RESUMEN
Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.
Asunto(s)
Ácido Eicosapentaenoico , Neuralgia , Proteínas de Transporte de Nucleótidos , Adenosina Trifosfato/metabolismo , Animales , Ácido Eicosapentaenoico/farmacología , Ácido Eicosapentaenoico/uso terapéutico , Humanos , Resistencia a la Insulina , Ratones , Neuralgia/tratamiento farmacológico , Neuralgia/genética , Nocicepción , Proteínas de Transporte de Nucleótidos/antagonistas & inhibidores , Proteínas de Transporte de Nucleótidos/genética , Proteínas de Transporte de Nucleótidos/metabolismoRESUMEN
Sulfur-based redox signaling has long attracted attention as critical mechanisms underlying the development of cardiac diseases and resultant heart failure. Especially, post-translational modifications of cysteine (Cys) thiols in proteins mediate oxidative stress-dependent cardiac remodeling including myocardial hypertrophy, senescence, and interstitial fibrosis. However, we recently revealed the existence of Cys persulfides and Cys polysulfides in cells and tissues, which show higher redox activities than Cys and substantially contribute to redox signaling and energy metabolism. We have established simple evaluation methods that can detect polysulfides in proteins and inorganic polysulfides in cells and revealed that polysulfides abundantly expressed in normal hearts are dramatically catabolized by exposure to ischemic/hypoxic and environmental electrophilic stress, which causes vulnerability of the heart to mechanical load. Accumulation of hydrogen sulfide, a nucleophilic catabolite of persulfides/polysulfides, may lead to reductive stress in ischemic hearts, and perturbation of polysulfide catabolism can improve chronic heart failure after myocardial infarction in mice. This review focuses on the (patho)physiological role of sulfur metabolism in hearts, and proposes that sulfur catabolism during ischemic/hypoxic stress has great potential as a new therapeutic strategy for the treatment of ischemic heart failure.
Asunto(s)
Cisteína , Insuficiencia Cardíaca , Sulfuro de Hidrógeno , Oxidación-Reducción , Sulfuros , Azufre , Insuficiencia Cardíaca/metabolismo , Animales , Humanos , Sulfuros/metabolismo , Azufre/metabolismo , Sulfuro de Hidrógeno/metabolismo , Cisteína/metabolismo , Estrés Oxidativo , Transducción de Señal , Procesamiento Proteico-Postraduccional , Ratones , Terapia Molecular Dirigida , Metabolismo Energético , Miocardio/metabolismoRESUMEN
The atrophic myocardium resulting from mechanical unloading and nutritional deprivation is considered crucial as maladaptive remodeling directly associated with heart failure, as well as interstitial fibrosis. Conversely, myocardial hypertrophy resulting from hemodynamic loading is perceived as compensatory stress adaptation. We previously reported the abundant presence of highly redox-active polysulfide molecules, termed supersulfide, with two or more sulfur atoms catenated in normal hearts, and the supersulfide catabolism in pathologic hearts after myocardial infarction correlated with worsened prognosis of heart failure. However, the impact of supersulfide on myocardial remodeling remains unclear. Here, we investigated the involvement of supersulfide metabolism in cardiomyocyte remodeling, using a model of adenosine 5'-triphosphate (ATP) receptor-stimulated atrophy and endothelin-1 receptor-stimulated hypertrophy in neonatal rat cardiomyocytes. Results revealed contrasting changes in intracellular supersulfide and its catabolite, hydrogen sulfide (H2S), between cardiomyocyte atrophy and hypertrophy. Stimulation of cardiomyocytes with ATP decreased supersulfide activity, while H2S accumulation itself did not affect cardiomyocyte atrophy. This supersulfide catabolism was also involved in myofibroblast formation of neonatal rat cardiac fibroblasts. Thus, unraveling supersulfide metabolism during myocardial remodeling may lead to the development of novel therapeutic strategies to improve heart failure.
