RESUMO
BACKGROUND & PROBLEMS: Patients with critical illnesses face an elevated risk of medical adhesive relation skin injuries (MARSI), which have negative, subsequent impacts on recovery and quality of healthcare. PURPOSE: The aim of this project was to decrease the incidence of MARSI in the surgical intensive care unit and to improve the accuracy of MARSI preventive care implementation. RESOLUTIONS: The intervention included the implementation of product-use cards, high-risk warning slogans, education programs, experience workshops, and a standard prevention-care protocol for MARSI. RESULTS: After project implementation, the incidence rate of MARSI decreased from 18.2% to 0%-9.3%, and the accuracy rate of preventive care increased from 38.6% to 95.5%. CONCLUSIONS: This project effectively reduced the incidence of skin injury and improved the quality of critical care. The skills related to the care and prevention of MARSI have been implemented throughout the hospital.
Assuntos
Adesivos , Dermatopatias , Adesivos/efeitos adversos , Cuidados Críticos , Humanos , Incidência , Unidades de Terapia Intensiva , Pele , Dermatopatias/cirurgiaRESUMO
BACKGROUND: There are limited effective drugs that can reach the brain to target brain tumors, in particular glioblastoma, which is one of the most difficult cancers to be cured from. Because the overexpression of the sigma-2 receptor is frequently reported in glioma clinical samples and associated with poor prognosis and malignancy, we herein studied the anti-tumor effect of the sigma-2 receptor agonist PB221 (4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperidine) on an anaplastic astrocytoma tumor model based on previous encouraging results in pancreatic cancer and neuroblastoma SK-N-SH cells. METHODS: The expression of the sigma-2 receptor, transmembrane protein 97 (TMEM97), in ALTS1C1 and UN-KC6141 cell lines was measured by RT-PCR and quantitative RT-PCR. The binding of sigma-2 receptor fluorescent ligands PB385 (6-[5-[3-(4-cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronaphthalen-5-yloxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine) and NO1 (2-{6-[2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one-5-yloxy]hexyl}-5-(dimethylamino)isoindoline-1,3-dione) was examined by flow cytometry and the fluorescent plate reader. The antitumor activity of PB221 was initially examined in the murine brain tumor cell line ALTS1C1 and then in the murine pancreatic cell line UN-KC6141. The potential therapeutic efficacy of PB221 for murine brain tumors was examined by in vitro migration and invasion assays and in vivo ectopic and orthotopic ALTS1C1 tumor models. RESULTS: The IC50 of PB221 for ALTS1C1 and UN-KC6141 cell lines was 10.61 ± 0.96 and 13.13 ± 1.15 µM, respectively. A low dose of PB221 (1 µM) significantly repressed the migration and invasion of ALTS1C1 cells, and a high dose of PB221 (20 µM) resulted in the apoptotic cell death of ALTS1C1 cells. These effects were reduced by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine, suggesting mitochondrial oxidative stress is involved. The in vivo study revealed that PB221 effectively retarded tumor growth to 36% of the control tumor volume in the ectopic intramuscular tumor model and increased the overall survival time by 20% (from 26 to 31 days) in the orthotopic intracerebral tumor model. CONCLUSIONS: This study demonstrates that the sigma-2 receptor agonist PB221 has the potential to be an alternative chemotherapeutic drug for brain tumors with comparable side effects as the current standard-of-care drug, temozolomide.
Assuntos
Astrocitoma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Membrana/genética , Naftalenos/farmacologia , Piperidinas/farmacologia , Tetra-Hidronaftalenos/farmacologia , Animais , Astrocitoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Masculino , Proteínas de Membrana/agonistas , Camundongos , Naftalenos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Piperidinas/uso terapêutico , Tetra-Hidronaftalenos/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The existence of a subsarcolemmal space with restricted diffusion for Na(+) in cardiac myocytes has been inferred from a transient peak electrogenic Na(+)-K(+) pump current beyond steady state on reexposure of myocytes to K(+) after a period of exposure to K(+)-free extracellular solution. The transient peak current is attributed to enhanced electrogenic pumping of Na(+) that accumulated in the diffusion-restricted space during pump inhibition in K(+)-free extracellular solution. However, there are no known physical barriers that account for such restricted Na(+) diffusion, and we examined if changes of activity of the Na(+)-K(+) pump itself cause the transient peak current. Reexposure to K(+) reproduced a transient current beyond steady state in voltage-clamped ventricular myocytes as reported by others. Persistence of it when the Na(+) concentration in patch pipette solutions perfusing the intracellular compartment was high and elimination of it with K(+)-free pipette solution could not be reconciled with restricted subsarcolemmal Na(+) diffusion. The pattern of the transient current early after pump activation was dependent on transmembrane Na(+)- and K(+) concentration gradients suggesting the currents were related to the conformational poise imposed on the pump. We examined if the currents might be accounted for by changes in glutathionylation of the ß1 Na(+)-K(+) pump subunit, a reversible oxidative modification that inhibits the pump. Susceptibility of the ß1 subunit to glutathionylation depends on the conformational poise of the Na(+)-K(+) pump, and glutathionylation with the pump stabilized in conformations equivalent to those expected to be imposed on voltage-clamped myocytes supported this hypothesis. So did elimination of the transient K(+)-induced peak Na(+)-K(+) pump current when we included glutaredoxin 1 in patch pipette solutions to reverse glutathionylation. We conclude that transient K(+)-induced peak Na(+)-K(+) pump current reflects the effect of conformation-dependent ß1 pump subunit glutathionylation, not restricted subsarcolemmal diffusion of Na(+).
Assuntos
Glutationa/metabolismo , Sarcolema/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Animais , Difusão , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Potássio/farmacologia , Conformação Proteica , Subunidades Proteicas/metabolismo , Coelhos , Sarcolema/efeitos dos fármacosRESUMO
FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na(+)/K(+)-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments. We measured FXYD3 expression, Na(+)/K(+)-ATPase activity and glutathionylation of the ß1 subunit of Na(+)/K(+)-ATPase, a reversible oxidative modification that inhibits the ATPase, in MCF-7 and MDA-MB-468 cells. Expression of FXYD3 was suppressed by transfection with FXYD3 siRNA. A colorimetric end-point assay was used to estimate cell viability. Apoptosis was estimated by caspase 3/7 (DEVDase) activation using a Caspase fluorogenic substrate kit. Expression of FXYD3 in MCF-7 breast cancer cells was ~eightfold and ~twofold higher than in non-cancer MCF-10A cells and MDA-MB-468 cancer cells, respectively. A ~50 % reduction in FXYD3 expression increased glutathionylation of the ß1 Na(+)/K(+)-ATPase subunit and reduced Na(+)/K(+)-ATPase activity by ~50 %, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the ß1 subunit of Na(+)/K(+)-ATPase and glutathionylation-induced inhibition of Na(+)/K(+)-ATPase. Treatment of MCF-7 and MDA-MB- 468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments upregulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na(+)/K(+)-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.
Assuntos
Neoplasias da Mama/genética , Sobrevivência Celular/genética , Doxorrubicina/farmacologia , Raios gama/uso terapêutico , Inativação Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , ATPase Trocadora de Sódio-Potássio/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Células MCF-7RESUMO
Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of ß3-adrenergic receptors (ß3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump ß1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump ß1-subunit and decrease in pump current. In vivo treatment with the ß3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump ß1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump ß1-subunit, which may reflect facilitation of deglutathionylation. In vivo ß3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes.
Assuntos
Agonistas de Receptores Adrenérgicos beta 3/uso terapêutico , Hiperglicemia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptor de Insulina/antagonistas & inibidores , Receptores Adrenérgicos beta 3/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Sequência de Aminoácidos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Dioxóis/farmacologia , Dioxóis/uso terapêutico , Hiperglicemia/tratamento farmacológico , Masculino , Dados de Sequência Molecular , Estresse Oxidativo/fisiologia , CoelhosRESUMO
Protein kinase C can activate NADPH oxidase and induce glutathionylation of the ß1-Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Coexposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between ß1-Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced ß1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47(phox) NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22(phox) subunit, and it decreased O2 (·-)-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47(phox) indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22(phox) and p47(phox) NADPH oxidase subunits and decrease ß1-Na(+)-K(+) pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in ß1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo.
