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1.
Cell ; 155(1): 160-71, 2013 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-24055366

RESUMEN

Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.


Asunto(s)
Respiración de la Célula , Transporte de Electrón , Membranas Mitocondriales/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/química , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , GTP Fosfohidrolasas/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/química , Mitocondrias/fisiología , Membranas Mitocondriales/química , Membranas Mitocondriales/ultraestructura , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Alineación de Secuencia
2.
FASEB J ; 38(11): e23716, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38847490

RESUMEN

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.


Asunto(s)
Movimiento Celular , Vesículas Extracelulares , Factores de Intercambio de Guanina Nucleótido , Transducción de Señal , beta Catenina , Proteínas de Unión al GTP rab5 , Humanos , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rab5/genética , beta Catenina/metabolismo , Vesículas Extracelulares/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Línea Celular Tumoral
3.
Br J Clin Pharmacol ; 2024 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-38970469

RESUMEN

AIMS: Dopamine beta-hydroxylase (DßH) inhibitors, like zamicastat, hold promise for treating pulmonary arterial hypertension. This study aimed to validate the mechanism of action of zamicastat by studying its effect on the overdrive of the sympathetic nervous system (SNS). METHODS: A single-centre, prospective, double-blind, randomized, placebo-controlled, crossover study evaluated the effect of 400 mg zamicastat in 22 healthy male subjects. Cold pressor test (CPT) was performed at screening and each treatment period on Days -1 and 10. Plasma and 24 h-urine levels of dopamine (DA), epinephrine (EPI) and norepinephrine (NE), and plasma DßH activity, were measured. RESULTS: Compared to placebo, zamicastat showed a - 4.62 mmHg decrease in systolic blood pressure during the cold stimulus vs. rest phases on Day 10 of CPT (P = .020). Zamicastat decreased mean arterial pressure response to cold stimulus during CPT (-2.62 mmHg; P = .025). At Day 10, zamicastat significantly increased plasma DA, before CPT (12.63 ng/L; P = .040) and after CPT (19.22 ng/L; P = .001) as well as the estimated plasma EPI change from baseline after CPT (P = .040). Inhibition of plasma DßH activity ranged from 19.8% to 25.0%. At Day 10, significant reductions in 24-h urinary excretion of EPI (P = .002) and NE (P = .001) were observed. Zamicastat Cτ geometric mean ± GSD ranged from 45.86 ± 1.46 ng/mL on Day 3 to 58.64 ± 1.52 ng/mL on Day 10, with moderate inter-individual variability (CV: 32.6%-36.6%). Steady state was already achieved on Day 6. CONCLUSIONS: Our results demonstrated the effect of zamicastat on the overdrive sympathetic response to cold stimulus, confirming its potential as SNS modulator.

4.
Am J Physiol Cell Physiol ; 324(5): C1028-C1038, 2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-36847442

RESUMEN

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and ß1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.


Asunto(s)
Enfermedades Inflamatorias del Intestino , Receptor Toll-Like 2 , Humanos , Adenosina Trifosfatasas/metabolismo , Células CACO-2 , Células Epiteliales/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Adaptadora de Señalización NOD2/farmacología , ARN Mensajero/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo , Receptor Toll-Like 7/metabolismo , Receptores Toll-Like/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo
5.
Biochem Biophys Res Commun ; 676: 207-212, 2023 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-37562221

RESUMEN

The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.


Asunto(s)
Histatinas , Osteogénesis , Ratones , Animales , Histatinas/farmacología , Microtomografía por Rayos X , Regeneración Ósea , Colágeno/metabolismo , Proteínas y Péptidos Salivales , Diferenciación Celular
6.
Nucleic Acids Res ; 49(9): 5230-5248, 2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-33956154

RESUMEN

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.


Asunto(s)
ADN Polimerasa gamma/genética , Proteasas ATP-Dependientes/metabolismo , Animales , Células Cultivadas , ADN Polimerasa gamma/metabolismo , Replicación del ADN , ADN Mitocondrial/análisis , Estabilidad de Enzimas/genética , Células HeLa , Holoenzimas/metabolismo , Humanos , Ratones , Proteínas Mitocondriales/metabolismo , Mutación
7.
Int J Mol Sci ; 24(2)2023 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-36674816

RESUMEN

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A>G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism.


