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1.
Front Physiol ; 13: 947537, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35991176

RESUMO

In acute lung injury (ALI), the NF-κB-mediated downregulation of Sox18 gene expression leads to the disruption of the pulmonary endothelial barrier. Previous studies have suggested that the action of NF-κB as a transcriptional repressor also requires the action of class I histone deacetylases (HDACs). Thus, the purpose of this study was to investigate and further delineate the mechanism of Sox18 repression during lipopolysaccharide (LPS) induced ALI. Using selective inhibitors and specific siRNA-driven depletion of HDACs 1-3 in human lung microvascular endothelial cells (HLMVEC) we were able to demonstrate a critical role for HDACs 1 and 2 in the LPS-mediated repression of Sox18 gene expression and the loss of endothelial monolayer integrity. Moreover, our data demonstrate that HDAC1 associates with a transcription-repressive complex within the NF-κB-binding site of Sox18 promoter. Further, we were able to show that the selective inhibitor of HDAC1, tacedinaline, significantly reduced the endothelial permeability and injury associated with LPS challenge in the mouse lung. Taken together, our data demonstrate, for the first time, that transcription repressors HDACs 1 and 2 are involved in pathological mechanism of ALI and can be considered as therapeutic targets.

2.
Front Physiol ; 13: 1066515, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36620216

RESUMO

Mechanical strain contributes to ventilator-induced lung injury (VILI) through multi-factorial and complex mechanisms that remain unresolved. Prevailing evidence suggests that the loss of pulmonary endothelial tight junctions (TJs) plays a critical role. TJs are dynamically regulated by physiologic and hemodynamic forces to stabilize the endothelial barrier. The transcription factor sex-determining region Y-box (SOX)-18 is important in regulating blood vessel development and vascular permeability through its ability to regulate the transcription of Claudin-5, an endothelial TJ protein. Previously, we demonstrated that SOX18 expression is increased by shear stress in the pulmonary endothelium. Therefore, in this study, we investigated how mechanical strain mediated through cyclic stretch affects the SOX18/Claudin-5 regulatory axis. Our data demonstrate that SOX18 and Claudin-5 are downregulated in human lung microvascular endothelial cells (HLMVEC) exposed to cyclic stretch and the mouse lung exposed to high tidal mechanical ventilation. Overexpression of SOX18 reduced the loss of Claudin-5 expression in HLMVEC with cyclic stretch and preserved endothelial barrier function. Additionally, overexpression of Claudin-5 in HLMVEC ameliorated barrier dysfunction in HLMVEC exposed to cyclic stretch, although SOX18 expression was not enhanced. Finally, we found that the targeted overexpression of SOX18 in the pulmonary vasculature preserved Claudin-5 expression in the lungs of mice exposed to HTV. This, in turn reduced lung vascular leak, attenuated inflammatory lung injury, and preserved lung function. Together, these data suggest that enhancing SOX18 expression may prove a useful therapy to treat patients with ventilator-induced lung injury.

3.
Sci Rep ; 10(1): 18078, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093565

RESUMO

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, disrupts the alveolar-capillary barrier, triggering pulmonary vascular leak thus inducing acute lung injury (ALI). Extracellular purines, adenosine and ATP, protected against ALI induced by purified LPS. In this study, we investigated whether these purines can impact vascular injury in more clinically-relevant E.coli (non-sterile LPS) murine ALI model. Mice were inoculated with live E. coli intratracheally (i.t.) with or without adenosine or a non-hydrolyzable ATP analog, adenosine 5'-(γ-thio)-triphosphate (ATPγS) added intravenously (i.v.). After 24 h of E. coli treatment, we found that injections of either adenosine or ATPγS 15 min prior or adenosine 3 h after E.coli insult significantly attenuated the E.coli-mediated increase in inflammatory responses. Furthermore, adenosine prevented weight loss, tachycardia, and compromised lung function in E. coli-exposed mice. Accordingly, treatment with adenosine or ATPγS increased oxygen saturation and reduced histopathological signs of lung injury in mice exposed to E. coli. Lastly, lung-targeting gene delivery of adenosine or ATPγS downstream effector, myosin phosphatase, significantly attenuated the E. coli-induced compromise of lung function. Collectively, our study has demonstrated that adenosine or ATPγS mitigates E. coli-induced ALI in mice and may be useful as an adjuvant therapy in future pre-clinical studies.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Trifosfato de Adenosina/análogos & derivados , Adenosina/farmacologia , Escherichia coli/patogenicidade , Pneumonia Bacteriana/complicações , Vasodilatadores/farmacologia , Lesão Pulmonar Aguda/etiologia , Trifosfato de Adenosina/farmacologia , Marcadores de Afinidade/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
Am J Respir Cell Mol Biol ; 58(5): 614-624, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29115856

