Anti-Trypanosoma cruzi action of a new benzofuran derivative based on amiodarone structure

Chagas disease is a neglected tropical affection caused by the protozoan parasite Trypanosoma cruzi. There is no current effective treatment since the only two available drugs have a limited efficacy and produce side effects. Thus, investigation efforts have been directed to the identification of new drug leads. In this context, Ca2+ regulating mechanisms have been postulated as targets for antiparasitic compounds, since they present paramount differences when compared to host cells. Amiodarone is an antiarrhythmic with demonstrated trypanocidal activity acting through the disruption of the parasite intracellular Ca2+ homeostasis. We now report the effect of a benzofuran derivative based on the structure of amiodarone on T. cruzi. This derivative was able to inhibit the growth of epimastigotes in culture and of amastigotes inside infected cells, the clinically relevant phase. We also show that this compound, similarly to amiodarone, disrupts Ca2+ homeostasis in T. cruzi epimastigotes, via two organelles involved in the intracellular Ca2+ regulation and the bioenergetics of the parasite. We demonstrate that the benzofuran derivative was able to totally collapse the membrane potential of the unique giant mitochondrion of the parasite and simultaneously produced the alkalinization of the acidocalcisomes. Both effects are evidenced by a large increase in the intracellular Ca2+ concentration of T. cruzi.Fil: Pinto Martinez, Andrea. instituto de Estudios Avanzados; VenezuelaFil: Hernández Rodríguez, Vanessa. instituto de Estudios Avanzados; VenezuelaFil: Rodríguez Durán, Jessica Jenireth. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. instituto de Estudios Avanzados; VenezuelaFil: Hejchman, Elżbieta. Medical University of Warsaw; PoloniaFil: Benaim, Gustavo. Universidad Central de Venezuela; Venezuel

Venezuela's humanitarian crisis, resurgence of vector-borne diseases, and implications for spillover in the region.

In the past 5-10 years, Venezuela has faced a severe economic crisis, precipitated by political instability and declining oil revenue. Public health provision has been affected particularly. In this Review, we assess the impact of Venezuela's health-care crisis on vector-borne diseases, and the spillover into neighbouring countries. Between 2000 and 2015, Venezuela witnessed a 359% increase in malaria cases, followed by a 71% increase in 2017 (411 586 cases) compared with 2016 (240 613). Neighbouring countries, such as Brazil, have reported an escalating trend of imported malaria cases from Venezuela, from 1538 in 2014 to 3129 in 2017. In Venezuela, active Chagas disease transmission has been reported, with seroprevalence in children (<10 years), estimated to be as high as 12·5% in one community tested (n=64). Dengue incidence increased by more than four times between 1990 and 2016. The estimated incidence of chikungunya during its epidemic peak is 6975 cases per 100 000 people and that of Zika virus is 2057 cases per 100 000 people. The re-emergence of many vector-borne diseases represents a public health crisis in Venezuela and has the possibility of severely undermining regional disease elimination efforts. National, regional, and global authorities must take action to address these worsening epidemics and prevent their expansion beyond Venezuelan borders

Phosphorylation of calmodulin: Functional implications

Journal now known as FEBS Journal.-- Open Access content older than 1 year.Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein-serine/threonine and protein-tyrosine kinases. Casein kinase II and myosin light-chain kinase are two of the well established protein-serine/threonine kinases implicated in this process. On the other hand, within the protein-tyrosine kinases involved in the phosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein-tyrosine kinases, such as several members of the Src family kinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We will emphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation-mediated changes of CaM activity, its action on CaM-dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.The work in the authors laboratories was financed by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF99-0052), the Agencia Española de Cooperación Internacional (2002 CN0013) and Consejería de Educación y Cultura de la Comunidad de Madrid (08.1/0027/2001–1) to AV; grants from the Fondo Nacional de Ciencia, Tecnología e Innovación del Ministerio de Ciencia y Tecnología de Venezuela (S1-99000058 and G-2001000637) and Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (C-03-10-33-4798/00) to GB; and a grant from the Spanish Ministry of Education (Programa de Cooperación con Iberoamérica) to A. V. and G. B.Peer Reviewe

La CA2+-ATPasa de la membrana plasmática como enzima clave en la homeostasis intracelular del calcio. Estimulación por etanol y otros efectores