Asunto(s)
Mitocondrias , ATPasas de Translocación de Protón Mitocondriales , Animales , Humanos , Ratones , Adenosina Trifosfato/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , ADN Mitocondrial/genética , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mutación , Fenotipo , Respiración
8.
Am J Physiol Gastrointest Liver Physiol ; 323(5): G410-G419, 2022 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-36040119

RESUMEN

Disproportionate activation of pattern recognition receptors plays a role in inflammatory bowel disease (IBD) pathophysiology. Diarrhea is a hallmark symptom of IBD, resulting at least in part from an electrolyte imbalance that may be caused by changes in potassium channel activity. We evaluated the impact of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) stimulation on potassium conductance of the basolateral membrane in human intestinal epithelial cells (IECs) and the role of potassium channels through electrophysiological assays under short-circuit current in Ussing chambers. TLRs and NOD2 were stimulated using specific agonists, and potassium channels were selectively blocked using triarylmethane-34 (TRAM-34), adenylyl-imidodiphosphate (AMP-PNP), and BaCl2. Potassium conductance of the basolateral membrane decreased upon activation of TLR2, TLR4, and TLR7 in T84 cells (means ± SE, -11.2 ± 4.5, -40.4 ± 7.2, and -19.4 ± 5.9, respectively) and in Caco-2 cells (-13.1 ± 5.7, -55.7 ± 7.4, and -29.1 ± 7.2, respectively). In contrast, activation of TLR5 and NOD2 increased basolateral potassium conductance, both in T84 cells (18.0 ± 4.1 and 18.4 ± 2.8, respectively) and in Caco-2 cells (21.2 ± 8.4 and 16.0 ± 3.6, respectively). TRAM-34 and AMP-PNP induced a decrease in basolateral potassium conductance upon TLR4 stimulation in both cell lines. Both KCa3.1- and Kir6-channels appear to be important mediators of this effect in IECs and could be potential targets for therapeutic agent development.NEW & NOTEWORTHY This study highlights that PRRs stimulation directly influences K+-channel conductance in IECs. TLR-2, -4, -7 stimulation decreased K+ conductance, whereas TLR5 and NOD2 stimulation had the opposite effect, leading to an increase of it instead. This study reports for the first time that KCa3.1- and Kir6-channels play a role in K+ transport pathways triggered by TLR4 stimulation. These findings suggest that KCa3.1- and Kir6-channels modulation may be a potential target for new therapeutic agents in IBD.


Asunto(s)
Enfermedades Inflamatorias del Intestino , Receptor Toll-Like 2 , Humanos , Receptor Toll-Like 2/metabolismo , Células CACO-2 , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 5/metabolismo , Adenilil Imidodifosfato/metabolismo , Adenilil Imidodifosfato/farmacología , Receptor Toll-Like 7/metabolismo , Células Epiteliales/metabolismo , Canales de Potasio/metabolismo , Receptores Toll-Like/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Potasio/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Electrólitos/metabolismo , Electrólitos/farmacología , Proteína Adaptadora de Señalización NOD2/metabolismo
9.
Chembiochem ; 23(20): e202200166, 2022 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-35843872

RESUMEN

BIA 10-2474 is a time-dependent inhibitor of fatty acid amide hydrolase (FAAH) that was under clinical development for the treatment of neurological conditions when the program was terminated after one subject died and four were hospitalized with neurological symptoms during a first-in-human clinical study. The present work describes the mechanism of FAAH inhibition by BIA 10-2474 as a target-specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme's active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on-site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme's catalytic nucleophile to form a covalent enzyme-inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10-2474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre-orienting and stabilizing the inhibitor in a near-reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.


Asunto(s)
Amidohidrolasas , Inhibidores Enzimáticos , Humanos , Amidohidrolasas/química , Inhibidores Enzimáticos/química , Serina/química , Imidazoles , Nitrógeno
10.
Mov Disord ; 37(11): 2272-2283, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36054562