RESUMO

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Lipopolissacarídeos , Pulmão/irrigação sanguínea , NF-kappa B/metabolismo , Edema Pulmonar/metabolismo , Fatores de Transcrição SOXF/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Sítios de Ligação , Células Cultivadas , Claudina-5/genética , Claudina-5/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Ácido Peroxinitroso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/genética , Edema Pulmonar/patologia , Fatores de Transcrição SOXF/genética , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
5.
Am J Physiol Lung Cell Mol Physiol ; 313(2): L218-L229, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28495854

RESUMO

Tuberculosis (TB) is one of the oldest known human diseases and is transmitted by the bacteria Mycobacterium tuberculosis (Mtb). TB has a rich history with evidence of TB infections dating back to 5,800 bc TB is unique in its ability to remain latent in an individual for decades, with the possibility of later reactivation, causing widespread systemic symptoms. Currently, it is estimated that more than one-third of the world's population (~2 billion people) are infected with Mtb. Prolonged periods of therapy and complexity of treatment regimens, especially in active infection, have led to poor compliance in patients being treated for TB. Therefore, it is vitally important to have a thorough knowledge of the pathophysiology of Mtb to understand the disease progression, as well as to develop novel diagnostic tests and treatments. Alveolar macrophages represent both the primary host cell and the first line of defense against the Mtb infection. Apoptosis and autophagy of macrophages play a vital role in the pathogenesis and also in the host defense against Mtb. This review will outline the role of these two cellular processes in defense against Mtb with particular emphasis on innate immunity and explore developing therapies aimed at altering host responses to the disease.


Assuntos
Apoptose/imunologia , Autofagia/imunologia , Tuberculose/imunologia , Animais , Humanos , Imunidade Celular/imunologia , Imunidade Inata/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia
6.
PLoS One ; 10(3): e0119918, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25786132

RESUMO

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Lipopolissacarídeos/efeitos adversos , Óxido Nítrico Sintase Tipo III/deficiência , Lesão Pulmonar Aguda/induzido quimicamente , Amidoidrolases/metabolismo , Animais , Citocinas/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Knockout , Testes de Função Respiratória , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP
7.
J Cell Physiol ; 229(11): 1802-16, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24677020

RESUMO

Shear stress secondary to increased pulmonary blood flow (PBF) is elevated in some children born with congenital cardiac abnormalities. However, the majority of these patients do not develop pulmonary edema, despite high levels of permeability inducing factors. Previous studies have suggested that laminar fluid shear stress can enhance pulmonary vascular barrier integrity. However, little is known about the mechanisms by which this occurs. Using microarray analysis, we have previously shown that Sox18, a transcription factor involved in blood vessel development and endothelial barrier integrity, is up-regulated in an ovine model of congenital heart disease with increased PBF (shunt). By subjecting ovine pulmonary arterial endothelial cells (PAEC) to laminar flow (20 dyn/cm(2) ), we identified an increase in trans-endothelial resistance (TER) across the PAEC monolayer that correlated with an increase in Sox18 expression. Further, the TER was also enhanced when Sox18 was over-expressed and attenuated when Sox18 expression was reduced, suggesting that Sox18 maintains the endothelial barrier integrity in response to shear stress. Further, we found that shear stress up-regulates the cellular tight junction protein, Claudin-5, in a Sox18 dependent manner, and Claudin-5 depletion abolished the Sox18 mediated increase in TER in response to shear stress. Finally, utilizing peripheral lung tissue of 4 week old shunt lambs with increased PBF, we found that both Sox18 and Claudin-5 mRNA and protein levels were elevated. In conclusion, these novel findings suggest that increased laminar flow protects endothelial barrier function via Sox18 dependent up-regulation of Claudin-5 expression.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Pulmão/fisiopatologia , Fatores de Transcrição SOXF/metabolismo , Resistência ao Cisalhamento , Estresse Mecânico , Animais , Proliferação de Células , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Artéria Pulmonar/patologia , Ovinos , Proteínas de Junções Íntimas/metabolismo , Regulação para Cima
8.
J Biol Chem ; 289(11): 7948-61, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24469460