Assuntos
Miócitos Cardíacos/enzimologia , Estresse Oxidativo/fisiologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , CoelhosRESUMO
BACKGROUND & PROBLEMS: Incontinence associated dermatitis (IAD) is a common skin disorder in critical patients who suffer from fecal incontinence. Symptoms of IAD include pain, redness, swelling, and, in some cases, secondary infections. IAD is thus a major problem faced in critical nursing care. The incidence of incontinence associated dermatitis averaged 34.72% at the intensive care unit in our hospital from October to December 2013. The factors that we identified as associated with IAD included: (1) Nurses: incorrect nursing care and insufficient IAD-related knowledge among nurses; (2) DEVICES: lack of skin barrier products and the use of diapers with poor air ventilation; (3) Regulations: lack of IAD care standards and lack of auditing oversight; (4) Patient problems: skin edema, incontinence, and medication use. PURPOSE: To decrease the incidence of IAD from 34.72% to 22%. METHODS: The authors searched the Cochrane, PubMed, CINAHI, and Nursing Reference Center databases for relevant articles that were published from 2000 to 2014. Fifty studies were identified and four evidence-based references were selected for follow-up assessment. Levels of evidence were at levels 2 and 3. We designed an intervention that: (1) set a high-risk notice to increase awareness; (2) held six in-service training programs; (3) developed nursing standards and designed e-learning education lessons that specifically targeted reducing the incidence of IAD; (4) established a regular audit system. RESULT: The incidence of IAD decreased from 34.72% pretest to 19.8% posttest (posttest period: April - October 2014). CONCLUSION: This project effectively reduced the incidence of IAD in high-risk patients in our intensive care unit. The authors established evidence-based interventions that significantly improved patient outcomes.
Assuntos
Dermatite/prevenção & controle , Diarreia/complicações , Incontinência Fecal/complicações , Dermatite/etiologia , Humanos , Incidência , Unidades de Terapia IntensivaRESUMO
Liver cirrhosis is a critical state in the natural course of hepatocellular carcinoma (HCC). We sought to investigate the potential of in-depth proteomics to reveal plasma protein signatures that reflect common networks/pathways of liver cirrhosis, and to determine whether the cirrhosis-related signature in plasma is linked to the development of HCC among hepatitis B virus (HBV) carriers. We first compared plasma protein profiles using a 174-antibody microarray system between three groups of HBV carriers with different Child's grades of cirrhosis, which revealed a panel of 45 differentially expressed proteins with a high accuracy for discriminating Child's B/C. Ingenuity Pathway Analysis identified two main up-regulated networks connecting the 45 proteins that were most enriched for genes in the pathway of hepatic stellate cell activation. A parsimonious subset of 11 pathway-based proteins was then selected for quantification to correlate with HCC risk among 49 HCC cases and 50 controls in a nested case-control study within a 16-yr follow-up cohort of HBV carriers. A high risk score derived from a principal component analysis, which was used to extract the cluster structure of the 11 proteins, was associated with HCC (odds ratio = 4.83, 95% confidence interval: 1.26-18.56) even after adjustment for viral and clinical variables, implying the involvement of a pattern of coordinated proteins. Stepwise logistic regression on the 11 proteins revealed ICAM-2 as an independent predictor for HCC. These findings may give further insight into the pathobiology of hepatocarcinogenesis, allow testing of the cirrhosis-related plasma protein signature as a potential predictive biomarker for HCC.
