ABSTRACT
BACKGROUND: Ischemia/reperfusion injury (IRI) has been shown to cause endothelial glycocalyx (EG) damage.Whether the hypoxic/ischemic insult or the oxidative and inflammatory stress of reperfusion plays a greater part in glycocalyx damage is not known. Furthermore, the mechanisms by which IRI causes EG damage have not been fully elucidated. The aims of this study were to determine if hypoxia alone or hypoxia/reoxygenation (H/R) caused greater damage to the glycocalyx, and if this damage was mediated by reactive oxygen species (ROS) and Ca signaling. METHODS: Human umbilical vein endothelial cells were cultured to confluence and exposed to either normoxia (30 minutes), hypoxia (2% O2 for 30 minutes), or H/R (30 minutes hypoxia followed by 30 minutes normoxia). Some cells were pretreated with ROS scavengers TEMPOL, MitoTEMPOL, Febuxostat, or Apocynin, or with the Ca chelator BAPTA or Ca channel blockers 2-aminoethoxydiphenyl borate, A967079, Pyr3, or ML204. Intracellular ROS was quantified for all groups. Endothelial glycocalyx was measured using fluorescently tagged wheat germ agglutinin and imaged with fluorescence microscopy. RESULTS: Glycocalyx thickness was decreased in both hypoxia and H/R groups, with the decrease being greater in the H/R group. TEMPOL, MitoTEMPOL, BAPTA, and 2-aminoethoxydiphenyl borate prevented loss of glycocalyx in H/R. The ROS levels were likewise elevated compared with normoxia in both groups, but were increased in the H/R group compared with hypoxia alone. BAPTA did not prevent ROS production in either group. CONCLUSION: In our cellular model for shock, we demonstrate that although hypoxia alone is sufficient to produce glycocalyx loss, H/R causes a greater decrease in glycocalyx thickness. Under both conditions damage is dependent on ROS and Ca signaling. Notably, we found that ROS are generated upstream of Ca, but that ROS-mediated damage to the glycocalyx is dependent on Ca.
Subject(s)
Calcium Signaling/physiology , Endothelium/pathology , Glycocalyx/pathology , Reperfusion Injury/physiopathology , Shock/physiopathology , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Calcium Signaling/drug effects , Cell Hypoxia/physiology , Cell Line , Chelating Agents/pharmacology , Endothelium/cytology , Free Radical Scavengers/pharmacology , Human Umbilical Vein Endothelial Cells , Humans , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Reperfusion Injury/pathology , Shock/pathologyABSTRACT
Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.
Subject(s)
Enzyme Inhibitors/pharmacology , Thiolester Hydrolases/antagonists & inhibitors , Amino Acid Sequence , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Protein Conformation, alpha-Helical/drug effects , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/metabolismABSTRACT
Introducción. La contaminación en el hogar puede desempeñar un papel importante en el desarrollo y exacerbación del asma infantil, sobre todo entre niños genéticamente predispuestos. Objetivos. Evaluar la asociación entre la presencia de contaminantes biológicos intradomiciliarios y síntomas indicativos de asma, en preescolares de Bucaramanga, Colombia. Materiales y métodos. Se hizo un estudio de corte transversal analítico, en menores de 7 años, en dos zonas de la ciudad con niveles diferentes de contaminación atmosférica por material particulado. Se evaluaron los síntomas respiratorios indicativos de asma y los contaminantes intradomiciliarios mediante cuestionarios previamente validados, las mediciones biológicas de ácaros y hongos por métodos de laboratorio estandarizados, y se usó el modelo log binomial para el análisis multivariado, que permitió evaluar asociaciones mediante la estimación de las razones de prevalencia. Resultados. Participaron 678 niños, con edad media de 42 meses. La prevalencia de síntomas respiratorios indicativos de asma fue 8,0 % (IC95%: 5,6-9,6), sin diferencias significativas entre las dos zonas de contaminación extradomiciliaria. El modelo binomial mostró que los síntomas de asma se asociaron con la presencia de: ácaros [razón de prevalencia (RP)=1,78; IC95%: 1,0-3,0] y Acremonium sp. (RP=6,2; IC95%: 3,8-10,0), y antecedentes de neumonía (RP=4,0; IC95%: 2,5-6,4), de rinitis alérgica (RP=1,9; IC95%: 1,25-3,1), de prematuridad (RP=3,4; IC95%: 1,7-6,5), de padres con asma (RP=2,6; IC95%: 1,4-5,0) y de tenencia de mascotas (RP=0,4; IC95%: 0,2-0,9).Conclusiones. La exposición a contaminantes biológicos intramurales (ácaros y hongos), los antecedentes personales de prematuridad, neumonía o rinitis y el antecedente familiar de asma, incrementan la probabilidad de presentación de síntomas indicativos de asma bronquial en la población estudiada.
