RESUMO
Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.
Assuntos
Escherichia coli/metabolismo , Transdução de Sinais , Aerobiose , Anaerobiose , Sequência de Bases , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilaminas/metabolismo , Metilaminas/farmacologia , Oxigênio/metabolismo , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Trimethylamine N-oxide (TMAO) is a novel risk factor for atherosclerosis, and its underlying regulatory mechanisms are under intensive investigation. Inflammation-related vascular endothelial damage is the major driver in atherogenic process. Pyroptosis, a type of proinflammatory programmed cell death, has been proved to promote the initiation and progression of atherosclerosis. In our study, we found that TMAO triggered endothelial cells excessive mitophagy, thereby facilitating pyroptosis. This process is mediated by the upexpression of phosphatidylethanolamine acyltransferase (LPEAT). These findings provide insights into TMAO-induced vascular endothelial cell damage and suggest that LPEAT may be a valuable target for the prevention and treatment of atherosclerosis.
Assuntos
Aterosclerose , Células Endoteliais , Humanos , Células Endoteliais/metabolismo , Piroptose , Mitofagia , Metilaminas/farmacologia , Metilaminas/metabolismo , Aterosclerose/metabolismoRESUMO
The role of G-quadruplex (G4) in cellular processes can be investigated by the covalent modification of G4-DNA using alkylating reagents. Controllable alkylating reagents activated by external stimuli can react elegantly and selectively. Herein, we report a chemical activation system that can significantly boost the reaction rate of methylamine-protected vinyl-quinazolinone (VQ) derivative for the alkylation of G4-DNA. The two screened activators can transform low-reactive VQ-NHR' to highly reactive intermediates following the Michael addition mechanism. This approach expands the toolbox of activable G4 alkylating reagents.
Assuntos
Quadruplex G , Metilaminas , Quinazolinonas , Alquilação , Quadruplex G/efeitos dos fármacos , Metilaminas/química , Metilaminas/farmacologia , Metilaminas/síntese química , Quinazolinonas/química , Quinazolinonas/farmacologia , Quinazolinonas/síntese química , Humanos , Estrutura Molecular , DNA/química , Compostos de Vinila/química , Compostos de Vinila/farmacologiaRESUMO
Previous studies have revealed the microbial metabolism of dietary choline in the gut, leading to its conversion into trimethylamine (TMA). Polymethoxyflavones (PMFs), exemplified by tangeretin, have shown efficacy in mitigating choline-induced cardiovascular inflammation. However, the specific mechanism by which these compounds exert their effects, particularly in modulating the gut microbiota, remains uncertain. This investigation focused on tangeretin, a representative PMFs, to explore its influence on the gut microbiota and the choline-TMA conversion process. Experimental results showed that tangeretin treatment significantly attenuated the population of CutC-active bacteria, particularly Clostridiaceae and Lactobacillus, induced by choline chloride in rat models. This inhibition led to a decreased efficiency in choline conversion to TMA, thereby ameliorating cardiovascular inflammation resulting from prolonged choline consumption. In conclusion, tangeretin's preventive effect against cardiovascular inflammation is intricately linked to its targeted modulation of TMA-producing bacterial activity.
Assuntos
Arterite , Flavonas , Microbioma Gastrointestinal , Ratos , Animais , Colina/metabolismo , Metilaminas/farmacologia , Metilaminas/metabolismo , Bactérias/metabolismo , Inflamação/tratamento farmacológicoRESUMO
Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide but still lacks specific treatment modalities. The gut microbiota and its metabolites have been shown to be intimately involved in NAFLD development, participating in and regulating disease progression. Trimethylamine N-oxide (TMAO), a metabolite highly dependent on the gut microbiota, has been shown to play deleterious regulatory roles in cardiovascular disease, but the relationship between it and NAFLD lacks validation from basic experiments. This research applied TMAO intervention by constructing fatty liver cell models in vitro to observe its effect on fatty liver cells and potential key genes and performed siRNA interference on the gene to verify the action. The results showed that TMAO intervention promoted the appearance of more red-stained lipid droplets in Oil-red O staining results, increased triglyceride (TG) levels and increased mRNA levels of liver fibrosis-related genes, and also identified one of the key genes, keratin17 (KRT17) via transcriptomics. Following the reduction in its expression level, under the same treatment, there were decreased red-stained lipid droplets, decreased TG levels, decreased indicators of impaired liver function as well as decreased mRNA levels of liver fibrosis-related genes. In conclusion, the gut microbiota metabolite TMAO could promote lipid deposition and fibrosis process via the KRT17 gene in fatty liver cells in vitro.