RESUMO

The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [(3)H]cGMP binding assay. Our study shows that the nitration of Tyr(247) and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , GMP Cíclico/química , Miócitos de Músculo Liso/metabolismo , Nitrogênio/química , Tirosina/química , Adulto , Animais , Aorta/citologia , Doenças Cardiovasculares/metabolismo , Domínio Catalítico , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Modelos Moleculares , Ácido Peroxinitroso/química , Ligação Proteica , Ovinos , Adulto Jovem
9.
Am J Respir Cell Mol Biol ; 50(3): 614-25, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24134589

RESUMO

Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA), together with exaggerated oxidative/nitrative stress, contributes to the development of ALI in mice exposed to LPS. Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown, and was the focus of this study. In human lung microvascular endothelial cells, DDAH II overexpression prevented the LPS-dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also attenuated the endothelial barrier disruption associated with LPS exposure. Similarly, in vivo, we demonstrated that the targeted overexpression of DDAH II in the pulmonary vasculature significantly inhibited the accumulation of ADMA and the subsequent increase in oxidative/nitrative stress in the lungs of mice exposed to LPS. In addition, augmenting pulmonary DDAH II activity before LPS exposure reduced lung vascular leak and lung injury and restored lung function when DDAH activity was increased after injury. Together, these data suggest that enhancing DDAH II activity may prove a useful adjuvant therapy to treat patients with ALI.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Amidoidrolases/metabolismo , Células Endoteliais/enzimologia , Terapia Genética , Lipopolissacarídeos , Pulmão/irrigação sanguínea , Microvasos/enzimologia , Edema Pulmonar/prevenção & controle , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/genética , Amidoidrolases/genética , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Líquido da Lavagem Broncoalveolar/química , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Humanos , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Estresse Oxidativo , Ácido Peroxinitroso/metabolismo , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/enzimologia , Edema Pulmonar/genética , Superóxidos/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima
10.
Compr Physiol ; 3(3): 1011-34, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23897679

RESUMO

The pathogenesis of pulmonary hypertension is a complex multifactorial process that involves the remodeling of pulmonary arteries. This remodeling process encompasses concentric medial thickening of small arterioles, neomuscularization of previously nonmuscular capillary-like vessels, and structural wall changes in larger pulmonary arteries. The pulmonary arterial muscularization is characterized by vascular smooth muscle cell hyperplasia and hypertrophy. In addition, in uncontrolled pulmonary hypertension, the clonal expansion of apoptosis-resistant endothelial cells leads to the formation of plexiform lesions. Based upon a large number of studies in animal models, the three major stimuli that drive the vascular remodeling process are inflammation, shear stress, and hypoxia. Although, the precise mechanisms by which these stimuli impair pulmonary vascular function and structure are unknown, reactive oxygen species (ROS)-mediated oxidative damage appears to play an important role. ROS are highly reactive due to their unpaired valence shell electron. Oxidative damage occurs when the production of ROS exceeds the quenching capacity of the antioxidant mechanisms of the cell. ROS can be produced from complexes in the cell membrane (nicotinamide adenine dinucleotide phosphate-oxidase), cellular organelles (peroxisomes and mitochondria), and in the cytoplasm (xanthine oxidase). Furthermore, low levels of tetrahydrobiopterin (BH4) and L-arginine the rate limiting cofactor and substrate for endothelial nitric oxide synthase (eNOS), can cause the uncoupling of eNOS, resulting in decreased NO production and increased ROS production. This review will focus on the ROS generation systems, scavenger antioxidants, and oxidative stress associated alterations in vascular remodeling in pulmonary hypertension.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Antioxidantes/uso terapêutico , Modelos Animais de Doenças , Sequestradores de Radicais Livres/metabolismo , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Estresse Oxidativo/fisiologia
11.
Protein Expr Purif ; 79(2): 271-6, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21600289

