Distinct Roles for Wnt-4 and Wnt-11 During Retinoic Acid-Induced Neuronal Differentiation.
Elizalde C, Campa VM, Caro M, Schlangen K, Aransay AM, Del Mar Vivanco M, Kypta RM.
Cell biology and Stem Cells Unit, CIC bioGUNE, 48160 Derio, Spain.
Stem Cells. 2010 Nov 23. [Epub ahead of print]
Abstract
Retinoic acid and Wnt/β-catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA-2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited β-catenin/Tcf activity in NTERA-2 cells but not in 2102Ep cells. Inhibition occurred downstream of β-catenin but did not involve competition between retinoic acid receptors and β-catenin for binding to p300 or Tcf-4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA-2 cells induced the expression of Wnt-4 and Wnt-11, both of which were able to inhibit β-catenin/Tcf activity. Wnt-4 and Wnt-11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6 and GATA6. Gene silencing of Wnt-4, but not Wnt-11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5 and NEUROD1, suggesting that Wnt-4 promotes early neuronal differentiation. Gene expression analysis of NTERA-2 cells stably overexpressing Wnt-11 suggested that Wnt-11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt-11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid-induced neuronal differentiation.
PMID: 21104974 [PubMed - as supplied by publisher]
Crystal Violet: as easy as it gets
Uno de los protocolos más sencillos para estudiar el crecimiento celular es plaquear células a baja densidad, cultivarlas durante varios días y realizar una tinción del cultivo con cristal violeta:
- Remove media
- Wash with PBSx1
- Add staining solution (20% EtOH, 0.4% PFA, 0.05% Crytal Violet in PBSx1)
- Incubate 1h RT, wash 2x PBSx1, remove PBS completely and take pictures
En la imagén podeis observar cuatro pocillos de una placa de 6-wells teñida con crystal violeta y fotografiada con una camara digital compacta con la ayuda de un tripode (para mantener el mismo enfoque en diferentes placas).
Una vez fotografiadas las células, el cristal violeta se puede disolver en etanol y medir la densidad optica a 540nm
Autor: Víctor M. Campa.
Vector Designer
En la imagen un mapa del plasmido pSM2c vacio (Openbyosystems) realizado con el programa vector designer. Se muestran los sitios de corte recomendados por el fabricante para comprobar el plasmido.
Vector DesignerTM es una herramienta online proporcionada de manera gratuita por invitrogen previo registro en su página web que resulta muy útil para organizar nuestras secuencias de DNA/RNA/proteínas u oligos.
Es un software básico, pero a la vez sencillo(nada que ver con vectorNTi) con el que se pueden realizar todas las tareas normalmente necesarias en un laboratorio de biología molecular.
Importar (import) secuencias en formato GeneBank, EMBL, FASTA o nucleotidos sin más. Una vez importada la secuencia se puede indicar si es linear o circular (Edit>Properties>Molecule) asi como otras muchas características.
Buscar secuencias específicas (right click on Sequence Pane)
Insertar secuencias en un punto específico (Edit>Insert Sequence…) o borrar secuencias (seleccionar secuencia > Edit > Delete sequence). Con lo que se pueden modificar los plásmidos seg
Realizar análisis de restricción (Tools>Restriction Map Search), realizar BLAST (Tools> BLAST search), diseñar oligos (Tools>Oligo Perfect).
Se pueden añadir a la secuencia todo tipo de elementos e indicar donde se encuentran (genes, LTR, promotores, etc, etc) Edit>add feature… y en la ventana que se habre indicar el comienzo y final Start X, End Y, el tipo elemento y su nombre.
Las secuencias y mapas construidos se pueden exportar como archivos con formato FASTA, EMBL o Genebank y también como imágenes GIF (Molecule>Export to File/GIF).
http://www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Designers/VectorDesigner.html
Autor: Victor M. Campa
Cardiac Development of Human Embryonic Stem Cells
Maria Barcova, Victor M. Campa, and Mark Mercola.
Human Stem Cell Manual: A Laboratory Guide. Academic Press, Elsevier Inc. (2007). Chapter 18.
ISBN: 978-0-1237-0465-8
Introduction
Human embryonic stem cells (hESCs) offer an unprecedented opportunity to study human cardiomyogenesis and to produce a wide array of human cardiomyogenic cells for use in research applications and, potencially, for treatment of heart disease. hESCs can produce atrial and ventricular myocytes, as well as cells of the central and peripheral conducting systems in the heart. Despise recent advances, the possibility for successful clinical translation requires a concerted research effort because current producction of cardiomyocytes from hESCs is far too inefficient and the methods must still be developed for integrating ESC-derived cardiomyocytes into damages tissue sa that they are compatible with host function and able to restore heart function.
AUTOMATED CALCIUM MEASUREMENTS IN LIVE CARDIOMYOCYTES
David Charlot1,2, Victor Campa1, Behrad Azimi1,2, Mark Mercola1, Randall Ingermanson 3, Patrick M. McDonough3, and Jeffrey H. Price1,3.