Assuntos
Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Humanos , Fibrose , Metilaminas/farmacologia , Metilaminas/metabolismo , Cirrose Hepática , LipídeosRESUMO
BACKGROUND: The aim of this study is to investigate the effect of trimethylamine (TMA) and trimethylamine-n-oxide (TMAO) on the contractility of human umbilical artery and the possible mechanisms involved. METHODS: Vasoactive responses to TMA and TMAO on human umbilical artery rings were measured in isolated organ baths. Cumulative dose-response curves for TMA and TMAO were obtained before and after incubation with atropine, yohimbine, prazosin, indomethacin, verapamil, and Ca+2 -free Krebs-Henselite solution. RESULTS: Administration of cumulative TMA and TMAO resulted in dose-dependent contraction at concentrations ranging from 10 to 100 mM on human umbilical artery rings. TMA-induced contractions were more potent than TMAO-induced contractions (TMA: -logEC50 = 1.00 ± 0.02, TMAO: -logEC50 = 0.57 ± 0.02). Contraction responses to TMA were significantly lower in the presence of verapamil and in the absence of external Ca+2 (p < 0.001, p < 0.05, respectively). CONCLUSION: Our results showed that TMA and TMAO caused vasoconstriction in isolated human umbilical artery rings. Our findings also indicated that TMA but not TMAO-induced vasoconstriction was partially dependent on extracellular Ca2+ and calcium influx through L-type Ca2+ channels. Our results suggest that TMA and TMAO may have the potential to contribute to cardiovascular diseases through their direct effect on vascular contractility in human arteries.
Assuntos
Metilaminas , Artérias Umbilicais , Humanos , Metilaminas/administração & dosagem , Metilaminas/farmacologia , Óxidos , Artérias Umbilicais/efeitos dos fármacosRESUMO
In this study, the plausible role of trimethylamine N-oxide (TMAO), a microbiota metabolite, was investigated as a link between peripheral inflammation and the inflammation of the central nervous system using different cell lines. TMAO treatment favored the differentiation of adipocytes from preadipocytes (3T3-L1 cell line). In macrophages (RAW 264.7 cell line), which infiltrate adipose tissue in obesity, TMAO increased the expression of pro-inflammatory cytokines. The treatment with 200 µM of TMAO seemed to disrupt the blood-brain barrier as it induced a significant decrease in the expression of occludin in hCMECs. TMAO also increased the expression of pro-inflammatory cytokines in primary neuronal cultures, induced a pro-inflammatory state in primary microglial cultures, and promoted phagocytosis. Data obtained from this project suggest that microbial dysbiosis and increased TMAO secretion could be a key link between peripheral and central inflammation. Thus, TMAO-decreasing compounds may be a promising therapeutic strategy for neurodegenerative diseases.
Assuntos
Inflamação , Metilaminas , Humanos , Inflamação/metabolismo , Metilaminas/farmacologia , Metilaminas/metabolismo , Citocinas , Projetos de PesquisaRESUMO
BACKGROUND: The present study aimed to develop a novel protein-repellent and antibacterial polymethyl methacrylate (PMMA) dental resin with 2-methacryloyloxyethyl phosphorylcholine (MPC) and quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM), and to investigate the effects of water-aging for 6 months on the mechanical properties, protein adsorption, and antibacterial activity of the dental resin. METHODS: Four groups were tested: PMMA control; PMMA + 3% MPC; PMMA + 1.5% DMAHDM; and PMMA + 3% MPC + 1.5% DMADDM in acrylic resin powder. Specimens were water-aged for 1 d, 3 months, and 6 months at 37 â. Their mechanical properties were then measured using a three-point flexure test. Protein adsorption was measured using a micro bicinchoninic acid (BCA) method. A human saliva microcosm model was used to inoculate bacteria on water-aged specimens and to investigate the live/dead staining, metabolic activity of biofilms, and colony-forming units (CFUs). RESULTS: The flexural strength and elastic modulus showed a significant loss after 6 months of water-ageing for the PMMA control (mean ± SD; n = 10); in contrast, the new protein repellent and antibacterial PMMA resin showed no strength loss. The PMMA-MPC-DMAHDM-containing resin imparted a strong antibacterial effect by greatly reducing biofilm viability and metabolic activity. The biofilm CFU count was reduced by about two orders of magnitude (p < 0.05) compared with that of the PMMA resin control. The protein adsorption was 20% that of a commercial composite (p < 0.05). Furthermore, the PMMA-MPC-DMAHDM-containing resin exhibited a long-term antibacterial performance, with no significant difference between 1 d, 3 months and 6 months (p > 0.05). CONCLUSIONS: The flexural strength and elastic modulus of the PMMA-MPC-DMAHDM-containing resin were superior to those of the PMMA control after 6 months of water-ageing. The novel PMMA resin incorporating MPC and DMAHDM exhibited potent and lasting protein-repellent and antibacterial properties.
