Sensitivity of polyamine metabolism to glucose deprivation is increased in neuroblastoma cells with N-myc amplification

Ornithine-derived polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. Neuroblastoma, the most frequent extra-cranial solid tumor in children, harbors amplification of n-myc oncogene (which enhances polyamine metabolism) in 25% of the cases. In the present communication, the relevance of n-myc amplification in several metabolic features of human neuroblastoma cell lines is studied. A previously unknown linkage between glycolysis impairment and polyamine reduction, related to n-myc amplification, is unveiled. Results show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. Metabolism-targeted therapies are emerging as new approaches for cancer treatment. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and PA metabolism impairment leading to cell death described in the present work, and its apparent dependence on n-myc amplification in the case of neuroblastoma. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc amplified tumors.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work has been funded by Grants SAF2011-26518 (Ministerio de Economía y Competitividad, Spain), Excellence Projects CTS-1507 and CVI-06585 (Junta de Andalucía, Spain) and BIO-267 (fondos PAIDI, Junta de Andalucía, Spain). MVRP was the recipient of a FPU long-term fellowship (Ministerio de Educación, Cultura y Deporte, Spain) and a “III Plan Propio de Investigación” short-term fellowship (University of Málaga). CIBERER is an initiative of Instituto de Salud Carlos III. This communication has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

A role for antizyme inhibitor 2 in the biosynthesis and content of serotonin and histamine in mouse mast cells

Polyamines (putrescine, spermidine and spermine; PAs) are essential for the majority of living cells. Antizymes and antizyme inhibitors are key regulatory proteins of PA levels by affecting ornithine decarboxylase and PA uptake. In addition to PAs, mast cells (MC) synthesize and store in their granules histamine (Hia) and serotonin (5-HT), which are critical for their function. Our previous studies have indicated a metabolic interplay among PAs, Hia and 5-HT in this cell type. For instance, we showed that PAs affect Hia synthesis during early stages of IL-3-induced bone marrow cell differentiation into bone marrow derived MCs (BMMCs) and demonstrated that PAs are present in MC secretory granules and are important for granule homeostasis, including Hia storage and 5-HT levels. A few years ago, a novel antizyme inhibitor (AZIN2) was described whose expression is restricted to a few tissues and cell types including brain, testis and MCs. In MCs, it was recently proposed that AZIN2 could act as a local regulator of PA biosynthesis in association with 5-HT-containing granules and with 5-HT release following MC activation. To gain insight into the role of AZIN2 in the biosynthesis and storage of 5-HT and also Hia, we have generated BMMCs from both wild-type and transgenic mice with severe Azin2 hypomorphism, and have analyzed the content of PAs, 5-HT and Hia, and some elements of their metabolisms. Spermine and 5-HT levels were reduced in Azin2 hypomorphic BMMCs compared with wild-type controls, whereas the amount of Hia was increased. Accordingly, the level of tryptophan hydroxylase 1 (the key enzyme for 5-HT biosynthesis) was reduced and the amount of enzymatic activity of histidine decarboxylase (the enzyme responsible for Hia biosynthesis) was increased in Azin2 hypomorphic BMMCs. Taken together, our results show evidence that AZIN2 has an important role in the regulation of 5-HT and Hia biosynthesis and storage in MCsUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was supported by SAF2011-26518 (MINECO, Spain) and P10-CVI-6585 and Bio-267 (Junta de Andalucia, Spain). CIBERER is an iniciative of Instituto de Salud Carlos III (Spain)

A role for antizyme inhibitor 2 in the biosynthesis and content of histamine and serotonin in mouse mast cells

