RESUMEN
RN-9893, a TRPV4 antagonist identified by Renovis Inc., showcased notable inhibition of TRPV4 channels. This research involved synthesizing and evaluating three series of RN-9893 analogues for their TRPV4 inhibitory efficacy. Notably, compounds 1b and 1f displayed a 2.9 to 4.5-fold increase in inhibitory potency against TRPV4 (IC50 = 0.71 ± 0.21 µM and 0.46 ± 0.08 µM, respectively) in vitro, in comparison to RN-9893 (IC50 = 2.07 ± 0.90 µM). Both compounds also significantly outperformed RN-9893 in TRPV4 current inhibition rates (87.6 % and 83.2 % at 10 µM, against RN-9893's 49.4 %). For the first time, these RN-9893 analogues were profiled in an in vivo mouse model, where intraperitoneal injections of 1b or 1f at 10 mg/kg notably mitigated symptoms of acute lung injury induced by lipopolysaccharide (LPS). These outcomes indicate that compounds 1b and 1f are promising candidates for acute lung injury treatment.
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Lesión Pulmonar Aguda , Bencenosulfonamidas , Sulfonamidas , Canales Catiónicos TRPV , Relación Estructura-Actividad , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismo , Lesión Pulmonar Aguda/tratamiento farmacológico , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonamidas/síntesis química , Animales , Ratones , Humanos , Estructura Molecular , Relación Dosis-Respuesta a Droga , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BLRESUMEN
Acute lung injury (ALI) is characterized by endothelial barrier disruption and associated inflammatory responses, and transient receptor potential cation channel 6 (TRPC6)-mediated Ca2+ influx is critical for endothelial hyperpermeability. In this study, we investigated the role of TRPC6 in LPS-induced ALI, analyzed gene expression in WT and TRPC6-/- lungs using RNA sequencing, and explored the effects of TRPC6 in the LPS-induced hyperpermeability in human umbilical vein endothelial cells (HUVECs) to elucidate the underlying mechanisms. Intratracheal instillation of LPS caused edema in the mouse lungs. Deletion of TRPC6 reduced LPS-induced lung edema and decreased cell infiltration. RNA sequencing analysis suggested that downregulated cell adhesion molecules in TRPC6-/- lungs may be responsible for their resistance to LPS-induced injury. In addition, downregulation of TRPC6 significantly alleviated the LPS-induced decrease in eNOS expression in lung tissue as well as in HUVECs. Moreover, inhibition of TRPC6 with the channel antagonist larixyl led to a decrease in LPS-induced hyperpermeability and ROS production in HUVECs, which could be reversed by blocking eNOS. Our findings suggest that inhibition of TRPC6 ameliorates LPS-induced ALI, which may be achieved by acting on the cell adhesion molecule signaling pathway and participating in the regulation of eNOS levels in endothelial cells.
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Lesión Pulmonar Aguda , Canales de Potencial de Receptor Transitorio , Animales , Humanos , Ratones , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Edema/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Lipopolisacáridos/efectos adversos , Pulmón/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismoRESUMEN
The increased permeability of the lung microvascular endothelium is one critical initiation of acute lung injury (ALI). The disruption of vascular-endothelium integrity results in leakiness of the endothelial barrier and accumulation of protein-rich fluid in the alveoli. During ALI, increased endothelial-cell (EC) permeability is always companied by high frequency and amplitude of cytosolic Ca2+ oscillations. Mechanistically, cytosolic calcium oscillations include calcium release from internal stores and calcium entry via channels located in the cell membrane. Recently, numerous publications have shown substantial evidence that calcium-permeable channels play an important role in maintaining the integrity of the endothelium barrier function of the vessel wall in ALI. These novel endothelial signaling pathways are future targets for the treatment of lung injury. This short review focuses on the up-to-date research and provide insight into the contribution of calcium influx via ion channels to the disruption of lung microvascular endothelial-barrier function during ALI.