Assuntos
Polimetil Metacrilato , Água , Humanos , Antibacterianos/farmacologia , Biofilmes , Metacrilatos/farmacologia , Metilaminas/farmacologia , Polimetil Metacrilato/farmacologia , Proteínas , Água/farmacologia , Fatores de TempoRESUMO
BACKGROUND: Though drugs binding to serotonergic 5-HT2A receptors have long been claimed to influence human anxiety, it remains unclear if this receptor subtype is best described as anxiety promoting or anxiety dampening. Whereas conditioned fear expressed as freezing in rats is modified by application of 5-HT2A-acting drugs locally into different brain regions, reports on the effect of systemic administration of 5-HT2A receptor agonists and 5-HT2A antagonists or inverse agonists on this behavior remain sparse. METHODS: We assessed the possible impact of systemic administration of 5-HT2A receptor agonists, 5-HT2A receptor inverse agonists, and a selective serotonin reuptake inhibitor (SSRI)-per se or in combination-on the freezing displayed by male rats when re-exposed to a conditioning chamber in which they received foot shocks 7 days earlier. RESULTS: The 5-HT2A receptor agonists psilocybin and 25CN-NBOH induced a reduction in conditioned fear that was countered by pretreatment with 5-HT2A receptor inverse agonist MDL 100907. While both MDL 100907 and another 5-HT2A receptor inverse agonist, pimavanserin, failed to impact freezing per se, both compounds unmasked a robust fear-reducing effect of an SSRI, escitalopram, which by itself exerted no such effect. CONCLUSIONS: The results indicate that 5-HT2A receptor activation is not a prerequisite for normal conditioned freezing in rats but that this receptor subtype, when selectively over-activated prior to expression, exerts a marked fear-reducing influence. However, in the presence of an SSRI, the 5-HT2A receptor, on the contrary, appears to counter an anti-freezing effect of the enhanced extracellular serotonin levels following reuptake inhibition.
Assuntos
Comportamento Animal/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Medo/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Animais , Compostos Bicíclicos com Pontes/farmacologia , Fluorbenzenos/farmacologia , Ligantes , Masculino , Metilaminas/farmacologia , Piperidinas/farmacologia , Psilocibina/farmacologia , Ratos , Ratos Sprague-Dawley , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Trimethylamine (TMA), formed by intestinal microbiota, and its Flavin-Monooxygenase 3 (FMO3) product Trimethylamine-N-Oxide (TMAO), are potential modulators of host cardiometabolic phenotypes. High circulating levels of TMAO are associated with increased risk for cardiovascular diseases. We hypothesized that TMA/TMAO could directly change the vascular tone. Perivascular adipose tissue (PVAT) helps to regulate vascular homeostasis and may also possess FMO3. Thoracic aorta with(+) or without(-) PVAT, also + or - the endothelium (E), of male Sprague Dawley rats were isolated for measurement of isometric tone in response to TMA/TMAO (1nM-0.5 M). Immunohistochemistry (IHC) studies were done to identify the presence of FMO3. TMA and TMAO elicited concentration-dependent arterial contraction. However, at a maximally achievable concentration (0.2 M), contraction stimulated by TMA was of a greater magnitude (141.5 ± 16% of maximum phenylephrine contraction) than that elicited by TMAO (19.1 ± 4.03%) with PVAT and endothelium intact. When PVAT was preserved, TMAO-induced contraction was extensively reduced the presence (19.1 ± 4.03%) versus absence of E (147.2 ± 20.5%), indicating that the endothelium plays a protective role against TMAO-induced contraction. FMO3 enzyme was present in aortic PVAT, but the FMO3 inhibitor methimazole did not affect contraction stimulated by TMA in aorta + PVAT. However, the l-type calcium channel blocker nifedipine reduced TMA-induced contraction by â¼50% compared to the vehicle. Though a high concentration of these compounds was needed to achieve contraction, the findings that TMA-induced contraction was independent of PVAT and E and mediated by nifedipine-sensitive calcium channels suggest metabolite-induced contraction may be physiologically important.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Aorta Torácica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Metilaminas/farmacologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/fisiologia , Cálcio/fisiologia , Canais de Cálcio Tipo L/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Oxigenases/metabolismo , Oxigenases/fisiologia , Ratos Sprague-DawleyRESUMO