Polyamines (putrescine, spermidine and spermine; PAs) are required for the survival of the majority of living cells. Antizymes and antizyme inhibitors are key regulatory proteins of PA levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and PA uptake. In addition to PA, mast cells (MC) synthesize and store in their granules the biogenically active amines histamine (Hia) and serotonin (5-HT), which are of critical importance for their function. Previously, we have performed several studies in this cell type regarding the interplay between the metabolisms of PAs and Hia and 5-HT. Our results showed that PAs affect Hia synthesis during early stages of IL-3-induced bone marrow cell differentiation into bone marrow derived MCs (BMMCs) and demonstrated that PAs are present in MC secretory granules and are important for granule homeostasis, including Hia storage and 5-HT levels. A few years ago, a novel antizyme inhibitor (AZIN2) was described. In contrast to AZIN1, AZIN2 expression is restricted to a few tissues and cell types including brain, testis and MCs. In MCs, it was recently described that AZIN2 could act as a local regulator of PA biosynthesis in association with the 5-HT granule content and release. At present, our aim is to gain further insight into the role of AZIN2 in the biosynthesis, storage and release of both Hia and 5-HT. In this study, we have generated BMMCs from both wild-type and transgenic mice with severe Azin2 hypomorphism, and have analyzed the content of PAs, Hia and 5-HT, and some elements of their metabolisms. Both PAs and 5-HT levels were reduced in Azin2 hypomorphic BMMCs compared with wild-type controls, whereas the amount of Hia was increased. Accordingly, the level of tryptophan hydroxylase 1 (the key enzyme for 5-HT biosynthesis) was reduced and the amount of enzymatic activity of histidine decarboxylase (the enzyme responsible for histamine biosynthesis) was increased in Azin2 hypomorphic BMMCs. Taken together, our results show evidence that AZIN2 has an important role in the regulation of Hia and 5-HT biosynthesis and storage in MCs. Department of Molecular Biology and Biochemistry, and CIBER de Enfermedades Raras (CIBER-ER), Faculty of Sciences, University of Málaga, Málaga 29071, Spain. Corresponding author: I. Fajardo ([email protected]) This work was supported by SAF2011-26518 (MINECO, Spain) and P10-CVI-6585 and Bio-267 (Junta de Andalucia, Spain). CIBERER is an iniciative of Instituto de Salud Carlos III (Spain).Universidad de Málaga. Campus de Excelencia Internacional Andalucía-Tech. SAF2011-26518 (MINECO, Spain) and P10-CVI-6585 and Bio-267 (Junta de Andalucia, Spain. CIBERER is an iniciative of Instituto de Salud Carlos III (Spain)

Structural and functional interaction between polyamine related molecules and biological membranes

La comunicación describe el conocimiento actual sobre las interacciones de biomoléculas relacionadas con el metabolismo de poliaminas con las estructuras y funciones de las membranas biológicasChanges induced by PA on nucleic acid (NA) conformation and synthesis is proven to be a major reason for PA essentiality (1-3). However, PA interactions with other polyanions, for instance polyanionic membrane lipid bilayers and glyosaminoglycans have received less attention (3-4). The functional importance of these interactions still is an obscure but interesting area of cell and molecular biology, especially in mammalian cells for which specific PA transport systems are not fully characterized (5). In mammals, activity and turnover of the polyamine (PA) synthesis key enzyme is controlled by a set of proteins: Antizymes (OAZ1-3) and antizyme inhibitors (AZIN1 and 2). It is demonstrated that AOZ modulate polyamine uptake (6), and that PA transport to mitochondria is linked to the respiratory chain state and modulates mitochondrial permeability transition (7). Antizyme expression variants have been located in mitochondria, being proposed as a proapoptotic factor (7-8). AZIN 2 is only expressed in a reduced set of tissues that includes mast cells, where it is associated to mast cell granules membrane (9). This fact, together to the abnormalities observed in bone marrow derived mast cell granules when they are differentiated under restricted PA synthesis conditions (10 and unpublished results), point out to important roles of PA and their related proteins in structure and function of mast cell granules. We will also present novel biophysical results on tripartite interactions of PA that remark the interest of the characterization of PA interactions with lipid bilayers for biomedicine and biotechnology. Thus, the information reported in this paper integrates previously reported information with our still unpublished results, all indicating that PA and their related proteins also are important factors for structure and dynamics of biological membranes and their associated functions essential in human physiology; for instance, solute interchange with the environment (uptake and secretion), oxidative metabolism and apoptosis. The importance of these involved processes for human homeostasis claim for further research efforts. 1. Ruiz-Chica J, Medina MA, Sánchez-Jiménez F and Ramírez FJ (2001) Fourier Transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines. Biophysical J. 80:443-454. 2. Lightfoot HL, Hall J (2014) Endogenous polyamine function--the RNA perspective. Nucleic Acids Res. 42:11275-11290. 3. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol. 42:39-51. 4. Finger S, Schwieger C, Arouri A, Kerth A, Blume A (2014) Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance. Biol Chem. 395:769-778. 5. Poulin R, Casero RA, Soulet D. (2012) Recent advances in the molecular biology of metazoan polyamine transport. Amino Acids. 42:711-723. 6. Kahana C (2009) Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor. Essays Biochem. 4:47-61. 7. Agostinelli E, Marques MP, Calheiros R, Gil FP, Tempera G, Viceconte N, Battaglia V, Grancara S, Toninello A (2010) Polyamines: fundamental characters in chemistry and biology. Amino Acids 38:393-403. 8. Liu GY, Liao YF, Hsu PC, Chang WH, Hsieh MC, Lin CY, Hour TC, Kao MC, Tsay GJ, Hung HC (2006) Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases' cascade. Apoptosis 11:1773-1788. 9. Kanerva K, Lappalainen J, Mäkitie LT, Virolainen S, Kovanen PT, Andersson LC (2009). Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion. PLoS One 31:e6858. 10. García-Faroldi G, Rodríguez CE, Urdiales JL, Pérez-Pomares JM, Dávila JC, Pejler G, Sánchez-Jiménez F, Fajardo I (2010) Polyamines are present in mast cell secretory granules and are important for granule homeostasis. PLoS One 30:e15071.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Evaluation of metabolism and biosignaling in the angiogenic microenvironment as potential targets for therapeutic intervention

