from rat brain and the P19 teratocarcinoma cell line, which forms neurospheres in vitro, were used to analyze the effect of vitamin C in neural stem cells. Both cell types expressed functional SVCT2 in vitro, and ascorbic acid (AA) induced their neural differentiation, increased βIII-tubulin and SVCT2 expression, and amplified vitamin C uptake.
- Published
- 2013
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43. MCT2 expression and lactate influx in anorexigenic and orexigenic neurons of the arcuate nucleus.
- Author
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Cortes-Campos C, Elizondo R, Carril C, Martínez F, Boric K, Nualart F, and Garcia-Robles MA
- Subjects
- Agouti-Related Protein metabolism, Animals, Anorexia pathology, Cell Line, Tumor, Cells, Cultured, Lactic Acid, Male, Mice, Orexins, Pro-Opiomelanocortin metabolism, Protein Transport, Rats, Rats, Sprague-Dawley, Anorexia metabolism, Arcuate Nucleus of Hypothalamus pathology, Intracellular Signaling Peptides and Proteins metabolism, Monocarboxylic Acid Transporters metabolism, Neurons metabolism, Neuropeptides metabolism
- Abstract
Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ~60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.
- Published
- 2013
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44. Endocytic pathway of exogenous iron-loaded ferritin in intestinal epithelial (Caco-2) cells.
- Author
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Antileo E, Garri C, Tapia V, Muñoz JP, Chiong M, Nualart F, Lavandero S, Fernández J, and Núñez MT
- Subjects
- Adenine analogs & derivatives, Adenine pharmacology, Autophagy, Caco-2 Cells, Coated Pits, Cell-Membrane metabolism, Endocytosis, Humans, Lysosomes metabolism, Microscopy, Confocal, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Vesicular Transport Proteins metabolism, Ferritins metabolism, Intestinal Mucosa metabolism, Iron metabolism
- Abstract
Ferritin, a food constituent of animal and vegetal origin, is a source of dietary iron. Its hollow central cavity has the capacity to store up to 4,500 atoms of iron, so its potential as an iron donor is advantageous to heme iron, present in animal meats and inorganic iron of mineral or vegetal origin. In intestinal cells, ferritin internalization by endocytosis results in the release of its iron into the cytosolic labile iron pool. The aim of this study was to characterize the endocytic pathway of exogenous ferritin absorbed from the apical membrane of intestinal epithelium Caco-2 cells, using both transmission electron microscopy and fluorescence confocal microscopy. Confocal microscopy revealed that endocytosis of exogenous AlexaFluor 488-labeled ferritin was initiated by its engulfment by clathrin-coated pits and internalization into early endosomes, as determined by codistribution with clathrin and early endosome antigen 1 (EEA1). AlexaFluor 488-labeled ferritin also codistributed with the autophagosome marker microtubule-associated protein 1 light chain 3 (LC3) and the lysosome marker lysosomal-associated membrane protein 2 (LAMP2). Transmission electron microscopy revealed that exogenously added ferritin was captured in plasmalemmal pits, double-membrane compartments, and multivesicular bodies considered as autophagosomes and lysosomes. Biochemical experiments revealed that the lysosome inhibitor chloroquine and the autophagosome inhibitor 3-methyladenine (3-MA) inhibited degradation of exogenously added (131)I-labeled ferritin. This evidence is consistent with a model in which exogenous ferritin is internalized from the apical membrane through clathrin-coated pits, and then follows a degradation pathway consisting of the passage through early endosomes, autophagosomes, and autolysosomes.
- Published
- 2013
- Full Text
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45. Clinical and experimental approaches to knee cartilage lesion repair and mesenchymal stem cell chondrocyte differentiation.