BACKGROUND: Recent studies have shown that human and experimental alcohol-related liver disease (ALD) is robustly associated with dysregulation of bile acid homeostasis, which may in turn modulate disease severity. Pharmacological agents targeting bile acid metabolism and signaling may be potential therapeutics for ALD. METHODS: The potential beneficial effects of a gut-restricted apical sodium-dependent bile acid transporter (ASBT) inhibitor were studied in a chronic-plus-binge ALD mouse model. RESULTS: Blocking intestinal bile acid reabsorption by the gut-restricted ASBT inhibitor GSK2330672 attenuated hepatic steatosis and liver injury in a chronic-plus-binge ALD mouse model. Alcohol feeding is associated with intestinal bile acid accumulation but paradoxically impaired ileal farnesoid × receptor (FXR) function, and repressed hepatic cholesterol 7α-hydrolase (CYP7A1) expression despite decreased hepatic small heterodimer partner (SHP) and ileal fibroblast growth factor 15 (FGF15) expression. ASBT inhibitor treatment decreased intestinal bile acid accumulation and increased hepatic CYP7A1 expression, but further decreased ileal FXR activity. Alcohol feeding induces serum bile acid concentration that strongly correlates with a liver injury marker. However, alcohol-induced serum bile acid elevation is not due to intrahepatic bile acid accumulation but is strongly and positively associated with hepatic multidrug resistance-associated protein 3 (MRP4) and MRP4 induction but poorly associated with sodium-taurocholate cotransporting peptide (NTCP) expression. ASBT inhibitor treatment decreases serum bile acid concentration without affecting hepatocyte basolateral bile acid uptake and efflux transporters. CONCLUSION: ASBT inhibitor treatment corrects alcohol-induced bile acid dysregulation and attenuates liver injury in experimental ALD.
Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatias Alcoólicas/tratamento farmacológico , Fígado/efeitos dos fármacos , Metilaminas/uso terapêutico , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Simportadores/antagonistas & inibidores , Tiazepinas/uso terapêutico , Proteínas Angiogênicas/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Avaliação Pré-Clínica de Medicamentos , Fígado/metabolismo , Masculino , Metilaminas/farmacologia , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Tiazepinas/farmacologia , Transaminases/sangueRESUMO
Several classes of antimalarial drugs are currently available, although issues of toxicity and the emergence of drug resistant malaria parasites have reduced their overall therapeutic efficiency. Quinoline based antiplasmodial drugs have unequivocally been long-established and continue to inspire the design of new antimalarial agents. Herein, a series of mono- and bisquinoline methanamine derivatives were synthesised through sequential steps; Vilsmeier-Haack, reductive amination, and nucleophilic substitution, and obtained in low to excellent yields. The resulting compounds were investigated for in vitro antiplasmodial activity against the 3D7 chloroquine-sensitive strain of Plasmodium falciparum, and compounds 40 and 59 emerged as the most promising with IC50 values of 0.23 and 0.93 µM, respectively. The most promising compounds were also evaluated in silico by molecular docking protocols for binding affinity to the {001} fast-growing face of a hemozoin crystal model.