Asunto(s)
Sulfuro de Hidrógeno , Miocitos Cardíacos , Sulfuros , Remodelación Ventricular , Animales , Miocitos Cardíacos/metabolismo , Sulfuros/metabolismo , Sulfuros/farmacología , Sulfuro de Hidrógeno/metabolismo , Células Cultivadas , Adenosina Trifosfato/metabolismo , Ratas , Atrofia , Cardiomegalia/metabolismo , Cardiomegalia/patología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Animales Recién Nacidos , Ratas Sprague-DawleyRESUMEN
Smoking is one of the most serious risk factors for cardiovascular diseases. Although cigarette mainstream and sidestream smoke are significant contributors to increased cardiovascular mortality and morbidity, the underlying mechanism is still unclear. Here, we report that exposure of rat neonatal cardiomyocytes to cigarette smoke extract (CSE) induces mitochondrial hyperfission-mediated myocardial senescence. CSE leads to mitochondrial fission and reactive oxygen species (ROS) production through the complex formation between mitochondrial fission factor Drp1 and actin-binding protein, filamin A. Pharmacological perturbation of interaction between Drp1 and filamin A by cilnidipine and gene knockdown of Drp1 or filamin A inhibited CSE-induced mitochondrial hyperfission and ROS production as well as myocardial senescence. We previously reported that Drp1 activity is controlled by supersulfide-induced Cys644 polysulfidation. The redox-sensitive Cys644 was critical for CSE-mediated interaction with filamin A. The administration of supersulfide donor, Na2S3 also improved mitochondrial hyperfission-mediated myocardial senescence induced by CSE. Our results suggest the important role of Drp1-filamin A complex formation on cigarette smoke-mediated cardiac risk and the contribution of supersulfide to mitochondrial fission-associated myocardial senescence.
Asunto(s)
Fumar Cigarrillos , Miocitos Cardíacos , Animales , Ratas , Filaminas , Mitocondrias , Especies Reactivas de OxígenoRESUMEN
We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Canal Catiónico TRPC6 , Animales , Humanos , Ratones , Colon/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Intestinos , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismoRESUMEN
Lipid droplet (LD) accumulation in hepatocytes is one of the major symptoms associated with fatty liver disease. Mitochondria play a key role in catabolizing fatty acids for energy production through ß-oxidation. The interplay between mitochondria and LD assumes a crucial role in lipid metabolism, while it is obscure how mitochondrial morphology affects systemic lipid metabolism in the liver. We previously reported that cilnidipine, an already existing anti-hypertensive drug, can prevent pathological mitochondrial fission by inhibiting protein-protein interaction between dynamin-related protein 1 (Drp1) and filamin, an actin-binding protein. Here, we found that cilnidipine and its new dihydropyridine (DHP) derivative, 1,4-DHP, which lacks Ca2+ channel-blocking action of cilnidipine, prevent the palmitic acid-induced Drp1-filamin interaction, LD accumulation and cytotoxicity of human hepatic HepG2 cells. Cilnidipine and 1,4-DHP also suppressed the LD accumulation accompanied by reducing mitochondrial contact with LD in obese model and high-fat diet-fed mouse livers. These results propose that targeting the Drp1-filamin interaction become a new strategy for the prevention or treatment of fatty liver disease.
Asunto(s)
Dihidropiridinas , Dinaminas , Gotas Lipídicas , Hígado , Animales , Dinaminas/metabolismo , Humanos , Gotas Lipídicas/metabolismo , Gotas Lipídicas/efectos de los fármacos , Ratones , Células Hep G2 , Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Dihidropiridinas/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Dinámicas Mitocondriales/efectos de los fármacos , Ratones Endogámicos C57BL , Dieta Alta en Grasa/efectos adversos , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacosRESUMEN
Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.
Asunto(s)
Insuficiencia Cardíaca , Infarto del Miocardio , Disfunción Ventricular Izquierda , Humanos , Ratones , Ratas , Animales , Infarto del Miocardio/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/prevención & control , Miocardio/metabolismo , Sulfuros/metabolismo , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/prevención & control , AzufreRESUMEN
Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Receptores Purinérgicos P2 , Animales , Humanos , Ratones , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , ARN Mensajero/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Insuficiencia Cardíaca , Animales , Reposicionamiento de Medicamentos , Humanos , Mamíferos , SARS-CoV-2RESUMEN
Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.
Asunto(s)
COVID-19 , NADPH Oxidasa 2 , Canales Catiónicos TRPC , Animales , Humanos , Ratas , Adenosina Trifosfato/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , Miocitos Cardíacos/metabolismo , NADPH Oxidasa 2/metabolismo , Unión Proteica , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Regulación hacia Arriba , Canales Catiónicos TRPC/metabolismoRESUMEN
The human myocardium contains robust cells that constantly beat from birth to death without being replaced, even when exposed to various environmental stresses. Myocardial robustness is thought to depend primarily on the strength of the reducing power to protect the heart from oxidative stress. Myocardial antioxidant systems are controlled by redox reactions, primarily via the redox reaction of Cys sulfhydryl groups, such as found in thioredoxin and glutathione. However, the specific molecular entities that regulate myocardial reducing power have long been debated. Recently, reactive sulfide species, with excellent electron transfer ability, consisting of a series of multiple sulfur atoms, i.e., Cys persulfide and Cys polysulfides, have been found to play an essential role in maintaining mitochondrial quality and function, as well as myocardial robustness. This review presents the latest findings on the molecular mechanisms underlying mitochondrial energy metabolism and the maintenance of quality control by reactive sulfide species and provides a new insight for the prevention of chronic heart failure.