- Author
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Montoya F, Martínez F, García-Robles M, Balmaceda-Aguilera C, Koch X, Rodríguez F, Silva-Álvarez C, Salazar K, Ulloa V, and Nualart F
- Subjects
- Chondrocytes cytology, Humans, Knee Injuries pathology, Tissue Engineering, Cartilage, Articular injuries, Cell Differentiation, Chondrocytes transplantation, Knee Injuries rehabilitation, Mesenchymal Stem Cell Transplantation methods, Regeneration physiology
- Abstract
Cartilage has poor regeneration capacity due to the scarcity of endogenous stem cells, its low metabolic activity and the avascular environment. Repair strategies vary widely, including microfracture, autologous or allogenic tissue implantation, and in vitro engineered tissues of autologous origin. However, unlike the advances that have been made over more than two decades with more complex organs, including vascular, cardiac or bone tissues, similar advances in tissue engineering for cartilage repair are lacking. Although the inherent characteristics of cartilage tissue, such as the lack of vascularity and low cellular diversity, suggest that it would be one of the more simple tissues to be engineered, its functional weight-bearing role and implant viability and adaptation make this type of repair more complex. Over the last decade several therapeutic approaches and innovative techniques show promise for lasting and functional regeneration of hyaline cartilage. Here we will analyze the main strategies for cartilage regeneration and discuss our experience.
- Published
- 2013
- Full Text
- View/download PDF
46. Glucose transporter 1 and monocarboxylate transporters 1, 2, and 4 localization within the glial cells of shark blood-brain-barriers.
- Author
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Balmaceda-Aguilera C, Cortés-Campos C, Cifuentes M, Peruzzo B, Mack L, Tapia JC, Oyarce K, García MA, and Nualart F
- Subjects
- Animals, Glucose Transporter Type 1 genetics, Monocarboxylic Acid Transporters genetics, Neuroglia metabolism, Sharks, Symporters genetics, Symporters metabolism, Blood-Brain Barrier cytology, Glucose Transporter Type 1 metabolism, Monocarboxylic Acid Transporters metabolism
- Abstract
Although previous studies showed that glucose is used to support the metabolic activity of the cartilaginous fish brain, the distribution and expression levels of glucose transporter (GLUT) isoforms remained undetermined. Optic/ultrastructural immunohistochemistry approaches were used to determine the expression of GLUT1 in the glial blood-brain barrier (gBBB). GLUT1 was observed solely in glial cells; it was primarily located in end-feet processes of the gBBB. Western blot analysis showed a protein with a molecular mass of 50 kDa, and partial sequencing confirmed GLUT1 identity. Similar approaches were used to demonstrate increased GLUT1 polarization to both apical and basolateral membranes in choroid plexus epithelial cells. To explore monocarboxylate transporter (MCT) involvement in shark brain metabolism, the expression of MCTs was analyzed. MCT1, 2 and 4 were expressed in endothelial cells; however, only MCT1 and MCT4 were present in glial cells. In neurons, MCT2 was localized at the cell membrane whereas MCT1 was detected within mitochondria. Previous studies demonstrated that hypoxia modified GLUT and MCT expression in mammalian brain cells, which was mediated by the transcription factor, hypoxia inducible factor-1. Similarly, we observed that hypoxia modified MCT1 cellular distribution and MCT4 expression in shark telencephalic area and brain stem, confirming the role of these transporters in hypoxia adaptation. Finally, using three-dimensional ultrastructural microscopy, the interaction between glial end-feet and leaky blood vessels of shark brain was assessed in the present study. These data suggested that the brains of shark may take up glucose from blood using a different mechanism than that used by mammalian brains, which may induce astrocyte-neuron lactate shuttling and metabolic coupling as observed in mammalian brain. Our data suggested that the structural conditions and expression patterns of GLUT1, MCT1, MCT2 and MCT4 in shark brain may establish the molecular foundation of metabolic coupling between glia and neurons.
- Published
- 2012
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47. MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction.
- Author
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Cortés-Campos C, Elizondo R, Llanos P, Uranga RM, Nualart F, and García MA
- Subjects
- Animals, Cells, Cultured, Ependyma chemistry, Ependyma cytology, Hypothalamus chemistry, Hypothalamus cytology, Metabolism, Rats, Rats, Sprague-Dawley, Ependyma metabolism, Glucose metabolism, Lactates metabolism, Monocarboxylic Acid Transporters analysis, Muscle Proteins analysis, Neuroglia metabolism, Neurons metabolism, Symporters analysis
- Abstract
Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.