Assuntos
Antimaláricos/farmacologia , Desenho de Fármacos , Metilaminas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Quinolinas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Relação Dose-Resposta a Droga , Metilaminas/síntese química , Metilaminas/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Sensibilidade Parasitária , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
In an attempt to search for new natural product-based antitumor agents, a series of novel (aryl)methyl-amine derivatives of dehydroabietic acid-based B ring-fused-thiazole were designed and synthesized. The primary bioassay showed that compounds 5r and 5s presented certain inhibitory activity against cancer cells, weak cytotoxic activity against normal cells, and inhibitory activity against PI3K/AKT/mTOR signaling pathway. The binding modes and the binding site interactions between the active compounds and the target proteins were predicted preliminarily by the molecular docking method.
Assuntos
Abietanos , Antineoplásicos , Metilaminas , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases , Tiazóis , Abietanos/química , Abietanos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Metilaminas/química , Metilaminas/farmacologia , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tiazóis/química , Tiazóis/farmacologiaRESUMO
Diet is a well-known risk factor of cardiovascular diseases (CVDs). Some microRNAs (miRNAs) have been described to regulate molecular pathways related to CVDs. Diet can modulate miRNAs and their target genes. Choline, betaine, and l-carnitine, nutrients found in animal products, are metabolized into trimethylamine n-oxide (TMAO), which has been associated with CVD risk. The aim of this study was to investigate TMAO regulation of CVD-related miRNAs and their target genes in cellular models of liver and macrophages. We treated HEPG-2, THP-1, mouse liver organoids, and primary human macrophages with 6 µM TMAO at different timepoints (4, 8, and 24 h for HEPG-2 and mouse liver organoids, 12 and 24 h for THP-1, and 12 h for primary human macrophages) and analyzed the expression of a selected panel of CVD-related miRNAs and their target genes and proteins by real-time PCR and Western blot, respectively. HEPG-2 cells were transfected with anti-miR-30c and syn-miR-30c. TMAO increased the expression of miR-21-5p and miR-30c-5p. PER2, a target gene of both, decreased its expression with TMAO in HEPG-2 and mice liver organoids but increased its mRNA expression with syn-miR-30c. We concluded that TMAO modulates the expression of miRNAs related to CVDs, and that such modulation affects their target genes.
Assuntos
Doenças Cardiovasculares/genética , Metilaminas/farmacologia , MicroRNAs/efeitos dos fármacos , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/fisiologia , Proteínas Circadianas Period/efeitos dos fármacos , Proteínas Circadianas Period/genética , Células THP-1RESUMO
Aberrant cellular Myc (c-Myc) is a common feature in the majority of human cancers and has been linked to oncogenic malignancies. Here, we developed a novel c-Myc-targeting compound, N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), and present evidence demonstrating its effectiveness in targeting c-Myc for degradation in human lung carcinoma. EMD exhibited strong cytotoxicity toward various human lung cancer cell lines, as well as chemotherapeutic-resistant patient-derived lung cancer cells, through apoptosis induction in comparison with chemotherapeutic drugs. The IC50 of EMD against lung cancer cells was approximately 60 µM. Mechanistically, EMD eliminated c-Myc in the cells and initiated caspase-dependent apoptosis cascade. Cycloheximide chase assay revealed that EMD tended to shorten the half-life of c-Myc by approximately half. The cotreatment of EMD with the proteasome inhibitor MG132 reversed its c-Myc-targeting effect, suggesting the involvement of ubiquitin-mediated proteasomal degradation in the process. We further verified that EMD strongly induced the ubiquitination of c-Myc and promoted protein degradation. c-Myc inhibition and apoptosis induction were additionally shown in hematologic malignant K562 cells, indicating the generality of the observed EMD effects. Altogether, we identified EMD as a novel potent compound targeting oncogenic c-Myc that may offer new opportunities for lung cancer treatment. SIGNIFICANCE STATEMENT: The deregulation of c-Myc is frequently associated with cancer progression. This study examined the effect of a new compound, N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), in targeting c-Myc in several lung cancer cell lines and drug-resistant primary lung cancer cells. EMD induced dramatic c-Myc degradation through a ubiquitin-proteasomal mechanism. The promising anticancer and c-Myc-targeted activities of EMD support its use in potential new approaches to treat c-Myc-driven cancer.