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RATIONALE: Even in antiretroviral therapy-treated patients, HIV continues to play a pathogenic role in cardiovascular diseases. A possible cofactor may be persistence of the early HIV response gene Nef, which we have demonstrated recently to persist in the lungs of HIV+ patients on antiretroviral therapy. Previously, we have reported that HIV strains with Nef, but not Nef-deleted HIV strains, cause endothelial proinflammatory activation and apoptosis. OBJECTIVE: To characterize mechanisms through which HIV-Nef leads to the development of cardiovascular diseases using ex vivo tissue culture approaches as well as interventional experiments in transgenic murine models. METHODS AND RESULTS: Extracellular vesicles derived from both peripheral blood mononuclear cells and plasma from HIV+ patient blood samples induced human coronary artery endothelial cells dysfunction. Plasma-derived extracellular vesicles from antiretroviral therapy+ patients who were HIV-Nef+ induced significantly greater endothelial apoptosis compared with HIV-Nef-plasma extracellular vesicles. Both HIV-Nef expressing T cells and HIV-Nef-induced extracellular vesicles increased transfer of cytosol and Nef protein to endothelial monolayers in a Rac1-dependent manner, consequently leading to endothelial adhesion protein upregulation and apoptosis. HIV-Nef induced Rac1 activation also led to dsDNA breaks in endothelial colony forming cells, thereby resulting in endothelial colony forming cell premature senescence and endothelial nitric oxide synthase downregulation. These Rac1-dependent activities were characterized by NOX2-mediated reactive oxygen species production. Statin treatment equally inhibited Rac1 inhibition in preventing or reversing all HIV-Nef-induction abnormalities assessed. This was likely because of the ability of statins to block Rac1 prenylation as geranylgeranyl transferase inhibitors were effective in inhibiting HIV-Nef-induced reactive oxygen species formation. Finally, transgenic expression of HIV-Nef in endothelial cells in a murine model impaired endothelium-mediated aortic ring dilation, which was then reversed by 3-week treatment with 5 mg/kg atorvastatin. CONCLUSIONS: These studies establish a mechanism by which HIV-Nef persistence despite antiretroviral therapy could contribute to ongoing HIV-related vascular dysfunction, which may then be ameliorated by statin treatment.
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Células Endoteliales/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Adulto , Anciano , Animales , Células Endoteliales/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and ß subunits. The high-affinity CaV2.2α1â CaVß3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1â CaVß3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVß3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.
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Bloqueadores de los Canales de Calcio/farmacología , Activación del Canal Iónico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacocinética , Femenino , Células HEK293 , Humanos , Transporte Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Ratones , Neuralgia/prevención & control , Nocicepción/efectos de los fármacos , Unión Proteica , Ratas , Ratas Sprague-Dawley , Bibliotecas de Moléculas Pequeñas/farmacocinéticaRESUMEN
Estrogen hormones play an important role in controlling glucose homeostasis and pancreatic ß-cell function. Despite the significance of estrogen hormones for regulation of glucose metabolism, little is known about the roles of endogenous estrogen metabolites in modulating pancreatic ß-cell function. In this study, we evaluated the effects of major natural estrogen metabolites, catechol estrogens, on insulin secretion in pancreatic ß-cells. We show that catechol estrogens, hydroxylated at positions C2 and C4 of the steroid A ring, rapidly potentiated glucose-induced insulin secretion via a nongenomic mechanism. 2-Hydroxyestrone, the most abundant endogenous estrogen metabolite, was more efficacious in stimulating insulin secretion than any other tested catechol estrogens. In insulin-secreting cells, catechol estrogens produced rapid activation of calcium influx and elevation in cytosolic free calcium. Catechol estrogens also generated sustained elevations in cytosolic free calcium and evoked inward ion current in HEK293 cells expressing the transient receptor potential A1 (TRPA1) cation channel. Calcium influx and insulin secretion stimulated by estrogen metabolites were dependent on the TRPA1 activity and inhibited with the channel-specific pharmacological antagonists or the siRNA. Our results suggest the role of estrogen metabolism in a direct regulation of TRPA1 activity with potential implications for metabolic diseases.
