At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

/pdf/free-radicals-in-the-physiological-control-of-cell-function-g3w3iyr0ph.pdf

Free Radicals in the Physiological Control of Cell Function

The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. Superoxide (O2•−) is the proximal mitochondrial ROS, and in the present review I outline the principles that govern O2•− production within the matrix of mammalian mitochondria. The flux of O2•− is related to the concentration of potential electron donors, the local concentration of O2 and the second-order rate constants for the reactions between them. Two modes of operation by isolated mitochondria result in significant O2•− production, predominantly from complex I: (i) when the mitochondria are not making ATP and consequently have a high Δp (protonmotive force) and a reduced CoQ (coenzyme Q) pool; and (ii) when there is a high NADH/NAD+ ratio in the mitochondrial matrix. For mitochondria that are actively making ATP, and consequently have a lower Δp and NADH/NAD+ ratio, the extent of O2•− production is far lower. The generation of O2•− within the mitochondrial matrix depends critically on Δp, the NADH/NAD+ and CoQH2/CoQ ratios and the local O2 concentration, which are all highly variable and difficult to measure in vivo. Consequently, it is not possible to estimate O2•− generation by mitochondria in vivo from O2•−-production rates by isolated mitochondria, and such extrapolations in the literature are misleading. Even so, the description outlined here facilitates the understanding of factors that favour mitochondrial ROS production. There is a clear need to develop better methods to measure mitochondrial O2•− and H2O2 formation in vivo, as uncertainty about these values hampers studies on the role of mitochondrial ROS in pathological oxidative damage and redox signalling.

/pdf/how-mitochondria-produce-reactive-oxygen-species-4spvs9r9mw.pdf

How mitochondria produce reactive oxygen species.

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis

Human mesenchymal stem cells modulate allogeneic immune cell responses

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2−•). Even though O2−• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments. We also discuss various physiological and pathological scenarios resulting from an increased steady state concentration of mitochondrial oxidants.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-7793.2003.00335.x

Mitochondrial formation of reactive oxygen species.

Regulation of erythropoietin gene expression.

The studies described in this paper were directed at an understanding of the regulation of heme synthesis during erythropoietin (epo)-induced erythroid differentiation. Although considerable work has been done on the regulation of heme synthesis in virally—transformed erythroid cell lines which were chemically induced to differentiate along the erythroid pathway1–4 our goal was to study the control of heme formation in normal bone marrow cells, the physiological site of epo action. Our earlier findings showing that heme synthesis is required for the epo-induced globin synthesis in marrow cells5, made this an important problem.

Studies of the Effect of Erythropoietin on Heme Synthesis

Over many years a standard concept of the regulation of normal erythropoiesis has developed. This concept involves the synthesis and secretion of erythropoietin (EPO) by a small subset of adult renall-4 or fetal liver cells.’ The secreted EPO in the circulation then acts on potential erythropoietic cells of the blood-forming system to induce red cell formation. Support for this view comes from a vast body of information, but recent evidence now indicates that the system may be more complex. Our studies of this problem were influenced by work with the transformed mouse cell lines IW32 and These cells secrete significant amounts of EPO constitutively but have an erythroid phenotype, having EPO receptors and synthesizing hemoglobin when treated with hemin or sodium butyrate.’ We have shown that IW32 cells have both a normal EPO gene and a rearranged and amplified gene.8The latter has been trans-located to near another, as yet, unidentified gene, which is constitutively expressed. We proposed that the abnormal EPO gene is expressed because of the proximity of this “active” gene.g The situation for NNlO cells is quite different. They have only a normal EPO gene, which we suggest, on the basis of gel-shift experiments,I0 is constitutively expressed because of the absence of the trans-acting repressor described in kidney nuclei extracts.” These transformed cells were postulated to be under autocrine control,’* but when we grew them in methylcellulose cultures in the presence of an excess of neutralizing anti-EPO, we found no effect of the antibody on colony number. If there were an autocrine mechanism regulating cell growth of IW32 and NNlO cells, it would have to be an internal one, with the synthesis and secretion of EPO and the expression of external receptors being epiphenomena unrelated to the regulation of cell proliferation. We tested the possibility of an internal autocrine mechanism using antisense oligomers corresponding to the 5‘ ends of exons I and I1 of the EPO gene. Both colony number and cell growth as measured by thymidine incorporation were suppressed by an antisense oligonucleotide (TABLES 1 and 2). We then tested the internal autocrine concept with normal mouse marrow cells and have presented data13 showing that colonies derived from multipotent cells of the marrow are significantly affected by antisense oligomers based on both

Internal Autocrine Regulation of the Early Stages of Hemopoiesisa

4 – Commitment in Blood Cell Differentiation: Erythropoietin as an Instructive Signal

SUMMARY Erythropoietin, a glycoprotein that induces normal erythrocyte development, has 16 to 18 sialic acid residues per mole. Desialation results in complete loss of biological activity when it is assayed in vivo. When the assay is done in vitro asialoerythropoietin has full activity, or when assayed at low levels of hormones is about three times more active than the native hormone. The loss of activity can be explained by the hepatic removal of asialoglycoproteins from the circulation (MORELL, A. G., IRVINE, R. A., STERNLIEB, I., SCHEINBERG, I. H., AND ASHWELL, G. (1968) J. Biol. Chem. 243, 155). Asialoorosomucoid or stachyose injected into assay animals act as competitors and permit 25 to 35 % of the original activity of erythropoietin to be seen in vivo after desialation. Treatment of asialoerythropoietin with galactose oxidase results in recovery of 45% of the original activity, confirming an earlier finding (LUKOWSKY, W. A., AND PAINTER, R. H. (1972) Can. J. Biochem. 50, 909). Asialoerythropoietin is more sensitive to heat denaturation and trypsin action than the native hormone. Our data indicate that terminal sialic acid is required for chemical and biological stability but not for action on target cells of the bone marrow. The asialohormone has enhanced activity towards the target cells. It has been known for some time that the role of sialic acid in the biological function of erythropoietin is paradoxical. Erythropoietin activity, when measured in tivo, is completely dependent on the presence of sialic acid (2), but when the assay is done in vitro, the asialohormone has full activity (3). Resolution of

Xiii. the role of sialic acid in erythropoietin action

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