Assuntos
Carcinoma Hepatocelular/etiologia , Hepatite B/complicações , Cirrose Hepática/sangue , Cirrose Hepática/complicações , Neoplasias Hepáticas/etiologia , Adulto , Biomarcadores/sangue , Proteínas Sanguíneas , Carcinoma Hepatocelular/sangue , Estudos de Casos e Controles , Análise por Conglomerados , Hepatite B/sangue , Humanos , Neoplasias Hepáticas/sangue , Masculino , Pessoa de Meia-Idade , Prognóstico , Mapeamento de Interação de Proteínas , Proteômica/métodos , Risco , Transdução de SinaisRESUMO
BACKGROUND: Real-world vaccine effectiveness following the third dose of vaccination against SARS-CoV-2 remains less investigated among people with HIV (PWH). METHODS: PWH receiving the third dose of BNT162b2 and mRNA-1273 (either 50- or 100-µg) were enrolled. Participants were followed for 180 days until the fourth dose of COVID-19 vaccination, SARS-CoV-2 infection, seroconversion of anti-nucleocapsid IgG, death, or loss to follow-up. Anti-spike IgG was determined every 1-3 months. RESULTS: Of 1427 participants undergoing the third-dose COVID-19 vaccination, 632 (44.3%) received 100-µg mRNA-1273, 467 (32.8%) 50-µg mRNA-1273, and 328 (23.0%) BNT162b2 vaccine and the respective rate of SARS-CoV-2 infection or seroconversion of anti-nucleocapsid IgG was 246.1, 280.8 and 245.2 per 1000 person-months of follow-up (log-rank test, p = 0.28). Factors associated with achieving anti-S IgG titers >1047 BAU/mL included CD4 count <200 cells/mm3 (adjusted odds ratio [aOR], 0.11; 95% CI, 0.04-0.31), plasma HIV RNA >200 copies/mL (aOR, 0.27; 95% CI, 0.09-0.80), having achieved anti-spike IgG >141 BAU/mL within 3 months after primary vaccination (aOR, 3.69; 95% CI, 2.68-5.07), receiving BNT162b2 vaccine as the third dose (aOR, 0.20; 95% CI, 0.10-0.41; reference, 100-µg mRNA-1273), and having previously received two doses of mRNA vaccine in primary vaccination (aOR, 2.46; 95% CI, 1,75-3.45; reference, no exposure to mRNA vaccine). CONCLUSIONS: PWH receiving different types of the third dose of COVID-19 vaccine showed similar vaccine effectiveness against SARS-CoV-2 infection. An additional dose with 100-µg mRNA-1273 could generate a higher antibody response than with 50-µg mRNA-1273 and BNT162b2 vaccine.
Assuntos
Vacina de mRNA-1273 contra 2019-nCoV , Anticorpos Antivirais , Vacina BNT162 , Vacinas contra COVID-19 , COVID-19 , Infecções por HIV , Imunoglobulina G , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Masculino , Feminino , COVID-19/prevenção & controle , COVID-19/imunologia , Anticorpos Antivirais/sangue , Pessoa de Meia-Idade , Infecções por HIV/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacina BNT162/administração & dosagem , Vacina BNT162/imunologia , SARS-CoV-2/imunologia , Imunoglobulina G/sangue , Adulto , Vacina de mRNA-1273 contra 2019-nCoV/imunologia , Vacina de mRNA-1273 contra 2019-nCoV/administração & dosagem , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinação/métodos , Eficácia de Vacinas , SoroconversãoRESUMO
Whole-cell patch-clamp measurements of the current, Ip, produced by the Na(+),K(+)-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in Ip over the extracellular Na(+) concentration range 0-50 mM. This is not predicted by the classical Albers-Post scheme of the Na(+),K(+)-ATPase mechanism, where extracellular Na(+) should act as a competitive inhibitor of extracellular K(+) binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K(+) ions into the cytoplasm. The increase in Ip is consistent with Na(+) binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K(+) to the cytoplasm, E2(K(+))2 â E1 + 2K(+). At normal physiological concentrations of extracellular Na(+) of 140 mM, it is to be expected that binding of Na(+) to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme's ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and ß-subunits of the enzyme.