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Estrógenos de Catecol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Canal Catiónico TRPA1/metabolismo , Animales , Células Cultivadas , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , RatasRESUMEN
A Gram stain-positive, non-spore-forming, non-motile and rod-shaped actinomycete, strain 5221T, was isolated from the sediment of a river collected at Ronggui in the Pearl River Delta, PR China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain formed a distinct lineage within the genus Brevibacterium and had the highest sequence similarity to Brevibacterium pityocampae Tp12T (96.7â%), followed by Brevibacterium daeguense 2C6-41T (96.5â%), Brevibacterium samyangense SST-8T (96.0â%) and Brevibacterium ravenspurgense 20T (95.9â%). The results of chemotaxonomic analyses, including detecting anteiso-C15â:â0, anteiso-C17â:â0, and C16â:â0 as the major cellular fatty acids, diphosphatidylglycerol, phosphatidylglycerol and three phosphoglycolipids as the polar lipids, MK-8(H2) as the major menaquinone, and a DNA G+C content of 72.4 mol%, supported that strain 5221T is a member of the genus Brevibacterium. Furthermore, low sequence similarities of 16S rRNA gene sequences, differences in fatty acid compositions and differential physiological characteristics such as enzyme activity and carbon sources utilization ability distinguished the isolate from its close relatives. Therefore, strain 5221T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium rongguiense sp. nov. is proposed, with the type strain 5221T (=GDMCC 1.1766T=KACC 21700T).
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Brevibacterium/clasificación , Sedimentos Geológicos/microbiología , Filogenia , Ríos/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , Brevibacterium/aislamiento & purificación , China , ADN Bacteriano/genética , Ácidos Grasos/química , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
BACKGROUND: The incidence of traumatic brain injuries (TBIs) is on the rise in the USA. Concussions, or mild TBIs without skull fracture, account for about 75% of all TBIs. Mild TBIs (mTBIs) lead to memory and cognitive deficits, headaches, intraocular pressure rises, axonal degeneration, neuroinflammation, and an array of cerebrovascular dysfunctions, including increased vascular permeability and decreased cerebral blood flow. It has been recently reported that besides vascular dysfunction in the cerebral circulation, mTBI may also cause a significant impairment of endothelial function in the systemic circulation, at least within mesenteric microvessels. In this study, we investigated whether mTBI affects endothelial function in aortas and determined the contribution of transient receptor potential canonical (TRPC) channels to modulating mTBI-associated endothelial dysfunction. METHODS: We used a model of closed-head mTBI in C57BL/6, 129S, 129S-C57BL/6-F2 mice, and 129S-TRPC1 and 129S-C57BL/6-TRPC6 knockout mice to determine the effect of mTBI on endothelial function in mouse aortas employing ex vivo isometric tension measurements. Aortic tissue was also analyzed using immunofluorescence and qRT-PCR for TRPC6 expression following mTBI. RESULTS: We show that in various strains of mice, mTBI induces a pronounced and long-lasting endothelial dysfunction in the aorta. Ablation of TRPC6 protects mice from mTBI-associated aortic endothelial dysfunction, while TRPC1 ablation does not impact brain injury-induced endothelial impairment in the aorta. Consistent with a role of TRPC6 activation following mTBI, we observed improved endothelial function in wild type control mice subjected to mTBI following 7-day in vivo treatment with larixyl acetate, an inhibitor of TRPC6 channels. Conversely, in vitro treatment with the pro-inflammatory endotoxin lipopolysaccharide, which activates endothelial TRPC6 in a Toll-like receptor type 4 (TLR4)-dependent manner, worsened aortic endothelial dysfunction in wild type mice. Lipopolysaccharide treatment in vitro failed to elicit endothelial dysfunction in TRPC6 knockout mice. No change in endothelial TRPC6 expression was observed 7 days following TBI. CONCLUSIONS: These data suggest that TRPC6 activation may be critical for inducing endothelial dysfunction following closed-head mTBI and that pharmacological inhibition of the channel may be a feasible therapeutic strategy for preventing mTBI-associated systemic endothelial dysfunction.