RESUMO

3',5' Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10µM) and varying ATP concentrations, PKG-1α had a maximal velocity (V(max)) of 5.02±0.25pmol/min/µg and a Michaelis-Menten constant (K(m)) of 11.78±2.68µM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.


Assuntos
Clonagem Molecular/métodos , Proteínas Quinases Dependentes de GMP Cíclico , GMP Cíclico/farmacologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteínas Recombinantes de Fusão , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Células Cultivadas , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/isolamento & purificação , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Ativação Enzimática , Escherichia coli , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico/farmacologia , Estresse Oxidativo , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transformação Bacteriana
12.
J Photochem Photobiol B ; 104(1-2): 154-7, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21393014

RESUMO

The redox potential of Q(A) in Photosystem II (PSII) from Thermosynechococcus elongatus was titrated monitoring chlorophyll fluorescence. A high potential form (E(m)=+60 ± 25 mV) was found in the absence of Mn(4)Ca, the active site for water oxidation. The low potential form (E(m)=-60 ± 48 mV), which is difficult to measure in conventional titration experiments, could be "locked in" by cross-linking the active enzyme. This indicates that the presence of Mn(4)Ca is relayed to the quinone site by significant structural changes in the protein. The presence of high and low potential forms agrees with what has been seen in plants, algae from our lab and in T. elongatus (Shibamoto et al., Biochemistry 48 (2009) 10682-10684). In the latter work, the potentials of Q(A) were shifted to lower potentials compared to other measurements. The redox potential of Q(A) in Mn-depleted PSII from spinach was titrated in the presence of redox mediators and the midpoint potential was shifted by 80 mV towards a more negative value compared to titrations without mediators. The lower values of the midpoint potential of the (Q(A)/Q(A)(-)) redox couple in the literature could be due to a perturbation due to a specific mediator.


Assuntos
Cianobactérias/enzimologia , Complexo de Proteína do Fotossistema II/fisiologia , Quinonas/química , Spinacia oleracea/enzimologia , Clorofila/química , Elétrons , Oxirredução , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Espectrometria de Fluorescência
13.
J Cell Physiol ; 226(12): 3104-13, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21351102

RESUMO

Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO(-) ) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO(-) donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24 h (with exogenous activation of cGMP) revealed increased ONOO(-) levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO(-) and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hipertensão Pulmonar/enzimologia , Óxido Nítrico/metabolismo , Artéria Pulmonar/enzimologia , Sistemas do Segundo Mensageiro , Vasodilatação , Administração por Inalação , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Ativação Enzimática , Sequestradores de Radicais Livres/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Metaloporfirinas/farmacologia , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Óxido Nítrico/administração & dosagem , Doadores de Óxido Nítrico/farmacologia , Ácido Peroxinitroso/metabolismo , Polietilenoglicóis/farmacologia , Processamento de Proteína Pós-Traducional , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Circulação Pulmonar , Ovinos , Espermina/análogos & derivados , Espermina/farmacologia , Superóxido Dismutase/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
14.
Physiol Plant ; 138(4): 463-73, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19947963

RESUMO

The function of cytochrome b559 (cyt b559) in photosystem II (PSII) was studied in a tobacco mutant in which the conserved phenylalanine at position 26 in the beta-subunit was changed to serine. Young leaves of the mutant showed no significant difference in chloroplast ultra structure or in the amount and activity of PSII, while in mature leaves the size of the grana stacks and the amount of PSII were significantly reduced. Mature leaves of the mutant showed a higher susceptibility to photoinhibition and a higher production of singlet oxygen, as shown by spin trapping electron paramagnetic resonance (EPR) spectroscopy. Oxygen consumption and superoxide production were studied in thylakoid membranes in which the Mn cluster was removed to ensure that all the cyt b559 was present in its low potential form. In thylakoid membranes, from wild-type plants, the larger fraction of superoxide production was 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive. This type of superoxide formation was absent in thylakoid membranes from the mutant. The physiological importance of the plastoquinol oxidation by cyt b559 for photosynthesis is discussed.


