Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes

Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.     

The supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.     

Synergistic effect of conjugated linolenic acid isomers against induced oxidative stress,inflammation and erythrocyte membrane disintegrity in rat model

Background

α-Eleostearic acid and punicic acid, two typical conjugated linolenic acid (CLnA) isomers present in bitter gourd and snake gourd oil respectively, exhibit contrasting cis-trans configuration which made them biologically important.

Methods

Rats were divided into six groups. Group 1 was control and group 2 was treated control. Rats in the groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0% respectively) while rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid respectively along with sodium arsenite by oral gavage once per day.

Results

Results showed that increase in nitric oxide synthase (NOS) activity, inflammatory markers expression, platelet aggregation, lipid peroxidation, protein oxidation, DNA damage and altered expression of liver X receptor-α (LXR-α) after arsenite treatment were restored with the supplementation of oils containing CLnA isomers. Altered activities of different antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and ferric reducing ability of plasma (FRAP) also restored after oil supplementation. Altered morphology and fluidity of erythrocyte membrane studied by atomic force and scanning electron microscopy, after stress induction were significantly improved due to amelioration in cholesterol/phospholipid ratio and fatty acid profile of membrane. Oils treatment also improved morphology of liver and fatty acid composition of hepatic lipid.

Conclusions

Overall two isomers showed synergistic antioxidant and anti-inflammatory effect against induced perturbations and membrane disintegrity.

General significance

Synergistic antioxidant and anti-inflammatory role of these CLnA isomers were established by this study.     

Multifaceted applications of nanomaterials in cell engineering and therapy

Nanomaterials with superior physiochemical properties have been rapidly developed and integrated in every aspect of cell engineering and therapy for translating their great promise to clinical success. Here we demonstrate the multifaceted roles played by innovatively-designed nanomaterials in addressing key challenges in cell engineering and therapy such as cell isolation from heterogeneous cell population, cell instruction in vitro to enable desired functionalities, and targeted cell delivery to therapeutic sites for prompting tissue repair. The emerging trends in this interdisciplinary and dynamic field are also highlighted, where the nanomaterial-engineered cells constitute the basis for establishing in vitro disease model; and nanomaterial-based in situ cell engineering are accomplished directly within the native tissue in vivo. We will witness the increasing importance of nanomaterials in revolutionizing the concept and toolset of cell engineering and therapy which will enrich our scientific understanding of diseases and ultimately fulfill the therapeutic demand in clinical medicine.