Assuntos
Sítio Alostérico , Miócitos Cardíacos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Potenciais de Ação , Regulação Alostérica , Animais , Miócitos Cardíacos/fisiologia , Potássio/metabolismo , Ligação Proteica , Coelhos , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
By the time it was appreciated that the positive inotropic effect of cardiac glycosides is due to inhibition of the membrane Na(+)-K(+) pump, glycosides had been used for treatment of heart failure on an empiric basis for ~200 years. The subsequent documentation of their lack of clinical efficacy and possible harmful effect largely coincided with the discovery that a raised Na(+) concentration in cardiac myocytes plays an important role in the electromechanical phenotype of heart failure syndromes. Consistent with this, efficacious pharmacological treatments for heart failure have been found to stimulate the Na(+)-K(+) pump, effectively the only export route for intracellular Na(+) in the heart failure. A paradigm has emerged that implicates pump inhibition in the raised Na(+) levels in heart failure. It invokes protein kinase-dependent activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and glutathionylation, a reversible oxidative modification, of the Na(+)-K(+) pump molecular complex that inhibits its activity. Since treatments of proven efficacy reverse the oxidative Na(+)-K(+) pump inhibition, the pump retains its status as a key pharmacological target in heart failure. Its role as a target is well integrated with the paradigms of neurohormonal abnormalities, raised myocardial oxidative stress and energy deficiency implicated in the pathophysiology of the failing heart. We propose that targeting oxidative inhibition of the pump is useful for the exploration of future treatment strategies. This article is part of a Special Issue entitled "Na(+)Regulation in Cardiac Myocytes".
Assuntos
Cardiotônicos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Cardiotônicos/uso terapêutico , Insuficiência Cardíaca/enzimologia , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Oxirredução , Fosfoproteínas/química , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
The widely reported stimulation of the cardiac Na(+)-K(+) pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na(+) levels in heart failure, yet blockade of the ß1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the ß1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the ß1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits and reduced myocardial O2(â¢-)-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the ß1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced ß1 subunit glutathionylation and increased Ip. The PKA-induced Na(+)-K(+) pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na(+) in the failing heart. This scheme is consistent with the efficacy of ß1 AR blockade in the treatment of heart failure.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Captopril/farmacologia , Colforsina/farmacologia , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Glutarredoxinas/metabolismo , Masculino , Metoprolol/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , NADPH Oxidases/metabolismo , Oxirredução , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/metabolismo , CoelhosRESUMO
Glutathionylation of cysteine 46 of the ß1 subunit of the Na(+)-K(+) pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K(+)·P(i) configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na(+)-K(+) pump cycle as described by the Albers-Post scheme. We measured ß1 subunit glutathionylation and function of Na(+)-K(+)-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na(+)-K(+)-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na(3) conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO(-)). Inhibition of Na(+)-K(+)-ATPase activity after exposure to ONOO(-) was greater when the enzyme had been in the E1Na(3) than the E2 conformation. We exposed myocytes to different extracellular K(+) concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K(+) concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO(-)-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na(+)-K(+) pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the ß1 subunit to glutathionylation depends on the conformational poise of the Na(+)-K(+) pump.
Assuntos
Glutationa/metabolismo , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Angiotensina II/farmacologia , Angiotensina II/fisiologia , Animais , Glutarredoxinas/metabolismo , Glutarredoxinas/fisiologia , Histidina/química , Imunoprecipitação , Rim/citologia , Masculino , Potenciais da Membrana , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Modelos Moleculares , Células Musculares/metabolismo , Oxirredução , Estresse Oxidativo , Técnicas de Patch-Clamp , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Potássio/farmacologia , Potássio/fisiologia , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Proteólise , Coelhos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , Suínos , Tripsina/químicaRESUMO
Inflammatory response plays an important role in myocardial ischaemia-reperfusion (IR) injury. Up-regulation of vascular cell adhesion molecule-1 (VCAM) contributes to this. We examined the feasibility of using intravenously administered VCAM-MPIO (microparticle iron oxide) to characterize VCAM expression patterns in myocardial IR injury. Myocardial ischemia was simulated by 30 min of transient ligation of the left coronary vessel in rats. Purified, monoclonal, rat-specific, mouse VCAM antibody coupled to MPIO was administered through the tail vein at 3 h post reperfusion and the rats were sacrificed 1 h later. High resolution 3D ex vivo MRI images were acquired at 9.4 Tesla. Extensive foci of signal voids were observed on T2*-weighted gradient-echo sequences, which corresponded to focal deposits of MPIOs observed in histological sections. The spatial density of the signal voids (expressed as a percentage of pixels below a threshold value) was increased in the peri-infarct zone compared with non-infarct zone (32.5 ± 4% vs. 13.9 ± 5%; n = 6; p < 0.05) and was substantially greater than the signal loss due to non-specific binding seen in rats administered IgG control MPIO (2.0 ± 1%; n = 6; p < 0.05). The VCAM-specific MPIO signal was also seen in myocardium and pericardium in segments remote from the IR injury, but not in rats undergoing a sham operation. In conclusion, molecular imaging in a model of myocardial IR injury is possible using high field MRI and VCAM-MPIOs and may provide novel insights beyond those achieved by standard histological and molecular analysis.
