Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Dysfunction of glutathione S-transferase leads to excess 4-hydroxy-2-nonenal and H(2)O(2) and impaired cytokine pattern in cultured keratinocytes and blood of vitiligo patients

Oxidative stress due to increased epidermal levels of H(2)O(2) with consequent inhibition of catalase activity is generally accepted as a leading cytotoxic mechanism of melanocyte loss in vitiligo. Keratinocyte-derived cytokines are considered key factors in the maintenance of melanocyte structure and functions. We hypothesized that abnormal redox control may lead to impaired cytokine production by keratinocytes, thus causing noncytotoxic defects in melanocyte proliferation and melanogenesis. We found significantly suppressed mRNA and protein expression of glutathione-S-transferase (GST) M1 isoform, and higher-than-normal levels of both 4-hydroxy-2-nonenal (HNE)-protein adducts and H(2)O(2) in the cultures of keratinocytes derived from unaffected and affected skin of vitiligo patients, and in their co-cultures with allogeneic melanocytes. GST and catalase activities, as well as glutathione levels, were dramatically low in erythrocytes, whilst HNE-protein adducts were high in the plasma of vitiligo patients. The broad spectrum of major cytokines, chemokines, and growth factors was dysregulated in both blood plasma and cultured keratinocytes of vitiligo patients, when compared to normal subjects. Exogenous HNE added to normal keratinocytes induced a vitiligo-like cytokine pattern, and H(2)O(2) overproduction accompanied by adaptive upregulation of catalase and GSTM1 genes, and transient inhibition of Erk1/2 and Akt phosphorylation. Based on these results, we suggest a novel GST-HNE-H(2)O(2)-based mechanism of dysregulation of cytokine-mediated keratinocyte-melanocyte interaction in vitiligo