RESUMEN

BACKGROUND: Inhibiting catechol-O-methyltransferase extends the plasma half-life of levodopa, potentially allowing physicians to optimize the levodopa regimen in patients with Parkinson's disease (PD) experiencing motor fluctuations. OBJECTIVES: To evaluate the effects of once-daily opicapone on levodopa plasma pharmacokinetics and motor response when added to two different levodopa dosing regimens. METHODS: A total of 24 patients with PD and motor fluctuations were enrolled in an exploratory, open-label, modified cross-over trial. Participants first received levodopa/carbidopa 500/125 mg (five intakes) for 2 weeks and were then randomly assigned (1:1) to levodopa/carbidopa 400/100 mg given over either four or five daily intakes plus opicapone 50 mg for an additional 2 weeks. Levodopa 12-hour pharmacokinetics was the primary outcome (ie, excluding the effect of last/evening levodopa/carbidopa intake), with motor complications evaluated as secondary outcomes. RESULTS: Over 12-hour pharmacokinetics and compared with five-intake levodopa/carbidopa 500/125 mg without opicapone, maximal levodopa concentrations were similar or nonsignificantly higher on both levodopa/carbidopa 400/100 mg regimens plus opicapone. Despite a 100 mg lower total levodopa/carbidopa daily dose, adding opicapone 50 mg at least doubled the levodopa plasma half-life and minimal concentrations, with a significant ≈30% increase in total exposure. The levodopa fluctuation index was only significantly lower for the five intakes plus opicapone regimen (difference of -71.8%; P < 0.0001). Modifications to levodopa pharmacokinetics were associated with decreased off time and increased on time. CONCLUSIONS: Combining opicapone 50 mg with a 100 mg lower daily dose of levodopa provides higher levodopa bioavailability with avoidance of trough levels. Despite the lower levodopa dose, modifying the levodopa pharmacokinetic profile with opicapone was associated with decreased off time and increased on time. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Asunto(s)
Levodopa , Enfermedad de Parkinson , Humanos , Antiparkinsonianos/efectos adversos , Carbidopa/uso terapéutico , Catecol O-Metiltransferasa , Inhibidores de Catecol O-Metiltransferasa/uso terapéutico , Levodopa/efectos adversos , Enfermedad de Parkinson/tratamiento farmacológico , Estudios Cruzados
11.
Br J Clin Pharmacol ; 88(10): 4540-4551, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35508762

RESUMEN

AIMS: The absorption, metabolism and excretion of opicapone (2,5-dichloro-3-(5-[3,4-dihydroxy-5-nitrophenyl]-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide), a selective catechol-O-methyltransferase inhibitor, were investigated. METHODS: Plasma, urine and faeces were collected from healthy male subjects following a single oral dose of 100 mg [14 C]-opicapone. The mass balance of [14 C]-opicapone and metabolic profile were evaluated. RESULTS: The recovery of total administered radioactivity averaged >90% after 144 hours. Faeces were the major route of elimination, representing 70% of the administered dose; 5% and 20% were excreted in urine and expired air, respectively. The Cmax of total radioactivity matched that of unchanged opicapone, whereas the total radioactivity remained quantifiable for a longer period, attributed to the contribution of opicapone metabolites, involving primarily 3-O-sulfate conjugation (58.6% of total circulating radioactivity) at the nitrocatechol ring. Other circulating metabolites, accounting for <10% of the radioactivity exposure, were formed by glucuronidation, methylation, N-oxide reduction and gluthatione conjugation. Additionally, various other metabolites resulting from combinations with the opicapone N-oxide reduced form at the 2,5-dichloro-4,6-dimethylpyridine 1-oxide moiety, including nitro reduction and N-acetylation, reductive opening and cleavage of the 1,2,4-oxadiazole ring and the subsequent hydrolysis products were identified, but only in faeces, suggesting the involvement of gut bacteria. CONCLUSION: [14 C]-opicapone was fully excreted through multiple metabolic pathways. The main route of excretion was in faeces, where opicapone may be further metabolized via reductive metabolism involving the 1,2,4-oxadiazole ring-opening and subsequent hydrolysis.


Asunto(s)
Inhibidores de Catecol O-Metiltransferasa , Oxadiazoles , Administración Oral , Inhibidores de Catecol O-Metiltransferasa/farmacocinética , Heces , Voluntarios Sanos , Humanos , Masculino , Oxadiazoles/farmacocinética
12.
BMC Neurol ; 22(1): 88, 2022 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-35279112