The "re-discovery" of Warburg effect at the turn of the present millennium has been a key determinant of the current renewed interest on cancer metabolism. In fact, metabolic reprogramming has been identified as one of the hallmarks of cancer. However, cancers grow in tight contact with non-tumoral accompanying cells and the surrounding extracellular matrix, as underlined by the concept of tumor microenvironment. Endothelial cells are key components of this tumor microenvironment, since they are requested for angiogenesis, another hallmark of cancer. In this complex system, rewiring of metabolism and signaling pathway in cancer, endothelial and other accompanying cell emerges as new potential targets for therapeutic intervention. In this communication, we will present the drug discovery and characterization approach of our group and our more recent results in this field, including new modeling with an evolutionary and ecological point of view.[Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communication has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Método de obtención de anticuerpos anti-hHDC y aplicaciones de los mismos

La presente invención proporciona un método para la obtención de anticuerpos, mono y/o policlonales, que interaccionan frente a la histidina descarboxilasa humana (hHDC), así como los polinucleótidos y polipéptidos necesarios para llevarlo a cabo. Del mismo modo, los usos de los mencionados anticuerpos, así como los kits de diagnóstico de los que formen parte también son objeto de la presente invención.REIVINDICACIONES: 1. Polinucleótido, capaz de codificar un polinpéptido capaz de generar de anticuerpos o fragmentos de los mismos específicos frente a hHDC, cuya secuencia es elegida del grupo: a.Secuencia que comprende SEQ ID 1 b.Secuencia que consiste en SEQ ID 1 o comprende fragmentos de ésta. c.Secuencia que difiera de las secuencias a o b debido a la degeneración del código genético. d.Secuencias que compartan al menos un 80%, 90%, 95% ó 98% de homología con cualquiera de las secuencias anteriores. 2. Polipéptido, según la reivindicación 1, capaz de generar anticuerpos específicos frente a hHDC y cuya secuencia es elegida del grupo: a.Secuencia que comprende SEQ ID 2 b.Secuencia que consiste en SEQ ID 2 o comprende fragmentos de ésta. c.Secuencia que comparta al menos un 80%, 90%, 95% ó 98% de homología la secuencia a o b. 3. Anticuerpos o fragmentos de los mismos específicos frente a hHDC, en donde dichos anticuerpos interaccionan específicamente frente cualquiera de los polipéptidos de la reivindicación 2. 4. Uso de cualquiera de los polipéptidos según la reivindicación 2 para la generación de anticuerpos o fragmentos de los mismos específicos frente a hHDC. 5. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por técnicas de generación de hibridomas. 6. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por la tecnología de "phage display". 7. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por inmunización de mamíferos no humanos. 8. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento. 9. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de la degeneración neurológica. 10. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de la anafilaxis y/o procesos alérgicos. 11. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento la degeneración de epitelios digestivos. 12. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento para el tratamiento de diferentes tipos de cáncer. 13. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de procesos infecciosos. 14. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3 para el diagnóstico in vitro de enfermedades. 15. Kit de diagnóstico que comprende la utilización de anticuerpos o fragmentos de los mismos según la reivindicación 3.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos III; Universidad de MálagaSolicitud de patent

