RESUMEN

Vascular graft surgeries are often conducted in trauma cases, which has increased the demand for scaffolds with good biocompatibility profiles. Biodegradable scaffolds resembling the extracellular matrix (ECM) of blood vessels are promising vascular graft materials. In the present study, polyurethane (PU) was blended with ECM proteins collagen and elastin (Col-El) and gelatin (Gel) to produce fibrous scaffolds by using the rotary jet spinning (RJS) technique, and their effects on in vitro properties were evaluated. Morphological and structural characterization of the scaffolds was performed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Micrometric fibers with nanometric rugosity were obtained. Col-El and Gel reduced the mechanical strength and increased the hydrophilicity and degradation rates of PU. No platelet adhesion or activation was observed. The addition of proteins to the PU blend increased the viability, adhesion, and proliferation of human umbilical vein endothelial cells (HUVECs). Therefore, PU-Col-El and PU-Gel scaffolds are promising biomaterials for vascular graft applications.

Asunto(s)

Bioprótesis , Poliuretanos , Prótesis Vascular , Matriz Extracelular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Poliuretanos/química , Poliuretanos/farmacología

RESUMEN

The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the amino acid residue hypusine, essential for its activity. Hypusine residue is produced by a posttranslational modification involving deoxyhypusine synthetase and deoxyhypusine hydroxylase. Herein, we aimed to describe the role of the alternative human isoform A on mitochondrial processes. Isoform A depletion modulates oxidative metabolism in association with the downregulation of mitochondrial biogenesis-related genes. Through positive feedback, it increases cell respiration leading to highly reactive oxygen species production, which impacts mitochondrial bioenergetics. These metabolic changes compromise mitochondrial morphology, increasing its electron density and fission, observed by transmission electron microscopy. This set of changes leads the cells to apoptosis, evidenced by increased DNA fragmentation and proapoptotic BAK protein content increase. Thus, we show that the alternative eIF5A isoform A is crucial for energy metabolism controlled by mitochondria and cellular survival.

Asunto(s)

Mitocondrias/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Apoptosis/fisiología , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Microscopía Electrónica de Transmisión , Factores de Iniciación de Péptidos/genética , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/genética , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Tubular polymer scaffolds based on tissue engineering techniques have been studied as potential alternatives for vascular regeneration implants. The blood vessels of the cardiovascular system are mainly fibrous, composed of collagen (Col) and elastin (El), and its inner layer consists of endothelial cells. In this work, Col and El were combined with polyurethane (PU), a biocompatible synthetic polymer, and rotary jet spinning, a new and highly productive technique, to produce fibrous scaffolds. The scaffolds produced at 18 000 rpm presented homogeneous, bead-free, and solvent-free fibers. The blend formation between PU-Col-El was identified by chemical composition analysis and enhanced the thermal stability up to 324°C. The hydrophilic nature of the scaffold was revealed by its low contact angle. Cell viability of human umbilical vein endothelial cells with the scaffold was proven for 72 hours. The combined strategy of rotary jet spinning with a polymer blend containing Col and El was verified as an effective and promising alternative to obtain tubular scaffolds for tissue engineering on a large-scale production.

Asunto(s)

Materiales Biocompatibles/química , Prótesis Vascular , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Colágeno/química , Elastina/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Poliuretanos/química

RESUMEN

The high capacity of the skeletal muscle to regenerate is due to the presence of muscle stem cells (MuSCs, or satellite cells). The E3 ubiquitin ligase Parkin is a key regulator of mitophagy and is recruited to mitochondria during differentiation of mouse myoblast cell line. However, the function of mitophagy during regeneration has not been investigated in vivo. Here, we have utilized Parkin deficient (Parkin-/-) mice to investigate the role of Parkin in skeletal muscle regeneration. We found a persistent deficiency in skeletal muscle regeneration in Parkin-/- mice after cardiotoxin (CTX) injury with increased area of fibrosis and decreased cross-sectional area (CSA) of myofibres post-injury. There was also a significant modulation of MuSCs differentiation and mitophagic markers, with altered mitochondrial proteins during skeletal muscle regeneration in Parkin-/- mice. Our data suggest that Parkin-mediated mitophagy plays a key role in skeletal muscle regeneration and is necessary for MuSCs differentiation.

Asunto(s)

Diferenciación Celular , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Desarrollo de Músculos , Músculo Esquelético/patología , Regeneración , Ubiquitina-Proteína Ligasas/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitofagia , Músculo Esquelético/metabolismo , Células Madre/citología

RESUMEN

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Asunto(s)

Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Péptidos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Silenciador del Gen , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de Masas , Factores de Iniciación de Péptidos/genética , Fosforilación , Reacción en Cadena de la Polimerasa , Unión Proteica , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2?Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

