Porphyrin Homeostasis Maintained by ABCG2 Regulates Self-Renewal of Embryonic Stem Cells

Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined.Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of gammaH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment.The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and gammaH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands

Heme and innate immunity: new insights for an old molecule

Hemolytic episodes such as sickle cell disease, malaria and ischemia-reperfusion occurrence are often associated to the statement of an inflammatory response which may develop or not to a chronic inflammatory status. Although these pathological states are triggered by distinct etiological agents, all of them are associated to high levels of free heme in circulation. In this review, we aim to focus the very recent achievements that have led to the statement of free heme as a proinflammatory molecule, which may play a central role during the onset and/or persistance of inflammation during these pathologies

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

Low complication rate and an increasing incidence of surgical repair of primary indirect sliding inguinal hernia.

The purpose of the present study was to explore the risk for complications and reoperations following open repairs for sliding groin hernias