Introduction. Indoor air pollution may play an important role in development and exacerbation of asthma in children.Objective. The association between the presence of indoor biological contaminants and respiratory symptoms related to asthma was assessed in preschool children. Materials and methods. This cross-sectional study was undertaken in Bucaramanga, Colombia, and included children <7 years of age living in two urban areas of with different levels of outdoor air pollution. The 678 children were an average of 3.5 years of age. Respiratory symptoms indicative of asthma and indoor air pollutants were assessed by previously validated questionnaires.. Biological samples potentially containing mites and fungi were collected by standardized laboratory methods. The log binomial regression model was used for multivariate analysis, using adjusted prevalence ratios (PR). Results. The prevalence of asthmatic respiratory symptoms was 8.0%; (95% C.I: 5.6-9.6), without significant differences between the two areas. Binomial model analysis showed that asthma symptoms were associated with mites (PR 1.78; 95% C.I. 1.0-3.0), Acremonium sp (PR 6.24; 95 C.I.: 3.8-10.0) and a history of child pneumonia (PR 4.0; 95% C.I. 2.5-6.4), allergic rhinitis (PR 1.9; 95% C.I.: 1.2-3.1), prematurity (PR 3.4; 95% C.I. 1.8-6.5), parents with asthma (PR 2.6; 95% C.I. 1.4-5.0) and pet ownership (PR 0.4; 95% C.I. 0.2-0.9). Conclusions. The indoor exposure to biological contaminants (dust mites and fungi), history of prematurity, pneumonia, rhinitis and family history of asthma increased the occurence of symptoms suggestive of asthma in young children.
Subject(s)
Humans , Air Pollution , Asthma , Child, Preschool , Breath TestsABSTRACT
INTRODUCTION: Indoorair pollution may play an important role in development and exacerbation of asthma in children. OBJECTIVE: The association between the presence of indoor biological contaminants and respiratory symptoms related to asthma was assessed in preschool children. Materials and methods. This cross-sectional study was undertaken in Bucaramanga, Colombia, and included children <7 years of age living in two urban areas of with different levels of outdoor air pollution. The 678 children were an average of 3.5 years of age. Respiratory symptoms indicative of asthma and indoor air pollutants were assessed by previously validated questionnaires.. Biological samples potentially containing mites and fungi were collected by standardized laboratory methods. The log binomial regression model was used for multivariate analysis, using adjusted prevalence ratios (PR). RESULTS: The prevalence of asthmatic respiratory symptoms was 8.0%; (95% C.I: 5.6-9.6), without significant differences between the two areas. Binomial model analysis showed that asthma symptoms were associated with mites (PR 1.78; 95% C.I. 1.0-3.0), Acremonium sp (PR 6.24; 95 C.I.: 3.8-10.0) and a history of child pneumonia (PR 4.0; 95% C.I. 2.5-6.4), allergic rhinitis (PR 1.9; 95% C.I.: 1.2-3.1), prematurity (PR 3.4; 95% C.I. 1.8-6.5), parents with asthma (PR 2.6; 95% C.I. 1.4-5.0) and pet ownership (PR 0.4; 95% C.I. 0.2-0.9). CONCLUSIONS: The indoor exposure to biological contaminants (dust mites and fungi), history of prematurity, pneumonia, rhinitis and family history of asthma increased the occurence of symptoms suggestive of asthma in young children.