Assuntos
Antineoplásicos/síntese química , Neoplasias Pulmonares/metabolismo , Metilaminas/síntese química , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células K562 , Neoplasias Pulmonares/tratamento farmacológico , Metilaminas/química , Metilaminas/farmacologia , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Ubiquitina/metabolismoRESUMO
Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment-mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.
Assuntos
Morte Celular , Regulação Enzimológica da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Diferenciação Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Citosol/metabolismo , Endoderma/citologia , Humanos , Concentração de Íons de Hidrogênio , Mesoderma/citologia , Metilaminas/farmacologia , Necrose , Fosforilação , Piridinas/farmacologiaRESUMO
Trimethylamine N-oxide (TMAO) is produced from the phosphatidylcholine metabolism of gut flora and acts as a risk factor of cardiovascular disease. However, the underlying mechanisms for its proatherogenic action remain unclear. This study aimed to observe the effect of TMAO on endothelial cell pyroptosis and explore the underlying mechanisms. Our results showed that TMAO promoted the progression of atherosclerotic lesions in apolipoprotein E-deficient (apoE-/- ) mice fed a high-fat diet. Pyroptosis and succinate dehydrogenase complex subunit B (SDHB) upregulation were detected in the vascular endothelial cells of apoE-/- mice and in cultured human umbilical vein endothelial cells (HUVECs) treated with TMAO. Overexpression of SDHB in HUVECs enhanced pyroptosis and impaired mitochondria and high reactive oxygen species (ROS) level. Pyroptosis in the SDHB overexpression of endothelial cells was inhibited by the ROS scavenger NAC. In summary, TMAO promotes vascular endothelial cell pyroptosis via ROS induced through SDHB upregulation, thereby contributing to the progression of atherosclerotic lesions.
Assuntos
Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Metilaminas/farmacologia , Piroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Free fatty acid (FFA) receptors FFA1 and FFA4 are omega-3 molecular targets in metabolic diseases; however, their function in cancer cachexia remains unraveled. We assessed the role of FFA1 and FFA4 receptors in the mouse model of cachexia induced by Lewis lung carcinoma (LLC) cell implantation. Naturally occurring ligands such as α-linolenic acid (ALA) and docosahexaenoic acid (DHA), the synthetic FFA1/FFA4 agonists GW9508 and TUG891, or the selective FFA1 GW1100 or FFA4 AH7614 antagonists were tested. FFA1 and FFA4 expression and other cachexia-related parameters were evaluated. GW9508 and TUG891 decreased tumor weight in LLC-bearing mice. Regarding cachexia-related end points, ALA, DHA, and the preferential FFA1 agonist GW9508 rescued body weight loss. Skeletal muscle mass was reestablished by ALA treatment, but this was not reflected in the fiber cross-sectional areas (CSA) measurement. Otherwise, TUG891, GW1100, or AH7614 reduced the muscle fiber CSA. Treatments with ALA, GW9508, GW1100, or AH7614 restored white adipose tissue (WAT) depletion. As for inflammatory outcomes, ALA improved anemia, whereas GW9508 reduced splenomegaly. Concerning behavioral impairments, ALA and GW9508 rescued locomotor activity, whereas ALA improved motor coordination. Additionally, DHA improved grip strength. Notably, GW9508 restored abnormal brain glucose metabolism in different brain regions. The GW9508 treatment increased leptin levels, without altering uncoupling protein-1 downregulation in visceral fat. LLC-cachectic mice displayed FFA1 upregulation in subcutaneous fat, but not in visceral fat or gastrocnemius muscle, whereas FFA4 was unaltered. Overall, the present study shed new light on FFA1 and FFA4 receptors' role in metabolic disorders, indicating FFA1 receptor agonism as a promising strategy in mitigating cancer cachexia.