RESUMEN

BACKGROUND: Optimisation of dopaminergic therapy may alleviate fluctuation-related pain in Parkinson's disease (PD). Opicapone (OPC) is a third-generation, once-daily catechol-O-methyltransferase inhibitor shown to be generally well tolerated and efficacious in reducing OFF-time in two pivotal trials in patients with PD and end-of-dose motor fluctuations. The OpiCapone Effect on motor fluctuations and pAiN (OCEAN) trial aims to investigate the efficacy of OPC 50 mg in PD patients with end-of-dose motor fluctuations and associated pain, when administered as adjunctive therapy to existing treatment with levodopa/dopa decarboxylase inhibitor (DDCi). METHODS: OCEAN is a Phase IV, international, multicentre, randomised, double-blind, placebo-controlled, parallel-group, interventional trial in PD patients with end-of-dose motor fluctuations and associated pain. It consists of a 1-week screening period, 24-week double-blind treatment period and 2-week follow-up period. Eligible patients will be randomised 1:1 to OPC 50 mg or placebo once daily while continuing current treatment with levodopa/DDCi and other chronic, stable anti-PD and/or analgesic treatments. The primary efficacy endpoint is change from baseline in Domain 3 (fluctuation-related pain) of the King's Parkinson's disease Pain Scale (KPPS). The key secondary efficacy endpoint is change from baseline in Domain B (anxiety) of the Movement Disorder Society-sponsored Non-Motor rating Scale (MDS-NMS). Additional secondary efficacy assessments include other domains and total scores of the KPPS and MDS-NMS, the Parkinson's Disease Questionnaire (PDQ-8), the MDS-sponsored Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Parts III and IV, Clinical and Patient's Global Impressions of Change, and change in functional status via Hauser's diary. Safety assessments include the incidence of treatment-emergent adverse events. The study will be conducted in approximately 140 patients from 50 clinical sites in Germany, Italy, Portugal, Spain and the United Kingdom. Recruitment started in February 2021 and the last patient is expected to complete the study by late 2022. DISCUSSION: The OCEAN trial will help determine whether the use of adjunctive OPC 50 mg treatment can improve fluctuation-associated pain in PD patients with end-of-dose motor fluctuations. The robust design of OCEAN will address the current lack of reliable evidence for dopaminergic-based therapy in the treatment of PD-associated pain. TRIAL REGISTRATION: EudraCT number 2020-001175-32 ; registered on 2020-08-07.


Asunto(s)
Enfermedad de Parkinson , Antiparkinsonianos , Catecol O-Metiltransferasa/uso terapéutico , Humanos , Oxadiazoles , Dolor/tratamiento farmacológico , Dolor/etiología , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/tratamiento farmacológico
13.
Nature ; 535(7613): 561-5, 2016 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-27383793

RESUMEN

Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.


Asunto(s)
Envejecimiento/genética , Núcleo Celular/genética , ADN Mitocondrial/genética , Variación Genética/genética , Metabolismo/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Envejecimiento/fisiología , Animales , Femenino , Genoma Mitocondrial/genética , Haplotipos , Insulina/metabolismo , Longevidad/genética , Masculino , Metabolismo/fisiología , Metabolómica , Ratones , Ratones Congénicos , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Obesidad/genética , Obesidad/metabolismo , Fenotipo , Proteómica , Especies Reactivas de Oxígeno/metabolismo , Acortamiento del Telómero , Transcriptoma , Respuesta de Proteína Desplegada
14.
Eur Neurol ; 85(5): 389-397, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35350024

RESUMEN

INTRODUCTION: The OPTIPARK study confirmed the effectiveness and safety of opicapone as adjunct therapy to levodopa in patients with Parkinson's disease (PD) and motor fluctuations under real-world conditions. The aim of this sub-analysis was to evaluate opicapone in the German patient cohort of OPTIPARK in order to provide country-specific data. METHODS: OPTIPARK was an open-label, single-arm study conducted in routine clinical practice across Germany and the UK. Patients with PD and motor fluctuations received once-daily opicapone 50 mg for 3 months in addition to levodopa. The primary endpoint was Clinicians' Global Impression of Change (CGI-C). Secondary assessments included Patients' Global Impressions of Change (PGI-C), Unified Parkinson's Disease Rating Scale (UPDRS) I-IV, Parkinson's Disease Questionnaire (PDQ-8), and Non-Motor Symptoms Scale (NMSS). This sub-analysis reports outcomes from the German patients only. RESULTS: Overall, 363 (97.6%) of the 372 patients included in the German cohort received ≥1 dose of opicapone and 291 (80.2%) completed the study. Improvements on CGI-C and PGI-C were reported by 70.8% and 76.3% of patients, respectively. UPDRS scores improved for activities of daily living during OFF time by -3.3 ± 4.5 points and motor scores during ON time by -5.3 ± 7.9 points. PDQ-8 and NMSS scores also demonstrated improvements. Treatment emergent adverse events considered at least possibly related to opicapone occurred in 37.7% of patients, with most being of mild or moderate intensity. CONCLUSION: Opicapone added to levodopa in patients with PD and motor fluctuations was effective and generally well tolerated in routine clinical practice across Germany.


