Plasma extracellular vesicle protein content for diagnosis and prognosis of global cardiovascular disease

Cardiovascular disease is a major public health problem worldwide. Its growing burden is particularly ominous in Asia, due to increasing rates of major risk factors such as diabetes, obesity and smoking. There is an urgent need for early identification and treatment of individuals at risk of adverse cardiovascular events. Plasma extracellular vesicle proteins are novel biomarkers that have been shown to be useful in the diagnosis, risk stratification and prognostication of patients with cardiovascular disease. Ongoing parallel biobank initiatives in European (the Netherlands) and Asian (Singapore) populations offer a unique opportunity to validate these biomarkers in diverse ethnic groups.     

Intermittent Hypoxia-Induced NF-κB and HO-1 Regulation in Human Endothelial EA.hy926 Cells

Intermittent hypoxia (IH) is a hallmark feature in obstructive sleep apnea (OSA) which is increasingly recognized as an independent risk factor for atherosclerosis. Oxidative stress, inflammation, and cell apoptosis are major pathological events initiating or accelerating atherogenesis. This study addressed whether IH would affect these proatherogenic factors in endothelial cells and the mechanistic pathways involved. EA.hy926 cells were exposed to intermittent normoxia or IH for different numbers of cycles (32, 64, or 96). IH exposure time-dependently raised cellular GSSG/GSH ratio, increased production of IL-6 and IL-8, and accelerated cell apoptosis and death, concurrent with activation of NF-κB and inhibition of Nrf2/HO-1 pathways. At 64 cycles, inhibition of NF-κB attenuated IH-induced cellular oxidative stress and accumulation of inflammatory cytokines in cell culture medium but aggravated IH-induced cell apoptosis, while stimulation of HO-1 suppressed IH-induced cellular oxidative stress and cell apoptosis without affecting accumulation of inflammatory cytokines in cell culture medium. We demonstrated that early stage of exposure to IH-induced oxidative and inflammatory stresses leading to acceleration of cell apoptosis via NF-κB and Nrf2/HO-1 pathways in endothelial cells, suggesting the potential mechanisms for IH-induced vascular pathogenesis, in resemblance to OSA.     

Vacuolar H+-translocating ATPases from plants: Structure,function, and isoforms

The vacuolar H⁺-translocating ATPase (V-type ATPase) plays a central role in the growth and development of plant cells. In a mature cell, the vacuole is the largest intracellular compartment, occupying about 90% of the cell volume. The proton electrochemical gradient (acid inside) formed by the vacuolar ATPase provides the primary driving force for the transport of numerous ions and metabolites against their electrochemical gradients. The uptake and release of solutes across the vacuolar membrane is fundamental to many cellular processes, such as osmoregulation, signal transduction, and metabolic regulation. Vacuolar ATPases may also reside on endomembranes, such as Golgi and coated vesicles, and thus may participate in intracellular membrane traffic, sorting, and secretion.Plant vacuolar ATPases are large complexes (400–650 kDa) composed of 7–10 different subunits. The peripheral sector of 5–6 subunits includes the nucleotide-binding catalytic and regulatory subunits of 70 and 60 kDa, respectively. Six copies of the 16-kDa proteolipid together with 1–3 other subunits make up the integral sector that forms the H⁺ conducting pathway. Isoforms of plant vacuolar ATPases are suggested by the variations in subunit composition observed among and within plant species, and by the presence of a small multigene family encoding the 16-kDa and 70-kDa subunits. Multiple genes may encode isoforms with specific properties required to serve the diverse functions of vacuoles and endomembrane compartments.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CAM Crassulacean acid metabolism - Nbd-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Bz-ATP 3-O-(4-benzoyl)benzolyadenosine 5-triphosphate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NEM N-ethylmaleimide - IP₃ inositol-1,4,5-triphosphate - H⁺-PPase H⁺-translocating pyrophosphatase - V-type vacuolar-type - P-type phosphorylated intermediate- or plasma membrane-type - F-type F₁F_o-type - V-ATPase vacuolar-type H⁺-ATPase     

In vitro and in silico evaluations of diarylpentanoid series as α-glucosidase inhibitor

A series of thirty-four diarylpentanoids derivatives were synthesized and evaluated for their α-glucosidase inhibitory activity. Eleven compounds (19, 20, 21, 24, 27, 28, 29, 31, 32, 33 and 34) were found to significantly inhibit α-glucosidase in which compounds 28, 31 and 32 demonstrated the highest activity with IC₅₀ values ranging from 14.1 to 15.1?µM. Structure-activity comparison shows that multiple hydroxy groups are essential for α-glucosidase inhibitory activity. Meanwhile, 3,4-dihydroxyphenyl and furanyl moieties were found to be crucial in improving α-glucosidase inhibition. Molecular docking analyses further confirmed the critical role of both 3,4-dihydroxyphenyl and furanyl moieties as they bound to α-glucosidase active site in different mode. Overall result suggests that diarylpentanoids with both five membered heterocyclic ring and polyhydroxyphenyl moiety could be a new lead design in the search of novel α-glucosidase inhibitor.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Probing the catalytic subunit of the tonoplast H+-ATPase from oat roots. Binding of 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole to the 72-kilodalton polypeptide

The purified tonoplast H+-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72, 60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-Cl preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)O-(2,4,6-trinitrophenyl) ATP. Nbd-Cl probably modified cysteinyl--SH or tyrosyl--OH groups, as dithiothreitol reversed both ATPase inactivation and [14C]Nbd-Cl binding to the 72-kDa subunit. The finding that N-ethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F1-ATPase. The antibody inhibited tonoplast ATPase and H+-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP- (or nucleotide-) binding site that may constitute the catalytic domain.     

Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H-Coupled Anion Transport

Chloride or nitrate decreased a pH gradient (measured as [¹⁴C]methylamine accumulation) in tonoplast-enriched vesicles. The ΔpH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H⁺-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K⁺ gradient and nigericin. 4,4′-Diisothiocyano-2,2′-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl⁻. Two possible models to account for this anion-dependent decrease of ΔpH are: (a) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ efflux from the vesicles via H⁺/anion symport systems on the tonoplast and (b) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ uptake into the vesicles via H⁺/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl⁻ and NO₃⁻ stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H⁺-coupled anion transport systems.