La enseñanza del metabolismo: retos y oportunidades

En el marco del Proyecto de Innovación Educativa de la Universidad de Málaga PIE15-163, cuya descripción y resultados incluimos, decidimos que esta era una excelente oportunidad para reflexionar acerca de la enseñanza del metabolismo y de poner por escrito dichas reflexiones en un libro. Quisimos y pudimos contar con la colaboración de buena parte de los compañeros del Departamento de Biología Molecular y Bioquímica que apoyaron con su firma el proyecto PIE15-163 y extendimos nuestra invitaciones a otros compañeros de dentro y fuera de la Universidad de Málaga. Del Departamento de Biología Molecular y Bioquímica de la Universidad de Málaga hemos recibido aportaciones de los catedráticos Victoriano Valpuesta Fernández, Ana Rodríguez Quesada y Antonio Heredia Bayona, los profesores titulares María Josefa Pérez Rodríguez, José Luis Urdiales Ruiz e Ignacio Fajardo Paredes y la investigadora postdoctoral y profesora sustituta interina Beatriz Martínez Poveda. De otros departamentos de la Universidad de Málaga hemos contado con las aportaciones de la catedrática del Departamento de Especialidades Quirúrgicas, Bioquímica e Inmunología Pilar Morata Losa, del catedrático del Departamento de Lenguajes y Ciencias de la Computación José Francisco Aldana Montes y los componentes de su grupo de investigación Khaos Ismael Navas Delgado, María Jesús García Godoy, Esteban López Camacho y Maciej Rybinski, del catedrático Ángel Blanco López, del Área de Conocimiento de Didáctica de las Ciencias Experimentales y del Doctor en Ciencias Químicas y actual doctorando del Programa de Doctorado "Educación y Comunicación Social" Ángel Luis García Ponce. De fuera de la Universidad de Málaga, hemos contado con las aportaciones del catedrático de la Universidad de La Laguna Néstor V. Torres Darias, de la catedrática de la Universitat de les Illes Balears Pilar Roca Salom y de sus compañeros los profesores Jorge Sastre Serra y Jordi Oliver, de los catedráticos de la Universidad de Granada Rafael Salto González y María Dolores Girón González y su colaborador el Dr. José Dámaso Vílchez Rienda, del profesor titular de la Universidad de Alcalá Ángel Herráez, del investigador postdoctoral de la Universidad de Erlangen (Alemania) Guido Santos y del investigador postdoctoral de la empresa Brain Dynamics Carlos Rodríguez Caso.Hemos estructurado los contenidos del libro en diversas secciones. La primera presenta el Proyecto en cuyo marco se ha gestado la iniciativa que ha conducido a la edición del presente libro. La segunda sección la hemos titulado "¿Qué metabolismo?" e incluye diversas aportaciones personales que reflexionan acerca de qué metabolismo debe conocer un graduado en Bioquímica, en Biología, en Química, en Farmacia o en Medicina, así como una aportación acerca de qué bioquímica estructural y enzimología son útiles y necesarias para un estudiante que vaya a afrontar el estudio del metabolismo. La tercera sección, "Bases conceptuales", analiza las aportaciones del aprendizaje colaborativo, el contrato de aprendizaje y el aprendizaje basado en la resolución de casos prácticos a la mejora del proceso enseñanza-aprendizaje dentro del campo de la Bioquímica y Biología Molecular, más concretamente en el estudio del metabolismo. La cuarta sección se titula "Herramientas", es la más extensa e incluye las diversas aportaciones centradas en propuestas concretas de aplicación relevantes y útiles para la mejora de la docencia-aprendizaje del metabolismo. Sigue una sección dedicada a presentar de forma resumida los "Resultados" del proyecto PIE15-163. El libro concluye con una "coda final" en la que se reflexiona acerca del aprendizaje de la Química a la luz de la investigación didáctica.Patrocinado por el Proyecto de Innovación Educativa de la Universidad de Málaga PIE15-16

An ecological model of invasion and metastasis

It has been argued that malignat tumours represent complex dynamic and self-organizing ecosystems. Furthermore, there is increasing evidence that collective cell migration occurs during invasion and metastasis. Here, we propose a mathematical model showing how cooperative behaviour could arise in heterogeneous tumour populations and why the emergence of such swarm-like patterns would confer advantageous properties to the spatio-temporal expansion of tumours. consequently, targeting such collectivity should be of interest for basic knowledge and clinical tratment of cancer, including rare tumours.[Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communication has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Turning around Cycles: An Approach Based on Selected Problems/Cases to Stimulate Collaborative Learning about Krebs and His Four Metabolic Cycles

Metabolism is a challenging subject for bioscience students due to the intrinsic complexity of the metabolic network, as well as that of the overlapping mechanisms of metabolic regulation. Collaborative learning based on a problem-based learning approach can help students to successfully learn and understand metabolism. In the present article, we propose a selection of exercises, problems, and cases aimed to focus students’ attention on the scientific work made by Sir Hans Krebs and his collaborators to elucidate four main metabolic cycles, as well as on the study of these cycles, their regulation, and their metabolic integration. The objectives, the tools, and the implementation of this proposal are described, and the results obtained during its first implementation with volunteer students enrolled in two courses on metabolic regulation at our university are presented and discussed. These volunteer students signed a learning contract and were randomly distributed in small groups (3–4 students each). Application of this collaborative learning activity to our classrooms has been very satisfactory, as evidenced by an improvement in the volunteers’ academic performance and a very positive perception by most of them, who declared to be “very satisfied” or “satisfied” with their experience and felt that they had learned more.This work was supported by the University of Málaga (Spain) with funds granted to the educational innovation projects PIE15-163, PIE17-145, and PIE19-057. The experimental work carried out by our group is supported by grants PID2019-105010RB-I00 and EDU2017-82197-P (Spanish Ministry of Science and Innovation), UMA18-FEDERJA-220 (Andalusian Government and FEDER), as well as PY20_00257 and funds from PAIDI group BIO 267 (Andalusian Government). Funding for open access charge: Universidad de Málaga / CBU