Asunto(s)
Levodopa , Enfermedad de Parkinson , Actividades Cotidianas , Antiparkinsonianos , Método Doble Ciego , Quimioterapia Combinada , Alemania , Humanos , Levodopa/uso terapéutico , Oxadiazoles , Enfermedad de Parkinson/tratamiento farmacológico
15.
J Bioenerg Biomembr ; 53(4): 489-498, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34159523

RESUMEN

Undue exposure to antimicrobials has led to the acquisition and development of sophisticated bacterial resistance mechanisms, such as efflux pumps, which are able to expel or reduce the intracellular concentration of various antibiotics, making them ineffective. Therefore, inhibiting this mechanism is a promising way to minimize the phenomenon of resistance in bacteria. In this sense, the present study sought to evaluate the activity of the Carvacrol (CAR) and Thymol (THY) terpenes as possible Efflux Pump Inhibitors (EPIs), by determining the Minimum Inhibitory Concentration (MIC) and the association of these compounds in subinhibitory concentrations with the antibiotic Norfloxacin and with Ethidium Bromide (EtBr) against strains SA-1199 (wild-type) and SA-1199B (overexpresses NorA) of Staphylococcus aureus. In order to verify the interaction of the terpenes with the NorA efflux protein, an in silico molecular modeling study was carried out. The assays used to obtain the MIC of CAR and THY were performed by broth microdilution, while the Efflux Pump inhibitory test was performed by the MIC modification method of the antibiotic Norfloxacin and EtBr. docking was performed using the Molegro Virtual Docker (MVD) program. The results of the study revealed that CAR and THY have moderate bacterial activity and are capable of reducing the MIC of Norfloxacin antibiotic and EtBr in strains of S. aureus carrying the NorA efflux pump. The docking results showed that these terpenes act as possible competitive NorA inhibitors and can be investigated as adjuvants in combined therapies aimed at reducing antibiotic resistance.


Asunto(s)
Cimenos/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Norfloxacino/uso terapéutico , Staphylococcus aureus/efectos de los fármacos , Timol/uso terapéutico , Cimenos/farmacología , Norfloxacino/farmacología , Timol/farmacología
16.
FASEB J ; 34(3): 4009-4025, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31990106

RESUMEN

Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of ß-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of ß-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the ß-catenin destruction complex allows nuclear accumulation of ß-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of ß-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3ß, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3ß, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the ß-catenin destruction complex leads to stabilization and nuclear accumulation of ß-catenin in oral dysplasia.


Asunto(s)
Apraxias/metabolismo , Núcleo Celular/metabolismo , beta Catenina/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7
17.
Crit Rev Toxicol ; 51(1): 65-75, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33528291

RESUMEN

In 2016, one subject died and four were hospitalized with neurological symptoms during a clinical trial with the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474. The present paper reviews the regulatory toxicology studies that were carried out to support the clinical trial application for BIA 10-2474. Animal studies complied with national and international standards including European regulatory guidelines (e.g. EEC Council Directive 75/318/EEC and subsequent amendments). The CNS effects seen in the rat and mouse appear to be common in rodents in such studies and do not in principle seem to be of the type to generate a signal. In the same way in non-human primates, insignificant alterations in the mesencephalon, and especially of the autonomic nervous system (Meissner's plexus in the bowel) in rodents and monkeys were observed in some animals treated with a high dose. Overall, these data, as well as the extensive additional data generated since the accident, support the conclusion that the tragic fatality that occurred during the clinical trial with BIA 10-2474 was unpredictable and that the mechanism responsible remains unknown, from a non-clinical toxicological perspective.


Asunto(s)
Óxidos N-Cíclicos/toxicidad , Inhibidores Enzimáticos/toxicidad , Piridinas/toxicidad , Administración Oral , Amidohidrolasas/antagonistas & inhibidores , Animales , Humanos , Ratones , Ratas
18.
Epilepsia ; 62(2): 542-556, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33452820

RESUMEN

OBJECTIVE: Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. METHODS: In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. RESULTS: We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 µM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. SIGNIFICANCE: These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.