Assuntos
Traumatismo por Reperfusão Miocárdica/patologia , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Anticorpos , Técnicas de Imagem Cardíaca , Compostos Férricos , Imageamento por Ressonância Magnética , Ratos , Ratos Sprague-DawleyRESUMO
Non-small cell lung cancer (NSCLC) is associated with a poor survival rate, even for patients with early-stage cancer. Identifying patients with pathological N0 NSCLC who may benefit from adjuvant chemotherapy treatment after surgery is essential. We conducted a retrospective cohort study used data from the Surveillance, Epidemiology, and End Results database and included 26,380 patients with pathological N0 NSCLC after surgery between January 2018, and December 2019. Among 26,380 patients, 24,273 patients received surgery alone and the other 2107 patients received surgery plus adjuvant chemotherapy. After 1:1 propensity score matching, both groups contained 2107 patients. Adjuvant chemotherapy did not show significantly better 24-month survival in T2aN0 NSCLC patients (83.41% vs. 82.91%, p = 0.067), although it did for T2bN0 patients (86.36% vs. 81.70%, p = 0.028). Poorly-differentiated NSCLC remained a high-risk factor for pT2N0, and adjuvant chemotherapy provided better 24-month survival after matching (86.36% vs. 81.70%, p = 0.029). In conclusion, when treating pN0 NSCLC, adjuvant chemotherapy had a beneficial effect when the tumor size was larger than 4 cm. The effect when the tumor size was between 3 and 4 cm was not remarkable. Poorly-differentiated NSCLC was a high-risk factor in the pT2N0 stage.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Estudos Retrospectivos , Estadiamento de Neoplasias , Quimioterapia Adjuvante/métodosRESUMO
BACKGROUND: Volume doubling time (VDT) has been proven to be a powerful predictor of lung cancer progression. In non-small cell lung cancer patients receiving sublobar resection, the discussion of correlation between VDT and surgery was absent. We proposed to investigate the surgical outcomes according to VDT. METHODS: We retrospectively studied 96 cases post sublobar resection from 2012 to 2018, collecting two chest CT scans preoperatively of each case and calculating the VDT. The receiver operating characteristic curve was constructed to identify the optimal cut-off point of VDTs as 133 days. We divided patients into two groups: VDT < 133 days and VDT ≥ 133 days. Univariable and multivariable analyses were performed for comparative purposes. RESULTS: Univariable and multivariable analyses revealed that the consolidation and tumor diameter ratio was the factor of overall survival (OS), and VDT was the only factor of disease-free survival (DFS). The five year OS rates of patients with VDTs ≥ 133 days and VDTs < 133 days, respectively, were 89.9% and 71.9%, and the five year DFS rates were 95.9% and 61.5%. CONCLUSION: As VDT serves as a powerful prognostic predictor and provides an essential role in planning surgical procedures, the evaluation of VDT preoperatively is highly suggested.