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Acetatos/uso terapéutico , Lesiones Traumáticas del Encéfalo/complicaciones , Endotelio Vascular , Naftalenos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Canales Catiónicos TRPC/antagonistas & inhibidores , Enfermedades Vasculares/etiología , Enfermedades Vasculares/prevención & control , Acetatos/farmacología , Animales , Aorta Torácica/fisiopatología , Traumatismos Cerrados de la Cabeza/complicaciones , Contracción Isométrica , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Naftalenos/farmacología , Fármacos Neuroprotectores/farmacología , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Receptor Toll-Like 4/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
Bacterial anaerobic respiration using an extracellular electron acceptor plays a predominant role in global biogeochemical cycles. However, the mechanisms of bacterial adaptation to the toxic organic pollutant as the extracellular electron acceptor during anaerobic respiration are not clear, which limits our ability to optimize the strategies for the bioremediation of a contaminated environment. Here, we report the physiological characteristics and the global gene expression of an ecologically successful bacterium, Shewanella decolorationis S12, when using a typical toxic organic pollutant, amaranth, as the extracellular electron acceptor. Our results revealed that filamentous shift (the cells stretched to fiber-like shapes as long as 18 µm) occurred under amaranth stress. Persistent stress led to a higher filamentous cell rate and decolorization ability in subcultural cells compared to parental strains. In addition, the expression of genes involved in cell division, the chemotaxis system, energy conservation, damage repair, and material transport in filamentous cells was significantly stimulated. The detailed roles of some genes with significantly elevated expressions in filamentous cells, such as the outer membrane porin genes ompA and ompW, the cytochrome c genes arpC and arpD, the global regulatory factor gene rpoS, and the methyl-accepting chemotaxis proteins genes SHD_2793 and SHD_0015, were identified by site-directed mutagenesis. Finally, a conceptual model was proposed to help deepen our insights into both the bacterial survival strategy when toxic organics were present and the mechanisms by which these toxic organics were biodegraded as the extracellular electron acceptors.IMPORTANCE Keeping toxic organic pollutants (TOPs) in tolerable levels is a huge challenge for bacteria in extremely unfavorable environments since TOPs could serve as energy substitutes but also as survival stresses when they are beyond some thresholds. This study focused on the underlying adaptive mechanisms of ecologically successful bacterium Shewanella decolorationis S12 when exposed to amaranth, a typical toxic organic pollutant, as the extracellular electron acceptor. Our results suggest that filamentous shift is a flexible and valid way to solve the dilemma between the energy resource and toxic stress. Filamentous cells regulate gene expression to enhance their degradation and detoxification capabilities, resulting in a strong viability. These novel adaptive responses to TOPs are believed to be an evolutionary achievement to succeed in harsh habitats and thus have great potential to be applied to environment engineering or synthetic biology if we could picture every unknown node in this pathway.
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Compuestos Azo/metabolismo , Colorantes/metabolismo , Shewanella/metabolismo , Anaerobiosis , Compuestos Azo/toxicidad , Proteínas Bacterianas/genética , Biodegradación Ambiental , Colorantes/toxicidad , Transporte de Electrón , Electrones , Oxidación-Reducción , Shewanella/efectos de los fármacos , Shewanella/genéticaRESUMEN
SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH3 sensitive, electrogenic H+ transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH4Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH4Cl when compared to wild type, indicating a decreased H+ permeability in mutants. All mutants exhibited significantly reduced H+ currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH4Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H+ flux properties.
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Proteínas de Transporte de Anión/metabolismo , Antiportadores/genética , Membrana Celular/metabolismo , Distrofias Hereditarias de la Córnea/genética , Mutación Puntual , Transporte de Proteínas/fisiología , Cloruro de Amonio/metabolismo , Animales , Proteínas de Transporte de Anión/genética , Línea Celular , Distrofias Hereditarias de la Córnea/metabolismo , Cricetinae , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , TransfecciónRESUMEN
Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.