Asunto(s)
Anticonvulsivantes/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Dibenzazepinas/farmacología , Epilepsia/fisiopatología , Interneuronas/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Adamantano/análogos & derivados , Adamantano/farmacología , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiopatología , Calcio/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epilepsia/inducido químicamente , Retroalimentación Fisiológica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Interneuronas/metabolismo , Potenciación a Largo Plazo , Agonistas Muscarínicos/toxicidad , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pilocarpina/toxicidad , Ratas , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo
19.
Biol Res ; 54(1): 3, 2021 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-33546773

RESUMEN

BACKGROUND: Testosterone regulates nutrient and energy balance to maintain protein synthesis and metabolism in cardiomyocytes, but supraphysiological concentrations induce cardiac hypertrophy. Previously, we determined that testosterone increased glucose uptake-via AMP-activated protein kinase (AMPK)-after acute treatment in cardiomyocytes. However, whether elevated glucose uptake is involved in long-term changes of glucose metabolism or is required during cardiomyocyte growth remained unknown. In this study, we hypothesized that glucose uptake and glycolysis increase in testosterone-treated cardiomyocytes through AMPK and androgen receptor (AR). METHODS: Cultured cardiomyocytes were stimulated with 100 nM testosterone for 24 h, and hypertrophy was verified by increased cell size and mRNA levels of ß-myosin heavy chain (ß-mhc). Glucose uptake was assessed by 2-NBDG. Glycolysis and glycolytic capacity were determined by measuring extracellular acidification rate (ECAR). RESULTS: Testosterone induced cardiomyocyte hypertrophy that was accompanied by increased glucose uptake, glycolysis enhancement and upregulated mRNA expression of hexokinase 2. In addition, testosterone increased AMPK phosphorylation (Thr172), while inhibition of both AMPK and AR blocked glycolysis and cardiomyocyte hypertrophy induced by testosterone. Moreover, testosterone supplementation in adult male rats by 5 weeks induced cardiac hypertrophy and upregulated ß-mhc, Hk2 and Pfk2 mRNA levels. CONCLUSION: These results indicate that testosterone stimulates glucose metabolism by activation of AMPK and AR signaling which are critical to induce cardiomyocyte hypertrophy.


Asunto(s)
Proteínas Quinasas Activadas por AMP , Glucosa/metabolismo , Miocitos Cardíacos , Receptores Androgénicos/metabolismo , Testosterona/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Células Cultivadas , Hipertrofia , Masculino , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Transducción de Señal
20.
Clin Exp Hypertens ; 43(5): 428-435, 2021 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-33688765

RESUMEN

Salt-inducible kinases (SIKs) represent a subfamily of AMPK family kinases. SIK1 has been shown to act as a mediator during the cellular adaptation to variations in intracellular sodium in a variety of cell types. SIK2, as an isoform of the SIK family, modulates various biological functions and acts as a signal transmitter in various pathways. To evaluate the role of both SIK1 and SIK2 isoforms in blood pressure (BP), body fluid regulation and cardiac hypertrophy development, we made use of constitutive sik1-/- (SIK1-KO), sik2-/- (SIK2-KO), double sik1-/-sik2-/- (double SIK1*2-KO) knockout and wild-type (WT) mice challenged to a standard (0.3% NaCl) or chronic high-salt (HS, 8% NaCl) diet intake for 12 weeks.Mice, under a standard diet intake, had similar and normal BP. On a chronic HS intake, SIK1-KO and double SIK1*2-KO mice showed increased BP, but not WT and SIK2-KO mice. A chronic HS intake led to the development of cardiac left ventricle hypertrophy (LVH) in normotensive WT and hypertensive SIK1-KO mice, but not in SIK2-KO mice. Double SIK1*2-KO mice under standard diet intake show normal BP but an increased LV mass. Remarkably, in response to a dietary stress condition, there is an increase in BP but LVH remained unchanged in double SIK1*2-KO mice.In summary, SIK1 isoform is required for maintaining normal BP in response to HS intake. LVH triggered by HS intake requires SIK2 isoform and is independent of high BP.


Asunto(s)
Cardiomegalia/fisiopatología , Hipertensión/fisiopatología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Glucemia/metabolismo , Presión Sanguínea , Peso Corporal , Cardiomegalia/sangre , Hipertensión/sangre , Pruebas de Función Renal , Lípidos/sangre , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Isoformas de Proteínas/metabolismo , Cloruro de Sodio Dietético
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