- Published
- 2011
- Full Text
- View/download PDF
48. Up-regulation of the vitamin C transporter SVCT2 upon differentiation and depolarization of myotubes.
- Author
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Low M, Sandoval D, Morales B, Nualart F, and Henríquez JP
- Subjects
- Animals, Ascorbic Acid, Cell Polarity, Cells, Cultured, Chick Embryo, Chickens, Muscle Development, Muscle Fibers, Skeletal chemistry, Myoblasts, Sodium-Coupled Vitamin C Transporters, Cell Differentiation, Muscle Fibers, Skeletal cytology, Organic Anion Transporters, Sodium-Dependent biosynthesis, Symporters biosynthesis, Up-Regulation
- Abstract
In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
49. Glial glucokinase expression in adult and post-natal development of the hypothalamic region.
- Author
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Millán C, Martínez F, Cortés-Campos C, Lizama I, Yañez MJ, Llanos P, Reinicke K, Rodríguez F, Peruzzo B, Nualart F, and García MA
- Subjects
- Age Factors, Animals, Animals, Newborn, Cell Differentiation genetics, Glucokinase genetics, Hypothalamus cytology, Neuroglia cytology, Rats, Rats, Sprague-Dawley, Gene Expression Regulation, Developmental, Gene Expression Regulation, Enzymologic, Glucokinase biosynthesis, Hypothalamus enzymology, Hypothalamus growth & development, Neuroglia enzymology
- Abstract
It has recently been proposed that hypothalamic glial cells sense glucose levels and release lactate as a signal to activate adjacent neurons. GK (glucokinase), the hexokinase involved in glucose sensing in pancreatic beta-cells, is also expressed in the hypothalamus. However, it has not been clearly determined if glial and/or neuronal cells express this protein. Interestingly, tanycytes, the glia that cover the ventricular walls of the hypothalamus, are in contact with CSF (cerebrospinal fluid), the capillaries of the arcuate nucleus and adjacent neurons; this would be expected for a system that can detect and communicate changes in glucose concentration. Here, we demonstrated by Western-blot analysis, QRT-PCR [quantitative RT-PCR (reverse transcription-PCR)] and in situ hybridization that GK is expressed in tanycytes. Confocal microscopy and immuno-ultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of beta1-tanycytes. Furthermore, GK expression increased in these cells during the second week of post-natal development. Based on this evidence, we propose that tanycytes mediate, at least in part, the mechanism by which the hypothalamus detects changes in glucose concentrations.
- Published
- 2010
- Full Text
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50. Iron supply determines apical/basolateral membrane distribution of intestinal iron transporters DMT1 and ferroportin 1.
- Author
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Núñez MT, Tapia V, Rojas A, Aguirre P, Gómez F, and Nualart F
- Subjects
- Animals, Caco-2 Cells, Cation Transport Proteins genetics, Cell Polarity, Chlorides, Ferric Compounds metabolism, Ferrous Compounds administration & dosage, Ferrous Compounds metabolism, Humans, Kinetics, Microscopy, Confocal, Microscopy, Fluorescence, Protein Transport, RNA Interference, Rats, Rats, Sprague-Dawley, Cation Transport Proteins metabolism, Cell Membrane metabolism, Duodenum metabolism, Intestinal Absorption, Intestinal Mucosa metabolism, Iron metabolism
- Abstract
Intestinal iron absorption comprises the coordinated activity of the influx transporter divalent metal transporter 1 (DMT1) and the efflux transporter ferroportin (FPN). In this work, we studied the movement of DMT1 and FPN between cellular compartments as a function of iron supply. In rat duodenum, iron gavage resulted in the relocation of DMT1 to basal domains and the internalization of basolateral FPN. Considerable FPN was also found in apical domains. In Caco-2 cells, the apical-to-basal movement of cyan fluorescent protein-tagged DMT1 was complete 90 min after the addition of iron. Steady-state membrane localization studies in Caco-2 cells revealed that iron status determined the apical/basolateral membrane distribution of DMT1 and FPN. In agreement with the membrane distribution of the transporters, (55)Fe flux experiments revealed inward and outward iron fluxes at both membrane domains. Antisense oligonucleotides targeted to DMT1 or FPN inhibited basolateral iron uptake and apical iron efflux, respectively, indicating the participation of DMT1 and FPN in these fluxes. The fluxes were regulated by the iron supply; increased iron reduced apical uptake and basal efflux and increased basal uptake and apical efflux. These findings suggest a novel mechanism of regulation of intestinal iron absorption based on inward and outward fluxes at both membrane domains, and repositioning of DMT1 and FPN between membrane and intracellular compartments as a function of iron supply. This mechanism should be complementary to those based in the transcriptional or translational regulation of iron transport proteins.
- Published
- 2010
- Full Text
- View/download PDF
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