1 The Burnham Institute for Medical Research, La Jolla, CA, 2 University of California in San Diego, La Jolla, CA, 3 Vala Sciences, Inc., San Diego, CA.
Biomedical Imaging: From Nano to Macro, 2008. ISBI 2008. 5th IEEE International Symposium on. 14/06/2008;
DOI: 10.1109/ISBI.2008.4540996
ABSTRACT
Heart failure due to hypertension, infarction, or other factors, is a leading killer of men and women in modern society and involves, at its base, a debilitating loss of cardiomyocytes. Recent studies indicate the feasibility of regenerating lost cardiomyocytes by transplanting embryonic stem cell-derived cardiomyocytes (ESCMs) or mobilizing resident stem cells. To realize the potential of stem-cell based therapies, we hypothesize that it will be extremely beneficial to develop technology and instruments for high throughput, high content screening (HCS) of drugs and genes for their ability to stimulate the formation of functional, contractile cardiomyocytes. Contractile activity is the primary physiological function of cardiomyocytes and abnormal contractility is potentially lethal. We discuss the first phase of the development of an instrument dedicated to distinguishing differentiated ESCMs from undifferentiated non-cardiac background cells using automated cell-by-cell quantification of contractile-calcium transients as the primary assay.
Notch activates cell cycle reentry and progression in quiescent cardiomyocytes.
Campa VM, Gutiérrez-Lanza R, Cerignoli F, Díaz-Trelles R, Nelson B, Tsuji T, Barcova M, Jiang W, Mercola M.
Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
J Cell Biol. 2008 Oct 6;183(1):129-41.
Abstract
The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jkappa (RBP-Jkappa)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jkappa. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.
PMID: 18838555 [PubMed - indexed for MEDLINE]PMCID: PMC2557048Free PMC Article
Polyinosinic acid induces TNF and NO production as well as NF-kappaB and AP-1 transcriptional activation in the monocytemacrophage cell line RAW 264.7.
Campa VM, Iglesias JM, Carcedo MT, Rodríguez R, Riera J, Ramos S, Lazo PS.
Instituto Universitario de Oncología del Principado de Asturias and Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33071 Oviedo, Spain.
Inflamm Res. 2005 Aug;54(8):328-37.
Abstract
OBJECTIVE: This study evaluates the poly inosinic acid (poly I)-induced activation in the murine monocytemacrophage cell line RAW 264.7, which led to an inflammatory phenotype.
MATERIAL: RAW 264.7, and WEHI 164 cell lines were used.
RESULTS: The activation process is characterized by the acquisition of a mature macrophage morphology and the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO). The activation by poly I has distinctive features. Thus, poly I induced an increase in nuclear factor kappaB (NF-kappaB) transcriptional activity due to a long-term degradation of inhibitory NF-kappaB (IkappaB) beta while lipopolysaccharide (LPS) induced the degradation of both IkappaBalpha and IkappaBbeta. Poly I also induced an increase in activator protein 1 (AP-1) transcriptional activity, possibly due to the activation of the mitogen activated protein kinases (MAPKs) ERK, Jun N terminal kinase (JNK) and p38. Dextran sulphate (DS) efficiently inhibited the activation induced by poly I including the production of the inflammatory mediators. Dextran sulphate also inhibited AP-1 and NF-kappaB transcriptional activities in poly I-stimulated cells. RAW 264.7 cells express macrophage scavenger receptor 1 (Msr1) type I and Msr1 type II that are differently up-regulated upon treatment with poly I.
CONCLUSIONS: The results presented demonstrate that the well-known blocker of scavenger receptors poly I activates macrophages to produce TNF and NO, triggering specific signal transduction pathways.
PMID: 16158333 [PubMed - indexed for MEDLINE]
Isolation and characterization of nudC from mouse macrophages, a gene implicated in the inflammatory response through the regulation of PAF-AH(I) activity.
Riera J, Rodríguez R, Carcedo MT, Campa VM, Ramos S, Lazo PS.
Departamento de Bioquímica y Biología Molecular and Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, 33071 Oviedo, Spain.
FEBS Lett. 2007 Jun 26;581(16):3057-62. Epub 2007 Jun 4.
Abstract
We report the characterization of a cDNA induced in mouse macrophages that encodes a 332-amino acid protein with extensive sequence identity with members of the mammalian nudC-like genes. The interaction between mNUDC and the regulatory beta subunit of platelet activating factor acetylhydrolase I (PAF-AH(I)) shown in this article indicates a new function of NUDC. Thus, we show that NUDC increases the catalytic activity of PAF-AH(I) and that this regulatory activity is located in the carboxyl terminal half of the protein which is highly conserved. This suggests a novel function for mammalian nudC-like genes as anti-inflammatory proteins.
PMID: 17555748 [PubMed - indexed for MEDLINE]
TNF triggers mitogenic signals in NIH 3T3 cells but induces apoptosis when the cell cycle is blocked.
Rodríguez R, Campa VM, Riera J, Carcedo MT, Ucker DS, Ramos S, Lazo PS.
Instituto Universitario de Oncología del Principado de Asturias and Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33071 Oviedo, Spain.
