Coenzyme A and protein CoAlation levels are regulated in response to oxidative stress and during morphogenesis in Dictyostelium discoideum.

Dictyostelium discoideum (D. discoideum) is a simple eukaryote with a unique life cycle in which it differentiates from unicellular amoebae into a fruiting body upon starvation. Reactive oxygen species (ROS) have been associated with bacterial predation, as well as regulatory events during D. discoideum development and differentiation. Coenzyme A (CoA) is a key metabolic integrator in all living cells. A novel function of CoA in redox regulation, mediated by covalent attachment of CoA to cellular proteins in response to oxidative or metabolic stress, has been recently discovered and termed protein CoAlation. In this study, we report that the level of CoA and protein CoAlation in D. discoideum are developmentally regulated, and correlate with the temporal expression pattern of genes implicated in CoA biosynthesis during morphogenesis. Furthermore, treatment of growing D. discoideum cells with oxidising agents results in a dose-dependent increase of protein CoAlation. However, much higher concentrations were required when compared to mammalian cells and bacteria. Increased resistance of D. discoideum to oxidative stress induced by H2O2 has previously been attributed to high levels of catalase activity. In support of this notion, we found that H2O2-induced protein CoAlation is significantly increased in CatA-deficient D. discoideum cells. Collectively, this study provides insights into the role of CoA and protein CoAlation in the maintenance of redox homeostasis in amoeba and during D. discoideum morphogenesis.

Rhinacanthus nasutus extracts prevent glutamate and amyloid-ß neurotoxicity in HT-22 mouse hippocampal cells: possible active compounds include lupeol, stigmasterol and ß-sitosterol.

The Herb Rhinacanthus nasutus (L.) Kurz, which is native to Thailand and Southeast Asia, has become known for its antioxidant properties. Neuronal loss in a number of diseases including Alzheimer's disease is thought to result, in part, from oxidative stress. Glutamate causes cell death in the mouse hippocampal cell line, HT-22, by unbalancing redox homeostasis, brought about by a reduction in glutathione levels, and amyloid-ß has been shown to induce reactive oxygen species (ROS) production. Here in, we show that ethanol extracts of R. nasutus leaf and root are capable of dose dependently attenuating the neuron cell death caused by both glutamate and amyloid-ß treatment. We used free radical scavenging assays to measure the extracts antioxidant activities and as well as quantifying phenolic, flavonoid and sterol content. Molecules found in R. nasutus, lupeol, stigmasterol and ß-sitosterol are protective against glutamate toxicity.

Cell Cycle Heterogeneity Can Generate Robust Cell Type Proportioning.

Cell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.

Protection of podocytes from hyperhomocysteinemia-induced injury by deletion of the gp91phox gene.

In this study, mice lacking the gp91(phox) gene were used to address the role of NADPH oxidase in hyperhomocysteinemia-induced podocyte injury. It was found that a folate-free diet increased plasma homocysteine levels, but failed to increase O(2)(-) production in the glomeruli from gp91(phox) gene knockout (gp91(-/-)) mice, compared with wild-type (gp91(+/+)) mice. Proteinuria and glomerular damage index (GDI) were significantly lower, whereas the glomerular filtration rate (GFR) was higher in gp91(-/-) than in gp91(+/+) mice when they were on the folate-free diet (urine albumin excretion, 21.23+/-1.88 vs 32.86+/-4.03 microg/24 h; GDI, 1.17+/-0.18 vs 2.59+/-0.49; and GFR, 53.01+/-4.69 vs 40.98+/-1.44 microl/min). Hyperhomocysteinemia-induced decrease in nephrin expression and increase in desmin expression in gp91(+/+) mice were not observed in gp91(-/-) mice. Morphologically, foot process effacement and podocyte loss due to hyperhomocysteinemia were significantly attenuated in gp91(-/-) mice. In in vitro studies of podocytes, homocysteine was found to increase gp91(phox) expression and O2(*)(-) generation, which was substantially inhibited by gp91(phox) siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A production was abolished by gp91(phox) siRNA or diphenyleneiodonium, a NADPH oxidase inhibitor. These results suggest that the functional integrity of NADPH oxidase is essential for hyperhomocysteinemia-induced podocyte injury and glomerulosclerosis.

NMDA receptor-mediated activation of NADPH oxidase and glomerulosclerosis in hyperhomocysteinemic rats.

This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague-Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O(2)(.-) production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O(2)(.-) production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O(2)(.-) generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis.