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The acidic peroxidoxin [also named thiol-specific antioxidant protein (TSA) or protector protein (PRP)], which plays a role in the response against oxidative stress, is one of the major proteins of red blood cells. In this work, we show that this protein is induced at early stages of erythroid differentiation prior to haemoglobin accumulation, which suggests that it may play a role at the erythroblast stage, where haemoglobinized, nucleated and genetically active cells are submitted to a maximally dangerous oxidative stress. The early accumulation of this protein has been demonstrated both on transformed cell systems and on normal differentiating human erythroid cells. This suggests that this protein may play an important role in the differentiation of the erythroid cells.

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The human pag gene product is an inhibitor of the cabl tyrosine kinase and belongs to a new family of proteins. We show here that higher levels of pag gene expression are observed following induction of proliferation and contact with compounds inducing oxidative stress such as diethyl maleate and sodium arsenate. A weaker overexpression is seen in a macrophage cell line using hydrogen peroxide or menadione as inducers. Pag gene expression increases in synchronized cells entering the S phase. This raises the possibility that elevated levels of pag counteract the cytostatic activity of abl. Treatment of growth arrested cells with diethyl maleate and sodium arsenate induces pag gene overexpression, independently of cell proliferation. Thus, enhanced pag gene expression occurs in two cellular events: proliferation and response to oxidative stress.z 1998 Federation of European Biochemical Societies.

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.

2',5'1Oligoadenylate synthetase has been purified from a rabbit reticulocyte lysate to a high degree of purity. The enzyme contained no detectable interfering activities that could degrade the nucleoside triphosphate substrate or the oligomeric products. Two basic properties of this enzyme have been examined: the elongation mechanism for the synthesis of oligoadenylates and the substrate specificity for nucleotides. Kinetic studies on the formation of different o igomeric intermediates show that the dimer pppA2'p5'A is the first product to accumulate in predominant proportion during the first period of reaction; the trimer and other longer oligomers appear after a lag phase. The amount of the trimer increases at the expense of the dimer. Preformed dimers and trimers added to the incubation mixture were readily incorporated into higher oligomers, suggesting the free access of these dimers and trimers to the active center after the onset of polymerization of ATP. The results indicate clearly that the enzyme catalyzes the de no-vo synthesis of the oligonucleotide from ATP and that the mechanism of elongation of the 2',5'-oligonucleotides catalyzed by the enzyme is not processive. Polymerization of a mixture of ATP and another nucleoside triphosphate shows that the enzyme is not only an ATP polymerase. The 2',5'-oligoadenylate synthetase is in fact a 2',5'-nucleotidyltransferase a catalyzes the formation of co-oligonucleotides. However, the purified reticulocyte enzyme catalyzed only the addition of one unit of GMP, UMP, CMP, 2'1dAMP, 3'-dAMP, dCMP, dGMP, or TMP to the 2'.OH end of a preformed oligoadenylate. A procedure for the separation of 2',5'-oligonucleotides with or without the 5'-triphosphate end also is described. (7) indicates that this enzyme also has a function independent of interferon.In this paper, we describe the reaction mechanism of the enzyme purified from rabbit reticulocytes and the synthesis of 2',5'-co-oligonucleotides. MATERIALS AND METHODSMaterials. All radioactive nucleotides were obtained from Amersham. All chemicals were the purest obtainable. Poly(I). poly(C) and poly(I)-poly(C)agarose were from Choay Institute (Paris, France). Calf intestine alkaline phosphatase was from Boehringer. Polyethyleneimine (Polymin P) was a gift from BASF. Cellulose MN 300 was from Macherey Nagel (Dfiren, Germany). Nuclease P1 was from Yamasa Shoyo Co (Tokyo, Japan).Purification of Oligo(A) Synthetase. Phenylhydrazine was injected into two rabbits for 5 days, according to Borsook et al. (19), and the blood was collected 2 days later, and the reticulocytes were isolated. The lysate (85 ml; 160 mg of protein per ml) obtained by osmotic shock was passed through a DEAEcellulose (DE-52) column (50 ml) equilibrated in 20 mM TrisHCl, pH 8.0/5 mM Mg(OAc)2/25 mM KCl/1 mM dithiothreitol/10% (vol/vol) glycerol (buffer A). The oligo(A) synthetase activity was recovered with the proteins not retained on the column (105 ml, 32 mg of protein per ml), and this filtrate was applied to a poly(I)-poly(C)-agarose colum...

The interferon induced enzyme 2'5' oligoadenylate synthetase produces 2'5' pppA(pA)n the first discovered natural nucleotide with a 2'5' linkage. We describe a direct assay of this enzyme based on separation by thin layer chromatography (TLC) of the substrate ATP and the products 2'5' pppA(pA)n (n larger than or equal to 1). This technique presents obvious advantages compared to the currently used methods. Moreover the enzyme uses other nucleotides as substrates forming co-oligonucleotides 2'5 pppA(pA)n pN (N = U,G,C,dA,dG,dT and dC). Additional procedures are described using different developing solvent systems for the separation of the core-2'5' oligonucleotides (2'5' A(pA)npN) containing AMP-residues entirely and those with another nucleotide at the 2' end.