RESUMEN

Sestrins and autophagy deficiencies are associated with several aging-related organic dysfunctions and metabolic disorders. Here we evaluate the effects of acute exercise on Sestrin 2 (Sesn2) protein content and autophagy markers in the skeletal muscle of experimental models of aging. Twenty-four months-old C57BL/6J male mice were submitted to a single bout of swimming exercise and the gastrocnemius muscle was evaluated by Western blot. Transcriptomic and phenotypic analysis were also performed by using strains of genetically-diverse BXD mice. The bioinformatics analysis showed a negative correlation between Sesn2 mRNA levels in the skeletal muscle and body weight gain, plasma triglycerides and fasting glucose and positive correlation with several autophagic markers in the muscle of BXD mice. Consistent with these findings, low levels of Sesn2 protein content were observed in the gastrocnemius muscle of C57BL/6J old mice when compared to young group. Interestingly, the acute aerobic exercise induced Sesn2 accumulation and modulated several markers of autophagy in the gastrocnemius muscle old mice, including unc-51-like kinase-1 (Ulk1) phosphorylation and the protein levels of Atg5, Atg7, p62 and LC3-II. Finally, exercise increased insulin sensitivity in old animals, as demonstrated by kITT. Taken together, these findings demonstrated the acutely, aerobic physical exercise recovers Sestrin 2 protein content and induces autophagy in the skeletal muscle of old mice, contributing with the improvement of insulin sensitivity an aging animal model.

Asunto(s)

Envejecimiento , Autofagia , Músculo Esquelético/fisiología , Proteínas Nucleares/metabolismo , Natación/fisiología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Biomarcadores , Regulación de la Expresión Génica , Glucosa/metabolismo , Insulina/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Peroxidasas , Fosforilación , Condicionamiento Físico Animal , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Clopidogrel is an essential antiplatelet drug used to prevent thrombosis complications associated with atherosclerosis. However, hepatotoxicity is a potential adverse effect related to clopidogrel therapy. Exosome-derived miRNAs may be useful for improved monitoring of drug response and hepatotoxicity risk. In the present study, the expression of several exosomal miRNAs (miR-26a-5p, miR-145-5p, miR-15b-5p, and miR-4701-3p) and cell-derived mRNA targets (PLOD2, SENP5, EIF4G2, HMGA2, STRADB, and TLK1) were evaluated in HepG2 cells treated with clopidogrel (6.25, 12.5, 25, 50, and 100 µM) for 24 and 48 h. Then, clopidogrel cytotoxicity was evaluated by analyzing DNA fragmentation and the cell cycle profile using flow cytometry. Differential expression of exosome-derived miRNAs and cell-derived mRNAs was analyzed by RT-qPCR. Exposure of HepG2 cells to high concentrations of clopidogrel (50 and 100 µM) for 24 h caused significant DNA fragmentation (17.6 and 44.4%, respectively; p < 0.05) and 48 h (26.8 and 48.9%, respectively; p < 0.05), indicating cellular toxicity. Upregulation of miR-26a-5p and downregulation of miR-15b-5p was observed in cells exposed to 100 µM clopidogrel for 24 and 48 h. The miR-26a-5p target mRNAs HMGA2, EIF4G2, STRADB, and SENP5 were downregulated in HepG2 cells following exposure to cytotoxic concentrations of clopidogrel (50 and 100 µM) for 24 h, and HMGA2 levels remained low after 48 h of treatment. TLK1, a target of miR-15b-5p, was downregulated by 50 and 100 µM clopidogrel at 24 h. In conclusion, our results suggest that exposure to high concentrations of clopidogrel modulates the expression of exosomal miR-26a-5p and miR-15b-5p and their target mRNAs in HepG2 cells. Dysregulation of these miRNAs maybe modulate the regulatory pathways involved in clopidogrel-induced liver injury.

RESUMEN

Clopidogrel is an essential antiplatelet drug used to prevent thrombosis complications associated with atherosclerosis. However, hepatotoxicity is a potential adverse effect related to clopidogrel therapy. Exosome-derived miRNAs may be useful for improved monitoring of drug response and hepatotoxicity risk. In the present study, the expression of several exosomal miRNAs (miR-26a-5p, miR-145-5p, miR-15b-5p, and miR-4701-3p) and cell-derived mRNA targets (PLOD2, SENP5, EIF4G2, HMGA2, STRADB, and TLK1) were evaluated in HepG2 cells treated with clopidogrel (6.25, 12.5, 25, 50, and 100 μM) for 24 and 48 h. Then, clopidogrel cytotoxicity was evaluated by analyzing DNA fragmentation and the cell cycle profile using flow cytometry. Differential expression of exosome-derived miRNAs and cell-derived mRNAs was analyzed by RT-qPCR. Exposure of HepG2 cells to high concentrations of clopidogrel (50 and 100 μM) for 24 h caused significant DNA fragmentation (17.6 and 44.4%, respectively; p < 0.05) and 48 h (26.8 and 48.9%, respectively; p < 0.05), indicating cellular toxicity...

Asunto(s)

Línea Celular , MicroARNs , Trombosis de las Arterias Carótidas

RESUMEN

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70-S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG-tagged p70-S6K1 and p54-S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high-scoring interactions. By a comparative approach, p70-S6K1 interacting proteins were predominantly related to "cytoskeleton" and "stress response," whereas p54-S6K2 interactome was more associated to "transcription," "splicing," and "ribosome biogenesis." Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.