Assuntos
Peso Corporal/efeitos dos fármacos , Caquexia/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Ácidos Docosa-Hexaenoicos/uso terapêutico , Receptores Acoplados a Proteínas G/metabolismo , Ácido alfa-Linolênico/uso terapêutico , Animais , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Caquexia/etiologia , Caquexia/metabolismo , Carcinoma Pulmonar de Lewis/complicações , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Metilaminas/farmacologia , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Transplante de Neoplasias , Fenilpropionatos/farmacologia , Propionatos/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Sulfonamidas/farmacologia , Xantenos/farmacologia , Ácido alfa-Linolênico/farmacologiaRESUMO
Adenosine triphosphate (ATP)-binding cassette subfamily A member 3 (ABCA3), a phospholipid transporter in lung lamellar bodies (LBs), is essential for the assembly of pulmonary surfactant and LB biogenesis. Mutations in the ABCA3 gene are an important genetic cause for respiratory distress syndrome in neonates and interstitial lung disease in children and adults, for which there is currently no cure. The aim of this study was to prove that disease causing misfolding ABCA3 mutations can be corrected in vitro and to investigate available options for correction. We stably expressed hemagglutinin (HA)-tagged wild-type ABCA3 or variants p.Q215K, p.M760R, p.A1046E, p.K1388N or p.G1421R in A549 cells and assessed correction by quantitation of ABCA3 processing products, their intracellular localization, resembling LB morphological integrity and analysis of functional transport activity. We showed that all mutant proteins except for M760R ABCA3 were rescued by the bithiazole correctors C13 and C17. These variants were also corrected by the chemical chaperone trimethylamine N-oxide and by low temperature. The identification of lead molecules C13 and C17 is an important step toward pharmacotherapy of ABCA3 misfolding-induced lung disease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/genética , Metilaminas/farmacologia , Mutação de Sentido Incorreto/efeitos dos fármacos , Deficiências na Proteostase/tratamento farmacológico , Deficiências na Proteostase/genética , Células A549 , Transportadores de Cassetes de Ligação de ATP/metabolismo , Humanos , Doenças Pulmonares Intersticiais/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estudo de Prova de Conceito , Dobramento de Proteína , Deficiências na Proteostase/metabolismoRESUMO
Cardiovascular disease is a major cause of morbidity and mortality among patients with chronic kidney disease (CKD). Trimethylamine-N-oxide (TMAO), a uremic metabolite that is elevated in the setting of CKD, has been implicated as a nontraditional risk factor for cardiovascular disease. While association studies have linked elevated plasma levels of TMAO to adverse cardiovascular outcomes, its direct effect on cardiac and smooth muscle function remains to be fully elucidated. We hypothesized that pathological concentrations of TMAO would acutely increase cardiac and smooth muscle contractility. These effects may ultimately contribute to cardiac dysfunction during CKD. High levels of TMAO significantly increased paced, ex vivo human cardiac muscle biopsy contractility (P < 0.05). Similarly, TMAO augmented contractility in isolated mouse hearts (P < 0.05). Reverse perfusion of TMAO through the coronary arteries via a Langendorff apparatus also enhanced cardiac contractility (P < 0.05). In contrast, the precursor molecule, trimethylamine (TMA), did not alter contractility (P > 0.05). Multiphoton microscopy, used to capture changes in intracellular calcium in paced, adult mouse hearts ex vivo, showed that TMAO significantly increased intracellular calcium fluorescence (P < 0.05). Interestingly, acute administration of TMAO did not have a statistically significant influence on isolated aortic ring contractility (P > 0.05). We conclude that TMAO directly increases the force of cardiac contractility, which corresponds with TMAO-induced increases in intracellular calcium but does not acutely affect vascular smooth muscle or endothelial function of the aorta. It remains to be determined if this acute inotropic action on cardiac muscle is ultimately beneficial or harmful in the setting of CKD.NEW & NOTEWORTHY We demonstrate for the first time that elevated concentrations of TMAO acutely augment myocardial contractile force ex vivo in both murine and human cardiac tissue. To gain mechanistic insight into the processes that led to this potentiation in cardiac contraction, we used two-photon microscopy to evaluate intracellular calcium in ex vivo whole hearts loaded with the calcium indicator dye Fluo-4. Acute treatment with TMAO resulted in increased Fluo-4 fluorescence, indicating that augmented cytosolic calcium plays a role in the effects of TMAO on force production. Lastly, TMAO did not show an effect on aortic smooth muscle contraction or relaxation properties. Our results demonstrate novel, acute, and direct actions of TMAO on cardiac function and help lay the groundwork for future translational studies investigating the complex multiorgan interplay involved in cardiovascular pathogenesis during CKD.