Inhibition of Macrophage Oxidative Stress Prevents the Reduction of ABCA-1 Transporter Induced by Advanced Glycated Albumin

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - Reversal by a chemical chaperone

ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes. (C) 2012 Elsevier Ltd. All rights reserved.

The Pst system of Streptococcus mutans is important for phosphate transport and adhesion to abiotic surfaces

The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate-binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild-type strain treated with an anti-PstS antibody. Nevertheless, a reduced ability to bind to saliva-coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild-type strains. Taken together, the present data indicate that the S.similar to mutans Pst system participates in phosphate uptake, cell growth and expression of virulence-associated traits.

Hemolymph ion regulation and kinetic characteristics of the gill (Na+, K+)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45 parts per thousand salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 +/- 22.1 mOsm kg(-1) H2O) at 45 parts per thousand but elevated compared to fresh-caught crabs (801.0 +/- 40.1 mOsm kg(-1) H2O). Hemolymph [Na+ (323.0 +/- 2.5 mmol L-1) and [Me2+) (34.6 +/- 1.0 mmol L-1) are hypo-regulated while [Ca2+] (22.5 +/- 0.7 mmol L-1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 +/- 0.5 mmol L-1) but hypo-regulated (6.2 +/- 0.7 mmol L-1) at 45 parts per thousand. Protein expression patterns are altered in the 45 parts per thousand-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase alpha-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 +/- 3.5 U mg(-1); K-0.5 = 7.07 +/- 0.01 mu mol L-1) and a low-affinity ATP binding site (Vm = 108.1 +/- 2.5 U mg(-1); K-0.5 = 0.11 +/- 0.3 mmol L-1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site-site interactions, but modulation by Na+ shows Michaelis-Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited approximate to 80% of total ATPase activity (K-I=464.7 +/- 23.2 mu mol L-1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1) mg(-1)) and 45 parts per thousand-acclimated crabs (around 154 nmol Pi min(-1) mg(-1)), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2-3-fold in 45 parts per thousand-acclimated crabs. (C) 2012 Elsevier Inc. All rights reserved.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.