Eur Cytokine Netw. 2007 Dec;18(4):172-80. Epub 2007 Nov 12.
Abstract
Tumor necrosis factor (TNF) is known to be a mediator of a variety of cellular responses including apoptotic death or proliferation depending on the target cell and the environmental conditions. We show here that TNF triggers both growth and death signals in NIH 3T3 murine fibroblasts. In cells arrested in G(0) by serum deprivation, TNF drives approximately 50% of them to enter the cell cycle, but kills the cells that remain quiescent. The presence of serum prevents toxic effects of TNF, suggesting that TNF can cooperate to drive cells through the cell cycle, but is unable to do so by itself and alternatively it triggers death signals in cells unable to proliferate. Interestingly, TNF induces a similar toxic effect in cells forced to stay at the G(1)/S border, S or M phases. We have explored the TNF apoptotic pathway in arrested cells. This mechanism is not due to the loss of the anti-apoptotic capacity of NFkappaB and is mediated by mitochondria since Bcl-2 overexpression partially inhibits cell death. There are, however, interesting differences in the kinetics of mitochondrial events which indicate that this form of sensitization to TNF leads to an apoptotic mechanism different from that observed after sensitization by RNA synthesis inhibition.
PMID: 17993451 [PubMed - indexed for MEDLINE]
Megabites and Bit Depth
Una imagen digital es una matriz de pixeles. En principio cuantos más pixeles tenga una imagén mayor resolución, hasta que la densidad de pixeles dobla a la capacidad de resolución del microscópio y por lo tanto se satisface el Teorema de Nyquist. Por lo tanto no siempre es mejor tener más megapíxeles ya que, sobretodo con objetivos potentes corremos el riesgo de oversampling. Pero no solo en número de pixeles es importante, también lo es y mucho el número de bits (bit depth) del archivo.
En microscopía de fluorescencia la imagen se capta mediante camaras en blanco y negro ya que tienen mayor sensibilidad que las camaras en color (el color se codifica posteriormente mediante un programa adecuado). De esta manera la fluorescencia emitida por la muestra queda registrada en una escala de grises. Debido a esto es de suma importancia asegurarse que no existe el temido Blead Trought de fluorescencia entre los distintos canales ya que si parte de la fluorescencia verde se cuela en el canal rojo la cmara no es capaz de identificarla como proveniente del canal verde y la codificaría en el canal rojo. Para ello hay que tener cuidado en las tinciones multiples y también incluir marcajes sencillos como controles.
Cada canal se captura como una imagén sin color con una escala de grises cuyo tamaño depende del número de bits de esta.
1 bit: 21 = 2 niveles de gris. (1 blanco, 2 negro).
2 bits: 22 = 4 niveles de gris.
8 bits: 28 = 256 niveles de gris.
14 bits: 214 = 16384 niveles de gris (camara del microscopio).
16 bits: 216 = 65536 nieveles de gris.
Al disminuir el número de bits de una imagen disminuye el tamaño de esta, pero también se pierde información ya que los valores intermedios se interpolan. Aumentar el número de bit no tiene sentido ya que solo aumenta el tamaño de los archivos sin aumentar la información contenida.
Las imágenes en blanco y negro se pueden combinar entre para formar las imágenes en color en un archivo RGB (rojo+verde=amarillo, rojo+azul=magentaen, verde+azul=cyan) en: Images>Color>Merge Channels….
Una imagen en color se puede dividir en los diferentes canales: Image>Color>Split Channels. Estos a su vez se pueden encender y apagar en Images>Color>Channels Tools… (Ten en cuenta que desde un archivo RGB solo se crea un composite con 8 bits por canal, para crear un composite con más bits por canal es necesario formarlo a partir de las imágenes originales; ver debajo).
RGB o TrueColor: 8 bits canal rojo, 8 bits verde y 8 bits azul (256x256x256 colores diferentes que son más de los que puede captar el ojo humano)
Este archivo RGB se puede convertir posteriormente a 8 bit color de manera que el número de colores queda reducido a 256 como máximo.
Por supuesto, al crear un archivo RGB o 8 bit color a partir de imágenes en monocromo de más de 8 bits se pierde información. Si no queremos perder esa información en el archivo final (lo que puede resultar interesante para cuantificación, pero no es necesario para presentación) siempre podemos generar un COMPOSITE (hay que seleccionar esta opción al combinar las imágenes en Images>Color>Merge Channels…. De esta manera tendremos una imagen en color en la que cada canal sigue manteniendo la profundidad de bits original.
Por último, hay que tener en cuenta que aunque para presentación en pantalla el formato RGB es el más adecuado, normalmente las impresoras tienen cuatro cartuchos de tinta con los colores cian, magenta, amarillo y negro (CMYK) por lo que lo ideal seria convertir las imágenes al entorno CMYK antes de imprimirlas.
Por último las imagenes originales deberiamos mantenerlas siempre en archivos .TIF (archivos de mayor tamaño y sin perdida de información), aunque a la hora de preparar presentaciones podemos generar archivos comprimidos del tipo .jpg o .gif.
Autor: Víctor M. Campa
