Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis.

Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.

D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy.

Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

Comparative genomic analysis of two novel plasmids from Acidithiobacillus ferrivorans strain PQ33 / Análisis genómico comparativo de dos nuevos plásmidos de la cepa PQ33 de Acidithiobacillus ferrivorans

Abstract Acidithiobacillus ferrivorans is a psychrotolerant acidophile capable of growing and oxidizing ferrous and sulphide substrates at low temperatures. To date, six genomes of this organism have been characterized; however, evidence of a plasmid in this species has been reported only once, whereby there is no conclusive role of the plasmids in the species. Herein, two novel plasmids of A. ferrivorans PQ33 were molecularly characterized and compared at a genomic scale. The genomes of two plasmids (12 kbp and 10 kbp) from A. ferrivorans PQ33 (NZ_LVZL01000000) were sequenced and annotated. The plasmids, named pAfPQ33-1 (NZ_CP021414.1) and pAfPQ33-2 (NZ_CP021415.1), presented 9 CDS and 13 CDS, respectively. In silico analysis showed proteins involved in conjugation (TraD, MobA, Eep and XerD), toxin-antitoxin systems (HicA and HicB), replication (RepA and DNA binding protein), transcription regulation (CopG), chaperone DnaJ, and a virulence gene (vapD). Furthermore, the plasmids contain sequences similar to phosphate-selective porins O and P and a diguanylate cyclase-phosphodiesterase protein. The presence of these genes suggests the possibility of horizontal transfer, a regulatory system of plasmid maintenance, and adhesion to substrates for A. ferrivorans species and PQ33. This is the first report of plasmids in this strain.

Resumen Acidithiobacillus ferrivorans es un acidófilo psicrotolerante capaz de hacer crecer y oxidar sustratos ferrosos y sulfurosos a bajas temperaturas. Hasta la fecha se han caracterizado seis genomas de este organismo; sin embargo, la evidencia de un plásmido en esta especie ha sido informado solo una vez, por lo que no hay un rol concluyente de los plásmidos en la especie. Aquí, dos plásmidos novedosos de A. ferrivorans PQ33 se caracterizaron molecularmente y se compararon a escala genómica. Se secuenciaron y anotaron los genomas de dos plásmidos (12 kpb y 10 kpb) de A. ferrivorans PQ33 (NZ_LVZL01000000). Los plásmidos, denominados pAfPQ33-1 (NZ_CP021414.1) y pAfPQ33-2 (NZ_CP021415.1), presentaron 9 CDS y 13 CDS, respectivamente. El análisis in silico mostró proteínas involucradas en la conjugación (TraD, MobA, Eep y XerD), sistemas de toxina-antitoxina (HicA y HicB), replicación (RepA y proteína de unión al ADN), regulación de la transcripción (CopG), chaperona DnaJ y un gen de virulencia (vapD). Además, los plásmidos contienen secuencias similares a las porinas selectivas de fosfato O y P y una proteína diguanilato ciclasa-fosfodiesterasa. La presencia de estos genes sugiere la posibilidad de transferencia horizontal, un sistema regulador de mantenimiento de plásmidos y adhesión a sustratos para especies de A. ferrivorans y PQ33. Este es el primer informe de plásmidos en esta cepa.

Actividad inhibitoria del crecimiento bacteriano por cobre nanoestructurado obtenido de minerales de la región Marañón: comparación con cobre comercial / Bacterial growth inhibitory activity for nanostructured copper minerals obtained from the Marañon region: comparison with commercial copper

En el presente trabajo se reporta la actividad inhibitoria del crecimiento bacteriano por nanopartículas de cobre cementado y de cobre comercial. Se utilizaron las cepas de Staphylococcus aureus ATCC 6538 (Gram positiva) y Escherichia coli ATCC 35218 (Gram negativa) para determinar el efecto inhibitorio mediante la concentración mínima inhibitoria de las nanopartículas diluidas en caldo de cultivo nutritivo y distribuidas en placas de ELISA. Las muestras de cobre cementado (obtenidas por procesos hidrometalúrgicos) y de cobre comercial fueron nanoestructuradas empleando un equipo de molienda mecánica. Los resultados indican que las nanopartículas de cobre comercial (a 2.5 horas de molienda) muestran acción inhibitoria del crecimiento de la cepa S. aureus y no así en la cepa E. coli. Asimismo, se determinó que la concentración mínima inhibitoria de la muestra de cobre comercial fue de 20 μg/mL frente a S. aureus. El cobre cementado (en su forma sólida y nanoestructurada) no mostró efecto inhibitorio del crecimiento en ninguna de las dos cepas estudiadas.