La Ca2+-ATPasa de la membrana plasmática (PMCA) es una enzima clave en la regulación de la concentración basal de Ca2+. Hemos demostrado que el etanol estimula la actividad de la Ca2+-ATPasa y el transporte de Ca2+ asociado. Cuando el etanol y la calmodulina (CaM) esta presentes simultáneamente, el efecto estimulatorio es aditivo, lo cual apoya que estos efectores actúan a través de distintos mecanismos. Mediante el uso de isoformas diferentes de la Ca2+-ATPasa se demostró que la variante PMCA2CI resultó ser mas sensible al efecto del etanol. Es Interesante resaltar que esta es la isoforma mas predominante en cerebro, cerebelo y tejido nervioso. Por otra parte, el fosfatidiletanol se forma mediante la transfosfatidilación de ciertos fosfolípidos cuando el etanol está presente, por una reacción que es catalizada por la fosfolipasa D. Este fosfolípido se acumula en la membrana de las células de mamífero luego del consumo de alcohol. Hemos demostrado que el fosfatidiletanol también estimula a la bomba de calcio de la membrana plasmática. Este fosfolípido incrementa la afinidad de la enzima por Ca2+ en mayor medida que la que se obtiene en presencia de CaM u otro fosfolípido acídico natural. Los esfingolípidos han sido reconocidos recientemente como importantes segundos mensajeros, actuando en varios sistemas en combinación con el Ca2+. En vista del hecho que la PMCA es estimulada por etanol, y tomando en cuenta que los esfingolípidos poseen grupos hidroxilo libres, decidimos estudiar el posible efecto de la ceramida y la esfingosina sobre esta bomba de calcio. Demostramos que la ceramida estimula a la Ca2+-ATPasa de una manera dosis-dependiente y de forma aditiva a la CaM y al etanol, cuando se compara con estos dos efectores añadidos separadamente. La ceramida afecta tanto la afinidad de la enzima por el Ca2+, como su Vmax. Mas aún, este esfingolípido también estimula el transporte de Ca2+ en vesículas invertidas de eritrocitos. Por lo contrario, la esfingosina, la cual se ha reportado en varios sistemas que actúa de manera antagónica a la ceramida, mostró un efecto inhibitorio sobre la Ca2+-ATPasa. Esta inhibición también se observó en presencia de CaM. Estos resultados tomados en conjunto sugieren que la ceramida y la esfingosina actúan antagónicamente sobre la PMCA, lo cual coincide con el efecto opuesto que estos esfingolípidos poseen frecuentemente en diferentes sistemas

Ca2+/calmodulin and apo-calmodulin both bind to and enhance the tyrosine kinase activity of c-Src

This is an open access article distributed under the terms of the Creative Commons Attribution License.Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2 +-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.This work was funded by grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494 & SAF2014-52048-R), the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), the CSIC program iCOOP+ 2014 (COOPA20053), and the European Commission (contract PITN-GA-2011-289033). SRS received funding from the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program FP7/2007-2013 under REA grant agreement n° PITN-GA-2011-289033. VS and GB were supported by fellowship and grants from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (03-00-6057-2005 & PG-03-8728-2013) and Fondo Nacional de Ciencia, Tecnología e Innovación (P-2011000884)Peer Reviewe

Comparative phosphorylation of calmodulin from trypanosomatids and bovine brain by calmodulin-binding protein kinases

Calmodulin (CaM), a major intracellular Ca2+ receptor protein, has been identified and partially characterized in several trypanosomatids. The amino acid sequences of CaM from Trypanosoma cruzi and Trypanosoma brucei are known, while that from Leishmania mexicana is not. CaM from T. cruzi contains 18 amino acid substitutions, as compared with CaM from bovine brain. In addition, CaM from bovine brain contains two tyrosine residues (Tyr-99 and Tyr-138), while CaM from T. cruzi only contains Tyr-138. In the present work we show that a monoclonal antibody developed against the carboxyl-terminal region of bovine brain CaM fails to recognize CaM from both T. cruzi and L. mexicana. CaM from both parasites and from bovine brain were phosphorylated in vitro by a preparation of CaM-binding protein kinases enriched in the epidermal growth factor (EGF) receptor. Phosphoamino acids analysis demonstrated EGF-dependent phosphorylation of tyrosine residues in bovine brain CaM, while only trace amounts of tyrosine phosphorylation were detected in CaM from both trypanosomatids. These results demonstrate that the EGF receptor tyrosine kinase targets Tyr-99, but not Tyr-138, as the single major phosphorylatable residue of CaM. On the other hand, and in contrast to bovine brain CaM, there is a significant phosphorylation of serine residues in CaM from trypanosomatids which is activated by the EGF receptor via a protein-serine/threonine kinase cascade. Copyright (C) 1998 Elsevier Science Inc.This work was supported by a Grant from the Ministerio de Educación y Ciencia, Programa de Cooperación Científica con Iberoamérica (Spain) to A.V. and G.B., Grants from the Comisión Interministerial de Ciencia y Tecnología (SAF392-93 and SAF96-0035), the Dirección General de Investigación Científica y Técnica (PR94-343), and the Consejería de Educación y Cultura de la Comunidad Autónoma de Madrid (AE16-94) (Spain) to A.V., and Grants from the CONICIT (RP-IV 110034 and S1-95000526) and from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (CDCH-C-03-10-3351/94) (Venezuela) to G.B.Peer Reviewe