Una propuesta de la biología de sistemas para el estudio de enfermedades raras

El grupo de Bases Moleculares de la Proliferación Celular de la Universidad de Málaga estudia patologías muy poco convencionales. Su poca incidencia en la población, menos de cinco casos por cada diez mil habitantes, las convierten hoy en un gran reto para la ciencia y la investigació

Aminooxi análogos de histidina: una posible estrategia para modular la histidina descarboxilasa humana.

Congreso anual de la Sociedad Española de Bioquímica y Biología MolecularLa histamina (Hia) es una amina biógena implicada en procesos fisiológicos esenciales (respuesta inmune, neurotransmisión y otros). El desequilibrio de su metabolismo juega un destacado papel en muchas patologías. El control de los efectos adversos de Hia se ejerce únicamente modulando sus receptores (H1R-H4R). En células humanas, Hia se sintetiza por descarboxilación de la L-His, en la reacción dependiente de piridoxal 5-fosfato (PLP) que cataliza la histidina decarboxilasa (HDC, EC 4.1.1.22). La baja tasa de expresión y la inestabilidad de HDC han dificultado su caracterización estructural y funcional, y por tanto el desarrollo de estrategias basadas en su inhibición. Nuestra experiencia derivada de estudios in silico (modelado y dinámica molecular) y experimentales con HDC, nos permitió abordar la búsqueda de nuevas posibilidades. Mediante tecnología de “barrido virtual” dedujimos que un inhibidor competitivo efectivo debería ser aquel capaz de enlazarse eficientemente con el PLP formando una estructura similar al intermediario PLP-substrato. Debería también conservar el grupo imidazol para reforzar la afinidad por el sitio catalítico. Ambas características podían conseguirse utilizando el compuesto 4(5)-aminooximetilimidazol (O-IMHA). Esta hipótesis se ha contrastado experimentalmente sobre extractos de HDC humana purificada. Hemos comprobado que O-IMHA es capaz de formar una PLP-oxima en el centro catalítico de la enzima humana. Los resultados indican que O-IMHA puede constituir una cabeza de síntesis prometedora de inhibidores de HDC. Aprovechando el conocimiento actual sobre la estructura de la enzima proponemos las configuraciones más probables del aducto en el sitio catalítico y podremos desarrollar nuevos derivados con potencial traslacional. SAF2011-26518 y CVI-06585. CIBERER (ISCIII)Universidad de Málaga. Campus Internacional Andalucia Tec

Polyamines and other biogenic amines at the molecular basis of low prevalence diseases

Ponencia Invitada en el 3rd International Conference on PolyaminesBiogenic amines are amino acid-derived compounds involved in the most important physiological functions: cell proliferation and differentiation, immunity, neurotransmission and neuroendocrine system, fertility. Diseases are the result of unrecovered alterations in the balance of, at least, one of our physiological network modules. In addition to the most common diseases, there are described more than 6000 low prevalent/rare diseases (RD), most of them related to the any of the physiological processes mentioned above. Thus, the pleitropy of biogenic amines suggest they must be involved in wide spectrum of human RDs. Putative solutions for these orphan patients need efforts to integrate useful molecular, clinical and pharmacological information from other similar, but more prevalent pathologies. The major aim of our group is to contribute to this relational and integrative effort (Reyes-Palomares et al., 2013, PMID: 23437198). Arginine-derived polyamines (putrescine, spermidine and spermine) are absolutely required for cell proliferation. Thus, polyamine overproduction is related to cancer proliferation and progression, including rare neoplasias (i.e.: neuroblastoma). Polyamine synthesis also is a clear target to fight against protozoan infections (rare diseases in Europe). Impaired polyamine synthesis has been related to epithelial pathologies as psoriasis and hairless phenotypes. It is proven that spermine synthase deficit is in the origin of Snyder-Robinson syndrome (Pegg, 2014, PMID:24395705). Histamine is derived from histidine. It is considered a neurotransmitter and immune mediator also having roles in cell life and death equilibrium. In a recent review, using text-mining technologies, we locate more than 25 immune, inflammatory, neurological and neuroendocrine rare pathologies having histamine (and other biogenic amines, as serotonin and dopamine) as relevant elements in their origins and/or evolution (Pino-Ángeles et al., 2012, PMID: 22435405). In addition, we should also consider the histamine role played in malignant pathologies as mastocytosis, rare gastrointestinal carcinomas and fertility (Sánchez-Jiménez et al., 2013, PMID: 22435405). The gaps in our knowledge still are considerable (specially at the level of membrane-related biomolecular interactions), and further integrative efforts (including in silico-assisted approaches) are claimed to fill them, as this information may contribute to development of new and more efficient strategies of prevention and therapy for many biomedical problems (both prevalent and rare diseases), of course, after the appropriate pre-clinical validation and clinical trial phases. Funded by Ministerio de Economía y Competitividad (Grant SAF2011-26518 and contract from AMER Consortium, CDTI), Plan Andaluz de Investigación y Desarrollo (CVI-06585). CIBERER is an initiative of Instituto de Salud Carlos III.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Funded by Ministerio de Economía y Competitividad (Grant SAF2011-26518 and contract from AMER Consortium, CDTI), Plan Andaluz de Investigación y Desarrollo (CVI-06585). CIBERER is an initiative of Instituto de Salud Carlos III

Network perspective of histamine related diseases

Histamine is the most pleiotropic biogenic amine. Produced and stored by a limited set of cells—histaminergic neurons, enterochromaffin-like cells, and mast cells—it broadcasts intercellular communication signals to a wide variety of cell types through its tissue-specific receptors. The many molecular interactions of these receptors and other mediators result in complex cellular networks whose alteration result in disease. Therefore, complex diseases map to modules of these cellular networks in the diseasomes. In this communication, we survey the histamine cellular networks to map the histamine diseasome, presenting a network view of the pleiotropy of histamine and its role in several complex diseases.A.A. Moya is a CIBERER fellow. 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"

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)

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"

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

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

Spatial and temporal patterns of Holocene precipitation change in the Iberian Peninsula

Precipitation is a key climate parameter of vegetation and ecosystems in the Iberian Peninsula. Here, we use a regional pollen-climate calibration model and fossil pollen data from eight sites from the Atlantic coast to southern Spain to provide quantitative reconstructions of annual precipitation trends and excursions and their regional patterns for the last 11 700 years. The Early Holocene (11 700 to 11 000 cal. a BP) was characterized by high precipitation values followed by a slowly declining trend until about 9000 cal. a BP in the south and about 8000 cal. a BP in the north. From 8000 to 6000 cal. a BP the reconstructed precipitation values are the highest in most records, especially in those located in the Mediterranean climatic region in the southern part of the peninsula, with maximum values nearly 100% higher than the modern reconstructed values. The results suggest a declining precipitation during the Late Holocene in the south, with a positive excursion at around 2500 cal. a BP, while in the north precipitation remained high until 500 cal. a BP. However, the Late Holocene climate reconstructions in the Iberian Peninsula are biased by intensifying human impact on vegetation. The statistical time series analyses using SiZer technique do not indicate any statistically significant high-frequency drought events in the region. In general, our results suggest regional differences in the precipitation patterns between the northern and southern parts of the peninsula, with a more distinct Middle Holocene period of high humidity in the south.Peer reviewe