In this paper, we report on the bacterial growth inhibitory activity of nanoparticles of cemented and commercial copper. Strains of Staphylococcus aureus ATCC 6538 (Gram positive) and Escherichia coli ATCC 35218 (Gram negative) were used to determine the inhibitory effect by the minimal inhibitory concentration of the nanoparticles diluted in nutrient culture broth and distributed in ELISA plates. The copper cements (obtained from hydrometallurgical processes) and the commercial one were nanostructured employing a mechanical milling equipment. The results indicate that commercial copper nanoparticles (after 2.5 hours of milling) show growth inhibitory action of S. aureus strain. However, in the case of E. coli strains no inhibitory action has been observed. It was also determined that the minimal inhibitory concentration of the commercial copper is 20 μg/mL against S. aureus. On the other hand, copper cements (in solid and nanostructured form) do not show inhibitory effects.

Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris.

BACKGROUND: Deschampsia antarctica shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the D. antarctica Cu/ZnSOD gene into Pichia pastoris and to characterize the heterologous protein. FINDINGS: The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a D. antarctica by cDNA library screening. This SOD gene was cloned in the expression vector pGAPZalphaA and successfully integrated into the genome of the yeast P. pastoris SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg. CONCLUSION: D. antarctica Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40 degrees C for 30 minutes and it is stable (80% of activity) between -20 degrees C and 20 degrees C. The recombinant SOD described in this study can be used in various biotechnological applications.

Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses.

BACKGROUND: The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a Deschampsia antarctica Desv. by cDNA library screening. The expression of SOD gene in the leaves of D. antarctica was determined by RT-PCR and its differential expression of gene transcripts in conditions of cold and UV radiation stresses was revealed by northern blot. FINDINGS: The molecular characterization shows that SOD cDNA is 709 bp in length, which translates an ORF of 152 amino acids that correspond to a protein of predicted molecular mass of 15 kDa. The assay shows that the expression of SOD gene increases when D. antarctica is acclimatised to 4 degrees C and exposed to UV radiation. These results indicate that the SOD gene of D. antarctica is involved in the antioxidative process triggered by oxidative stress induced by the conditions of environmental change in which they live. CONCLUSION: The present results allow us to know the characteristics of Cu/ZnSOD gene from D. antarctica and understand that its expression is regulated by cold and UV radiation.

La prueba de fijación del complemento en el diagnóstico de la histoplasmosis / Fixed complement test in histoplasmosis diagnosis

Se investigaron anticuerpos fijadores del complemento frente a los antígenos de Histoplasma Capsulatum, en el suero de 72 pacientes con síndrome respiratorio agudo y crónico, con signos anormales en radiografía de tórax, baciloscopía negativa y tratamiento antituberculoso. Resultaron 6 (8 por ciento) fijadores del complemento,, 6 (8 por ciento) anticomplementarios y 60 (84 por ciento) negativos. Dos pacientes mostraron títulos ò 1:4 frentes al antígeno micelial (AgM) y negativo al antígeno levaduriforme (AgL) y 4 pacientes mostraron títulos ó 1:8, frente a los dos antígenos. Un caso fue considerado como posible histoplasmosis "altamente sugestivo" y otro como histoplasmosis diseminada con evidencial histopatológica y cultural de histoplasmosis extrapulmonar. La edad de los pacientes de sueros positivos varió de 20-49 años y todos residieron temporalmente en zonas endémicas. Se comenta la utilidad clínica de los resultados y pruebas serológicas comunes.