On the mechanism underlying tellurite reduction by Aeromonas caviae ST dihydrolipoamide dehydrogenase.

The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K substitutions were individually transformed into Escherichia coli Δlpd. Cells expressing the modified genes exhibited decreased pyruvate dehydrogenase, dihydrolipoamide dehydrogenase and TR activity regarding that observed with the wild type A. caviae lpdA gene. In addition, cells expressing the altered lpdA genes showed increased oxidative stress levels and tellurite sensitivity than those carrying the wild type counterpart. The involvement of Cys residues in LpdA's TR activity was analyzed using specific inhibitors that interact with catalytic cysteines and/or disulfide bridges such as aurothiomalate, zinc or nickel. TR activity of purified LpdA was drastically affected by these compounds. Since LpdA belongs to the flavoprotein family, the involvement of the FAD/NAD(P)(+)-binding domain in TR activity was determined. FAD removal from purified LpdA results in loss of TR activity, which was restored with exogenously added FAD. Substitutions in E354, involved in FAD/NADH binding, resulted in low TR activity because of flavin loss. Finally, changing H322 (involved in NAD(+)/NADH binding) by tyrosine also resulted in altered TR activity.

SR-A regulates the inflammatory activation of astrocytes.

Scavenger receptor Class A (SR-A) participates in the regulation of inflammatory processes against pathogens and in inflammatory stimulation. We have recently demonstrated the presence of SR-A in astrocytes, but its participation in their inflammatory response is unknown. Astrocytes regulate neuroinflammation through the regulation of microglial cell activation and the production of cytokines, neurotrophic factors, and reactive species. Using astrocytes from SR-A(-/-) mice in culture, we assessed the participation of SR-A in their inflammatory activation, evaluating the activation of IκB/NF-κB and MAPK signaling pathways and the production of nitric oxide (NO) and IL-1ß in response to SR-A ligands. In SR-A(-/-) astrocytes, lipopolysaccharide (LPS) induced higher levels of NO and reduced levels of IL-1ß compared to SR-A(+/+) cells. In addition, SR-A(-/-) astrocytes had a reduced basal and LPS-stimulated JNK phosphorylation, and a delayed activation on IκB/NF-κB signaling pathway in response to LPS. Moreover, inhibition of the ERK pathway reduced NO production by SR-A(-/-) cells, suggesting that this signaling pathway modulated LPS-induced NO production, an effect that depended on the presence of SR-A. Our results suggest that SR-A participates in the modulation of signaling pathways involved in the production of soluble molecules implicated in the neuroinflammatory response.