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Calcio/metabolismo , Capsaicina/farmacología , Neuropatías Diabéticas/metabolismo , Ganglios Espinales/efectos de los fármacos , Neuronas/efectos de los fármacos , Dolor/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/patología , Modelos Animales de Enfermedad , Fluoresceínas/química , Colorantes Fluorescentes/química , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Regulación de la Expresión Génica , Glucosa/farmacología , Transporte Iónico/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Imagen Óptica , Dolor/genética , Dolor/fisiopatología , Técnicas de Placa-Clamp , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Análisis de la Célula Individual , Canales Catiónicos TRPV/genéticaRESUMEN
Transient receptor potential canonical type 5 (TRPC5) is a Ca2+-permeable cation channel that is highly expressed in the brain and is implicated in motor coordination, innate fear behavior, and seizure genesis. The channel is activated by a signal downstream of the G-protein-coupled receptor (GPCR)-Gq/11-phospholipase C (PLC) pathway. In this study we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. We report that Arg-593, a residue located in the E4 loop near the TRPC5 extracellular Gd3+ binding site, is critical for conferring the sensitivity to GPCR-Gq/11-PLC-dependent gating on TRPC5. Indeed, guanosine 5'-O-(thiotriphosphate) and GPCR agonists only weakly activate the TRPC5R593A mutant, whereas the addition of Gd3+ rescues the mutant's sensitivity to GPCR-Gq/11-PLC-dependent gating. Computer modeling suggests that Arg-593 may cross-bridge the E3 and E4 loops, forming the "molecular fulcrum." While validating the model using site-directed mutagenesis, we found that the Tyr-542 residue is critical for establishing a functional Gd3+ binding site, the Tyr-541 residue participates in fine-tuning Gd3+-sensitivity, and that the Asn-584 residue determines Ca2+ permeability of the TRPC5 channel. This is the first report providing molecular insights into the molecular mechanisms regulating the sensitivity to GPCR-Gq/11-PLC-dependent gating of a receptor-operated channel.
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Señalización del Calcio/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Gadolinio/farmacocinética , Activación del Canal Iónico/fisiología , Modelos Biológicos , Canales Catiónicos TRPC/metabolismo , Fosfolipasas de Tipo C/metabolismo , Sustitución de Aminoácidos , Animales , Señalización del Calcio/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Mutagénesis Sitio-Dirigida , Mutación Missense , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Canales Catiónicos TRPC/genética , Fosfolipasas de Tipo C/genéticaRESUMEN
Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2 , PIP2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes.
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Fenómenos Fisiológicos Celulares/genética , Activación del Canal Iónico/genética , Proteínas de la Membrana/genética , Fosfoinosítido Fosfatasas/genética , Canales Iónicos Sensibles al Ácido/genética , Animales , Exocitosis/genética , Humanos , Activación del Canal Iónico/fisiología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 4,5-Difosfato/genética , Dominios Proteicos/genéticaRESUMEN
A novel low-temperature regenerative hydroxy-functionalized hyperbranched polyamine adsorbent (0.16OH-HBPA) for CO2 capture was readily prepared using glutaraldehyde to cross-link amino-terminated hyperbranched polymers (HBP) and functionalized with glycidol, followed by the reduction of the imino groups of 0.16OH-HBPA to alkyl aminos using NaBH4. Here, the HBP has been prepared through the one-pot reaction between pentaethylenehexamine and methyl acrylate. The as-prepared 0.16OH-HBPA adsorbent showed a high adsorption capacity (4.05 mmol/g) for CO2 (concentration, 10%) in the presence of water at 25 °C, and the alkyl amino utilization efficiency reached 73%. More importantly, the CO2-adsorbed 0.16OH-HBPA showed excellent regenerative performance at low temperatures (85 °C, under pure CO2 gas) due to the introduced hydroxyl that can cooperatively adsorb CO2 via the amino groups to form stable carbamic acid. This process suppressed the formation of open-chain urea and cyclic urea and could overcome the disadvantages of high regeneration temperatures (≥90 °C, under pure inert gas) of CO2-adsorbed traditional solid amine adsorbents.