Assuntos
Grupo dos Citocromos b/metabolismo , Oxirredutases/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Plastoquinona/análogos & derivados , Grupo dos Citocromos b/genética , Espectroscopia de Ressonância de Spin Eletrônica , Fluorescência , Luz , Microscopia Eletrônica , Mutação , Oxirredução/efeitos da radiação , Oxirredutases/genética , Oxigênio/metabolismo , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastoquinona/metabolismo , Oxigênio Singlete/metabolismo , Tilacoides/enzimologia , Tilacoides/efeitos da radiação , Tilacoides/ultraestrutura , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/metabolismo
15.
J Biol Chem ; 284(45): 31174-80, 2009 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-19740740

RESUMO

Photoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b(6)f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant system.


Assuntos
Proteínas de Arabidopsis/genética , Expressão Gênica , Nicotiana/metabolismo , Estresse Oxidativo , Oxirredutases/genética , Proteínas de Arabidopsis/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Expressão Gênica/efeitos da radiação , Luz , Estresse Oxidativo/efeitos da radiação , Oxirredutases/metabolismo , Superóxidos/metabolismo , Tilacoides/genética , Tilacoides/metabolismo , Tilacoides/efeitos da radiação , Nicotiana/química , Nicotiana/genética , Nicotiana/efeitos da radiação
16.
J Parasitol ; 95(5): 1054-61, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19358622

RESUMO

The influence of ontological diet shifts on infracommunity nestedness was examined by comparing the infracommunity structure of Lepomis gulosus and Lepomis macrochirus at 2 localities in Par Pond, South Carolina, United States. Fill-constrained, occurrence-constrained, and abundance-constrained null models were used to evaluate the degree of nestedness. The presence-absence matrices from all 4 component communities had significantly fewer discrepancies than those produced by the fill-constrained model, and none had significantly fewer discrepancies than those produced by the occurrence-constrained model. Only the presence-absence matrix for the infracommunities of L. gulosus from the Cold Dam locality had significantly fewer discrepancies than those produced by the abundance-constrained null model. The nestedness of the 4 samples could not be distinguished from that expected under a hypothesis of passive sampling. A positive correlation between host size and total parasite abundance indicates the passive mechanism has a deterministic basis. Thus, even in the absence of habitat or diet shifts, nestedness can arise in infracommunities of freshwater fishes when older, larger fish have sampled more individuals from an uneven distribution of infective stages.


Assuntos
Doenças dos Peixes/parasitologia , Helmintíase Animal/parasitologia , Helmintos/classificação , Perciformes/parasitologia , Animais , Dieta/veterinária , Doenças dos Peixes/epidemiologia , Água Doce , Helmintíase Animal/epidemiologia , Helmintos/crescimento & desenvolvimento , Interações Hospedeiro-Parasita , Perciformes/fisiologia , South Carolina/epidemiologia
17.
J Biol Chem ; 282(17): 12492-502, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17327225

RESUMO

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, Q(A), in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of Q(A) by approximately -60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of Q(A). Thus the influence of the redox potential of Q(A) on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving Q(A) was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of Q(A) causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of Q(A). The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Elétrons , Complexo de Proteína do Fotossistema II/metabolismo , Plastoquinona/metabolismo , Oxigênio Singlete/metabolismo , Proteínas de Bactérias/genética , Cianobactérias/genética , Transporte de Elétrons/fisiologia , Cinética , Mutação de Sentido Incorreto , Oxirredução , Complexo de Proteína do Fotossistema II/genética
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