RESUMO
Efficacy of therapies that target the downstream nitric oxide (NO) pathway in pulmonary arterial hypertension (PAH) depends on the bioavailability of NO. Reduced NO level in PAH is secondary to "uncoupling" of endothelial nitric oxide synthase (eNOS). Stimulation of ß3 adrenergic receptors (ß3 ARs) may lead to the recoupling of NOS and therefore be beneficial in PAH. We aimed to examine the efficacy of ß3 AR agonism as a novel pathway in experimental PAH. In hypoxia (5 weeks) and Sugen hypoxia (hypoxia for 5 weeks + SU5416 injection) models of PAH, we examined the effects of the selective ß3 AR agonist CL316243. We measured echocardiographic indices and invasive right ventricular (RV)-pulmonary arterial (PA) hemodynamics and compared CL316243 with riociguat and sildenafil. We assessed treatment effects on RV-PA remodeling, oxidative stress, and eNOS glutathionylation, an oxidative modification that uncouples eNOS. Compared with normoxic mice, RV systolic pressure was increased in the control hypoxic mice (p < 0.0001) and Sugen hypoxic mice (p < 0.0001). CL316243 reduced RV systolic pressure, to a similar degree to riociguat and sildenafil, in both hypoxia (p < 0.0001) and Sugen hypoxia models (p < 0.03). CL316243 reversed pulmonary vascular remodeling, decreased RV afterload, improved RV-PA coupling efficiency and reduced RV stiffness, hypertrophy, and fibrosis. Although all treatments decreased oxidative stress, CL316243 significantly reduced eNOS glutathionylation. ß3 AR stimulation improved RV hemodynamics and led to beneficial RV-PA remodeling in experimental models of PAH. ß3 AR agonists may be effective therapies in PAH.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Camundongos , Animais , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Pulmonar/metabolismo , Citrato de Sildenafila/farmacologia , Citrato de Sildenafila/uso terapêutico , Artéria Pulmonar/metabolismo , Hemodinâmica , Agonistas Adrenérgicos beta/farmacologia , HipóxiaRESUMO
The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Proteínas de Membrana , Proteínas de Neoplasias/genéticaRESUMO
The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump ß(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that ß(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the ß(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the ß(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.
Assuntos
Glutationa/metabolismo , Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico/fisiologia , Células Cultivadas , Glutationa/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Miócitos Cardíacos/citologia , Proteínas de Neoplasias/genética , Oxirredução , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Coelhos , ATPase Trocadora de Sódio-Potássio/genética , XenopusRESUMO
BACKGROUND: Preeclampsia is a multifactorial cardiovascular disorder of pregnancy. If left untreated, it can lead to severe maternal and fetal outcomes. Hence, timely diagnosis and management of preeclampsia are extremely important. Biomarkers of oxidative stress are associated with the pathogenesis of preeclampsia and therefore could be indicative of evolving preeclampsia and utilized for timely diagnosis. In this study, we conducted a systematic review and meta-analysis to determine the most reliable oxidative stress biomarkers in preeclampsia, based on their diagnostic sensitivities and specificities as well as their positive and negative predictive values. METHODS: A systematic search using PubMed, ScienceDirect, ResearchGate, and PLOS databases (1900 to March 2021) identified nine relevant studies including a total of 343 women with preeclampsia and 354 normotensive controls. RESULTS: Ischemia-modified albumin (IMA), uric acid (UA), and malondialdehyde (MDA) were associated with 3.38 (95% CI 2.23, 4.53), 3.05 (95% CI 2.39, 3.71), and 2.37 (95% CI 1.03, 3.70) odds ratios for preeclampsia diagnosis, respectively. The IMA showed the most promising diagnostic potential with the positive predictive ratio (PPV) of 0.852 (95% CI 0.728, 0.929) and negative predictive ratio (NPV) of 0.811 (95% CI 0.683, 0.890) for preeclampsia. Minor between-study heterogeneity was reported for these biomarkers (Higgins' I2 = 0-15.879%). CONCLUSIONS: This systematic review and meta-analysis identified IMA, UA, and MDA as the most promising oxidative stress biomarkers associated with established preeclampsia. IMA as a biomarker of tissue damage exhibited the best diagnostic test accuracy. Thus, these oxidative stress biomarkers should be further explored in larger cohorts for preeclampsia diagnosis.