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OBJECTIVE: Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in apolipoprotein E null (ApoE-/-) mice. Because P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. APPROACH AND RESULTS: EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R floxed mice. Endothelial P2Y2R deficiency reduced endothelial nitric oxide synthase activity and significantly altered ATP- and UTP (uridine 5'-triphosphate)-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE-/- background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathologic examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration, and increased smooth muscle cell and collagen content, leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE-/- mice. Expression and proteolytic activity of matrix metalloproteinase-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE-/- mice. Furthermore, EC-specific P2Y2R deficiency inhibited nitric oxide production, leading to significant increase in smooth muscle cell migration out of aortic explants. CONCLUSIONS: EC-specific P2Y2R deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE-/- mice through impaired macrophage infiltration acting together with reduced matrix metalloproteinase-2 activity and increased smooth muscle cell migration.
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Aorta Torácica/metabolismo , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Placa Aterosclerótica , Receptores Purinérgicos P2Y2/deficiencia , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Movimiento Celular , Células Cultivadas , Colágeno/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Fibrosis , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/genética , Rotura Espontánea , Transducción de Señal , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Sphingomonads are isolated at exceptionally high frequency from organic polluted environments and assumed to be more hydrophobic than other Gram-negative bacteria. However, the potential roles of cell-surface hydrophobicity (CSH) in the cell survival in polluted environment, as well as the mechanisms underlying the CSH of sphingomonads, remain unclear. Sphingobium hydrophobicum C1T is a highly hydrophobic sphingomonad isolated from electronic-waste contaminated sediment. In this study, we found that exposure to the typical pollutants in electronic-waste contaminated sediment, such as the heavy metal ion Pb and the organic compound deca-brominated diphenyl ether (deca-BDE), resulted in the development of even higher CSH of the hydrophobic strain C1T; but no significant change was observed in the low CSH of its hydrophilic variant C2. The hydrophobic strain C1T achieved higher biomass yield in standing conditions and adsorbed more amounts of hydrophobic deca-BDE than its hydrophilic variant C2, suggesting that the high CSH potentially enhanced the adaptation of hydrophobic strain to colonize in sediment and adsorb hydrophobic nutrients. The identification of the bacterial cell-surface constituents showed that the high CSH of S. hydrophobicum was contributed greatly by outer-membrane proteins, particularly membrane transporters functioning as enhancers for nutrient uptake and stress sustainment. This study will enhance our understanding of the adaptive strategies of sphingomonads in contaminated environments. It will be of great importance to enhance the CSH of sphingomonads and utilize them in cleaning up the environment from organic pollution.
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Adaptación Biológica , Residuos Electrónicos , Interacciones Hidrofóbicas e Hidrofílicas , Sphingomonadaceae/fisiología , Propiedades de Superficie , Adsorción , Proteínas de la Membrana Bacteriana Externa/metabolismo , Microbiología Ambiental , Contaminantes Ambientales/metabolismo , Éteres Difenilos Halogenados/metabolismo , Plomo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Sphingomonadaceae/efectos de los fármacos , Sphingomonadaceae/aislamiento & purificaciónRESUMEN
The key component in bacteria-based biosensors is a transcriptional reporter employed to monitor induction or repression of a reporter gene corresponding to environmental change. In this study, we made a series of reporters in order to achieve highly sensitive detection of arsenite. From these reporters, two biosensors were developed by transformation of Escherichia coli DH5α with pLHPars9 and pLLPars9, consisting of either a high or low copy number plasmid, along with common elements of ArsR-luciferase fusion and addition of two binding sequences, one each from E. coli and Acidithiobacillus ferrooxidans chromosome, in front of the R773 ArsR operon. Both of them were highly sensitive to arsenite, with a low detection limit of 0.04 µM arsenite (~ 5 µg/L). They showed a wide dynamic range of detection up to 50 µM using high copy number pLHPars9 and 100 µM using low copy number pLLPars9. Significantly, they differ in metal specificity, pLLPars9 more specific to arsenite, while pLHPars9 to both arsenite and antimonite. The only difference between pLHPars9 and pLLPars9 is their copy numbers of plasmid and corresponding ratios of ArsR to its binding promoter/operator sequence. Therefore, we propose a working model in which DNA bound-ArsR is different from its free form in metal specificity.
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Arsenitos/metabolismo , Proteínas de Escherichia coli/genética , Dosificación de Gen , Genes Reporteros , Metales/metabolismo , Plásmidos/genética , Transactivadores/genética , Proteínas Bacterianas/genética , Técnicas Biosensibles , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras GenéticasRESUMEN
Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.