RESUMEN
Non-alcoholic steatohepatitis (NASH) is a disease that has progressed from non-alcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6 (TRPC3/6), reportedly participate in the development of fibrosis in cardiovascular and renal systems. We hypothesized that TRPC3/6 may also participate in NASH fibrosis. We evaluated the effects of TRPC3 or TRPC6 functional deficiency in a NASH mouse model using choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Wild-type (WT) and TRPC3 or TRPC6 gene-deficient (KO) mice were fed with CDAHFD or standard diet for 6 weeks. The CDAHFD-induced body weight loss in TRPC6 KO mice was significantly lower compared with WT mice with CDAHFD. CDAHFD treatment significantly increased TRPC3 mRNA expression level and tissue weight in WT liver, which were suppressed in TRPC3 KO mice. However, either systemic deletion of TRPC3 or TRPC6 failed to attenuate liver steatosis, inflammation and fibrosis. These results imply that TRPC3 and TRPC6 are unlikely to be involved in liver dysfunction and fibrosis of NASH model mice.
Asunto(s)
Cirrosis Hepática/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6/genética , Animales , Quimiocina CCL2/genética , Deficiencia de Colina/genética , Deficiencia de Colina/patología , Dieta Alta en Grasa , Inflamación/genética , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/patología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
About 60-85% of total phosphorus (P) in cereal crops is finally allocated to seeds, where it is required for seed development, germination and early growth. However, little is known about the molecular mechanisms underlying P allocation to seeds. Here, we found that two members (OsPHO1;1 and OsPHO1;2) of the PHO1 gene family are involved in the distribution of P to seeds in rice. Both OsPHO1;1 and OsPHO1;2 were localized to the plasma membrane and showed influx transport activities for inorganic phosphate. At the reproductive stage, both OsPHO1;1 and OsPHO1;2 showed higher expression in node I, the uppermost node connecting to the panicle. OsPHO1;1 was mainly localized at the phloem region of diffuse vascular bundles (DVBs) of node I, while OsPHO1;2 was expressed in the xylem parenchyma cells of the enlarged vascular bundles (EVBs). In addition, they were also expressed in the ovular vascular trace, the outer layer of the inner integument (OsPHO1;1) and in the nucellar epidermis (OsPHO1;2) of caryopses. Knockout of OsPHO1;2, as well as OsPHO1;1 to a lesser extent, decreased the distribution of P to the seed, resulting in decreased seed size and delayed germination. Taken together, OsPHO1;2 expressed in node I is responsible for the unloading of P from the xylem of EVBs, while OsPHO1;1 is involved in reloading P into the phloem of DVBs for subsequent allocation of P to seeds. Furthermore, OsPHO1;1 and OsPHO1;2 expression in the caryopsis is important for delivering P from the maternal tissues to the filial tissues for seed development.
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Grano Comestible/crecimiento & desarrollo , Oryza/crecimiento & desarrollo , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Membrana Celular/metabolismo , Grano Comestible/metabolismo , Germinación , Especificidad de Órganos , Oryza/metabolismo , Proteínas de Transporte de Fosfato/fisiología , Proteínas de Plantas/fisiologíaRESUMEN
Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl- A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT -/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.
RESUMEN
Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT-/- mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.
Asunto(s)
Adenosina Trifosfato/metabolismo , Infiltración Neutrófila , Neutrófilos/metabolismo , Proteínas de Transporte de Nucleótidos/metabolismo , Vesículas Secretoras/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Movimiento Celular/efectos de los fármacos , Adyuvante de Freund/farmacología , Regulación de la Expresión Génica , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Moduladores del Transporte de Membrana/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Neutrófila/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Proteínas de Transporte de Nucleótidos/antagonistas & inhibidores , Proteínas de Transporte de Nucleótidos/genética , Transporte de Proteínas/efectos de los fármacos , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/inmunologíaRESUMEN
Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/análisis , Fragmentos de Péptidos/análisis , Proteínas de Transporte Vesicular de Acetilcolina/análisis , Proteína 2 de Transporte Vesicular de Glutamato/análisis , Animales , Precipitación Química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 1 de Aminoácidos Excitadores/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Ratones , Mapeo Peptídico , Proteolisis , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ácido Tricloroacético/química , Tripsina/química , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Integration of the complete human herpesvirus 6 (HHV-6) genome into the telomere of a chromosome has been reported in some individuals (inherited chromosomally integrated HHV-6; iciHHV-6). Since the proportion of iciHHV-6-positive individuals with integration in chromosome 22 is high in Japan, we hypothesized a founder effect. In this study, we sought to elucidate the reason for the high proportion of viral integrations into chromosome 22. We analyzed six cases of iciHHV-6A and two cases of iciHHV-6B, including one iciHHV-6A case with a matched sample from a father and one iciHHV-6B case with a matched sample from a mother. In iciHHV-6A, the same copy numbers of viral telomeric repeat sequences (TRS) and the same five microsatellite markers were detected in both the index case and paternal sample. Moreover, the same five microsatellite markers were demonstrated in four cases and the same copy numbers of viral TRS were demonstrated in two pairs of two cases. The present microsatellite analysis suggested that the viral genomes detected in some iciHHV-6A patients were derived from a common ancestral integration.