Asunto(s)

Mapas de Interacción de Proteínas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Nucleofosmina , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Proteómica , Proteínas Quinasas S6 Ribosómicas 70-kDa/análisis , Transducción de Señal

RESUMEN

The S6K proteins are mTOR pathway effectors and accumulative evidence suggest that mTOR/S6K signaling contributes to several pathological conditions, such as diabetes, cancer and obesity. The activation of the mTOR/S6K axis stimulates protein synthesis and cell growth. S6K1 has two well-known isoforms, p70-S6K1 and p85-S6K1, generated by alternative translation initiation sites. A third isoform, named p31-S6K1, has been characterized as a truncated type of the protein due to alternative splicing, and reports have shown its important role in cancer. Studies involving S6K2 are scarce. This article aims to review what is new in the literature about these kinases and establish differences regarding their interacting proteins, activation and function, connecting their roles in the homeostasis of the cell and in pathological conditions.

Asunto(s)

Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Diabetes Mellitus/fisiopatología , Humanos , Neoplasias/patología , Obesidad/fisiopatología

RESUMEN

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.

Asunto(s)

Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Línea Celular , Ratones , Fibras Musculares Esqueléticas/citología , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Factores de Iniciación de Péptidos/antagonistas & inhibidores , Factores de Iniciación de Péptidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with N1-guanyl-1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

Asunto(s)

Diferenciación Celular , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Arginina/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatina/metabolismo , Conectina , Regulación de la Expresión Génica/efectos de los fármacos , Guanina/análogos & derivados , Guanina/farmacología , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Proteína MioD/metabolismo , Oxidación-Reducción/efectos de los fármacos , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Satélite del Músculo Esquelético/metabolismo , Células Madre/efectos de los fármacos , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Long-term memory, a persistent form of synaptic plasticity, requires translation of a subset of mRNA present in neuronal dendrites during a short and critical period through a mechanism not yet fully elucidated. Western blotting analysis revealed a high content of eukaryotic translation initiation factor 5A (eIF5A) in the brain of neonatal rats, a period of intense neurogenesis rate, differentiation and synaptic establishment, when compared to adult rats. Immunohistochemistry analysis revealed that eIF5A is present in the whole brain of adult rats showing a variable content among the cells from different areas (e.g. cortex, hippocampus and cerebellum). A high content of eIF5A in the soma and dendrites of Purkinje cells, key neurons in the control of motor long-term memory in the cerebellum, was observed. Detection of high eIF5A content was revealed in dendritic varicosities of Purkinje cells. Evidence is presented herein that a reduction of eIF5A content is associated to brain aging.

Asunto(s)

Envejecimiento , Encéfalo/metabolismo , Dendritas/metabolismo , Neuronas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Encéfalo/citología , Cerebelo/citología , Cerebelo/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Neuronas/citología , Factores de Iniciación de Péptidos/análisis , Células de Purkinje/citología , Células de Purkinje/metabolismo , Proteínas de Unión al ARN/análisis , Ratas , Ratas Wistar , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB(1) cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB(1) cannabinoid receptors. We find that hemopressin is a CB(1) receptor-selective antagonist, because it is able to efficiently block signaling by CB(1) receptors but not by other members of family A G protein-coupled receptors (including the closely related CB(2) receptors). Hemopressin also behaves as an inverse agonist of CB(1) receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressin's therapeutic potential. These results represent a demonstration of a peptide ligand for CB(1) cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB(1) receptors.

Asunto(s)

Agonismo Inverso de Drogas , Hemoglobinas/farmacología , Fragmentos de Péptidos/farmacología , Receptor Cannabinoide CB1/agonistas , Línea Celular , Humanos , Ligandos

RESUMEN

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P < 0.05). Lipovenus infusion failed to exert any effect on muscle force performance (P > 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P< 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.

Asunto(s)

Lípidos/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/genética , Estimulación Eléctrica , Ácidos Grasos no Esterificados/sangre , Glucosa-6-Fosfato/metabolismo , Infusiones Intravenosas , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ácido Láctico/sangre , Lípidos/administración & dosificación , Masculino , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxígeno/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteína Desacopladora 2 , Proteína Desacopladora 3

RESUMEN

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 murine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice.

Asunto(s)

Guanina/análogos & derivados , Melanoma/metabolismo , Melanoma/patología , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN , Femenino , Guanina/química , Guanina/farmacología , Melanoma/genética , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB1 cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB1 cannabinoid receptors. We find that hemopressin is a CB1 receptor-selective antagonist, because it is able to efficiently block signaling by CB1 receptors but not by other members of family A G protein-coupled receptors (including the closely related CB2 receptors). Hemopressin also behaves as an inverse agonist of CB1 receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressin's therapeutic potential. These results represent a demonstration of a peptide ligand for CB1 cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB1 receptors.

Asunto(s)

Animales , Ratas , Endocannabinoides , Inflamación/clasificación