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Enfermedad de la Arteria Coronaria/prevención & control , Vasos Coronarios/efectos de los fármacos , Síndrome Metabólico/tratamiento farmacológico , Antagonistas de Receptores de Mineralocorticoides/administración & dosificación , Espironolactona/administración & dosificación , Canales Catiónicos TRPC/efectos de los fármacos , Canal Catiónico TRPC6/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/fisiopatología , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Esquema de Medicación , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Síndrome Metabólico/fisiopatología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Porcinos , Porcinos Enanos , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Two facultative chemolithotrophic, nitrate-reducing thiosulfate-oxidizing strains, F43bT and F21, were isolated from the sulfide-rich anoxic sediment of an urban creek in Pearl River Delta, China. Both strains were Gram-negative, facultatively anaerobic, non-spore-forming and rod-shaped with a flagellum. Phylogenetic analyses of 16S rRNA genes and the thrC, recA, glnII and atpD housekeeping genes revealed that the type strain shared high sequence similarities to Ciceribacter lividus MSSRFBL1T, with 98.8, 90.9, 94.8, 95.4 and 96.1â% identity, respectively. In addition, the major isoprenoid quinone (ubiquinone Q-10) and the DNA G+C content (66.0 mol%) of the type strain were similar to those of Ciceribacter lividus MSSRFBL1T. These results strongly support the classification of strains F43bT and F21 into the genus Ciceribacter. However, these strains diverged markedly from strain MSSRFBL1T with respect to several physiological and biochemical properties such as their semi-translucent colonies and nitrate-reducing and simultaneous thiosulfate-oxidizing respiration. Furthermore, the predominant fatty acids of strain F43bT were summed feature 2 (C18â:â1ω9t and/or C18â:â1ω9c and/or C18â:â1ω11t), C14â:â0 3-OH, C18â:â0 and C16â:â0, and its polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidymonomethylethanolamine and an unidentified glycolipid, which represented another two significant differences from strain MSSRFBL1T. Importantly, the DNA-DNA relatedness between strain F43bT and MSSRFBL1T was only 47.7â%. Based on the aforementioned polyphasic taxonomic results, the two isolates are suggested to represent a novel species of the genus Ciceribacter, for which the name Ciceribacterthiooxidans sp. nov. is proposed; the type strain is F43bT (=CCTCC AB 2016062T=KCTC 52231T).
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Sedimentos Geológicos/microbiología , Filogenia , Rhizobiaceae/clasificación , Ríos/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , China , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Rhizobiaceae/genética , Rhizobiaceae/aislamiento & purificación , Análisis de Secuencia de ADN , Tiosulfatos/metabolismo , Ubiquinona/químicaRESUMEN
Increasing evidence has shown that the reaction of zero-valent iron [Fe(0)] by oxygen can produce strong oxidants and rapidly oxidize the tractable contaminants. However, Fe(0) is vulnerable to passivation in the presence of oxygen, which significantly decreases its surface reactivity towards the removal of refractory contaminants. Microorganisms capable of reducing ferric iron in the presence of oxygen are expected to overcome the limitation of Fe(0) passivation. However, no studies to date have shown that microorganisms are able to depassivate Fe(0) for the removal of recalcitrant compounds in the presence of oxygen. In this study, we demonstrated that the carotenoid-producing Sphingobium hydrophobicum C1 was able to significantly enhance the removal of deca-brominated diphenyl ether by depassivating Fe(0) and subsequently removing the newly formed metabolites under semi-aerobic conditions (> 4 mg/L oxygen). S. hydrophobicum C1 effectively depassivated Fe(0) and regenerated its reactivity by reducing ferric iron under semi-aerobic conditions. Some unique characteristics of S. hydrophobicum C1, including the presence of membrane-integrated carotenoids and certain cell proteins, were essential for the ferric iron reduction of S. hydrophobicum C1 in the presence of oxygen. Our results may provide new insights into the bioremediation of persistent pollutants and will contribute to future studies to enhance our understanding of microbial iron reduction.