Asunto(s)
Cromosomas Humanos Par 22/virología , Herpesvirus Humano 6/aislamiento & purificación , Infecciones por Roseolovirus/virología , Adulto , Cromosomas Humanos Par 22/genética , Femenino , Genoma Viral , Herpesvirus Humano 6/clasificación , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/fisiología , Humanos , Japón , Masculino , Secuencias Repetitivas de Ácidos Nucleicos , Infecciones por Roseolovirus/congénito , Infecciones por Roseolovirus/genética , Integración ViralRESUMEN
BACKGROUND AND AIM: Proton pump inhibitors are effective for the treatment of gastric ulcers after endoscopic submucosal dissection (ESD). However, the most excellent therapy is controversial. Vonoprazan, an active potassium-competitive acid blocker, has a strong gastric acid secretion inhibitory effect, but its efficacy for the treatment of post-ESD gastric ulcers is unclear. Herein, we aimed to determine the healing effect of vonoprazan on post-ESD gastric ulcers. METHODS: We carried out a prospective randomized controlled trial examining 92 patients who had undergone ESD for the treatment of gastric neoplasms between April 2015 and June 2016 at Machida Municipal Hospital. Patients were treated with 20 mg/day vonoprazan (V group) or 20 mg/day esomeprazole (E group) for 8 weeks. We evaluated the 8-week cure rate for artificial ulcers and any complications after ESD. RESULTS: A total of 80 patients (median age, 73.5 years; 71.3% male) were analyzed. Cure rate for the V group was significantly higher than that for the E group (94.9% [37/39] vs 78.0% [32/41], respectively; P = 0.049). In a multivariate analysis, only vonoprazan was correlated with ulcer healing (odds ratio = 6.33; 95% CI = 1.21-33.20; P = 0.029). Delayed bleeding was experienced only in the E group (7.3% [3/41]), but no significant difference compared with the V group was observed (P = 0.241). CONCLUSION: Vonoprazan was significantly superior to esomeprazole for the healing of post-ESD gastric ulcers and should be considered as a treatment of first choice.
Asunto(s)
Resección Endoscópica de la Mucosa/efectos adversos , Complicaciones Posoperatorias/tratamiento farmacológico , Inhibidores de la Bomba de Protones/uso terapéutico , Pirroles/uso terapéutico , Neoplasias Gástricas/cirugía , Úlcera Gástrica/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Anciano , Anciano de 80 o más Años , Esomeprazol/uso terapéutico , Femenino , Humanos , Masculino , Complicaciones Posoperatorias/etiología , Estudios Prospectivos , Úlcera Gástrica/etiologíaRESUMEN
Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 10(6) copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 10(6) copies/tube) in the multiplex PCR assay; however, 1 × 10(6) copies/tube of HHV-6A DNA did not affect the quantification of 1 × 10(4) copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R(2) = 0.913) and HHV-6B (R(2) = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 10(6) copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628-1635, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Herpesvirus Humano 6/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , ADN Viral/sangre , ADN Viral/genética , Herpesvirus Humano 6/genética , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Infecciones por Roseolovirus/virología , Especificidad de la Especie , Carga ViralRESUMEN
Probiotics have been shown to improve the condition of not only the human gastrointestinal tract but also the entire body. We found that quercetin enhances the anti-inflammatory activity of Bifidobacterium adolescentis, which is abundant in human intestines. Here, we assessed whether certain phytochemicals could enhance the anti-inflammatory activity of B. adolescentis. Bifidobacteria were anaerobically cultured with phytochemicals for 3 h, and the anti-inflammatory activity of the supernatants was estimated by testing their ability to inhibit nitric oxide (NO) production by lipopolysaccharide-stimulated RAW264 macrophages. Of the 55 phytochemicals tested, phloretin, (+)-taxifolin, and (-)-epigallocatechin gallate as well as quercetin-3-O-glucoside and quercetin-4'-O-glucoside were similar to quercetin in promoting NO suppression by B. adolescentis. In addition, the phytochemicals excluding quercetin increased the concentrations of lactic and acetic acids in the co-culture supernatants. These results suggest that some phytochemicals may activate the anti-inflammatory function of B. adolescentis.