Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins G␣ s and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10°C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.

The appearance of phosphatidylserine (PS) on the outer surface of red cells is an important signal for their uptake by macrophages. We report for the first time that procaspase 3 present in the anucleated mature human erythrocyte is activated under oxidative stress induced by t-butylhydroperoxide leading to impairment of the aminophospholipid translocase, PS externalization and increased erythrophagocytosis. This is the first report linking caspase 3 activation to inhibition of flippase activity and uptake of red cells by macrophages. ß

Modification of the cytidine in the first anticodon position of the AUA decoding tRNA Ile (tRNA Ile 2 ) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography-mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of tRNA Ile 2 adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the tRNA Ile 2 demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C þ , for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis tRNA Ile 2 and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNA Ile of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.agmatine | decoding | RNA modification | tRNA | wobble pairing T he genetic code table consists of sixteen four-codon boxes. In fourteen of the boxes, all four codons either specify the same amino acid or are split into two sets of two codons, with each set encoding a different amino acid. For example, the UUN box is split into UUU/UUC coding for phenylalanine and UUA/UUG coding for leucine. The wobble hypothesis of Crick proposes how a single phenylalanine tRNA with G in the first anticodon position can base pair with either U or C and a single leucine tRNA with a modified U (or 2-thioU) in the anticodon can base pair with either A or G (1-3). The two remaining boxes, UGN and AUN, are exceptions in that the UGN box is split into UGU/UGC coding for cysteine, UGG coding for tryptophan, and UGA being used as a stop codon, whereas the AUN box is split into AUU/AUC/AUA coding for isoleucine and AUG coding for methionine. The isoleucine codons AUU and AUC can be read by an isoleucine tRNA with G in the anticodon following the wobble pairing rules, but how the AUA codon is read specifically by a tRNA Ile without also reading the AUG codon has been a question of much interest over the years.Different organisms have developed different strategies for reading the AUA codon. Bacteria use a tRNA Ile with the anticodon LAU (L ¼ lysidine) (4-7). Lysidine is a modified cytidine in which the C2-oxo group of cytidine is replaced by lysine. Exactly how it base pairs with A but not with G is not established.Eukaryotes, on the other hand, con...

Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1␤ (IL-1␤) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1␤ release and dissected the signaling pathways leading to LPS-induced IL-1␤ secretion. We demonstrate that both the NF-B and the C/EBP␤-binding elements of the IL-1␤ promoter drive LPS-induced IL-1␤ gene expression. NF-B activation requires the classical TLR4-initiated signaling cascade leading to I B phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBP␤ activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBP␤, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBP␤. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1␤. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser 376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1␤ gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1␤ gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.

The N-terminal cytoplasmic domain of the anion exchanger 1 (AE1 or band 3) of the human erythrocyte associates with peripheral membrane proteins to regulate membrane-cytoskeleton interactions, with glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldolase, with the protein-tyrosine kinase p72 syk , with hemoglobin and with hemichromes. We have demonstrated that the N-terminal cytoplasmic domain of band 3 (CDB3) is a substrate of the apoptosis executioner caspase 3 (1). CDB3 has two non-conventional caspase 3 cleavage sites, TATD 45 and EQGD 205 (2). In vitro treatment of recombinant CDB3 with caspase 3 generated two fragments, which could be blocked by pretreatment with the caspase 3 inhibitor Z-DEVD-fmk Apoptosis is required to maintain the balance between cell proliferation and cell death. Nuclear collapse and DNA fragmentation are universal features of apoptosis in nucleated cells. However, the nucleus itself is not required for apoptosis, since apoptotic stimuli induce apoptotic morphological features in anucleate cells (1-3). The signaling events that culminate in some of the key plasma membrane changes associated with cell death in vivo, such as externalization of PS, 1 operate independently of the nucleus (3). While the process of apoptosis has been studied in-depth in nucleated cells, the role of apoptosis regulatory molecules in anucleate cells is poorly understood. The anucleate mature circulating human erythrocyte is cleared by macrophages after its 120-day life span. The senescent erythrocyte shares at least one feature in common with nucleated cells undergoing apoptosis. The externalization of PS on the outer leaflet of the red cells results in recognition by macrophages probably through a receptor that engages PS (4). We hypothesized that the circulating human erythrocyte potentially shares part of the apoptotic machinery of nucleated cells, and that this machinery may be triggered as a consequence of red cell senescence, pathology such as in diseases like sickle cell anemia, thalassemia, and oxidative insult to red cells. Proteases play a critical role in the expression of mammalian apoptosis. The caspases are a specialized family of cysteine-dependent aspartate-directed proteases (5-7). The caspases recognize a tetrapeptide sequence on their substrates with an aspartate residue at the fourth position. Caspase activation is a critical event in the onset of apoptosis (5). Human caspase 3 is perhaps the most universal apoptosis mediator. It is present in most mammalian cells (8). Its deletion by gene knockout blocks neuronal death during brain development with consequent lethality (9). Studies in our laboratory have demonstrated that caspase 3 is present in mature human erythrocytes and activated by oxidative stress (10). Berg et al. (11) have also demonstrated that caspases 3 and 8 are present in mature human erythrocytes.The cytoskeleton of an apoptotic cell undergoes profound changes. Underlying these changes is the cleavage of many cytoskeletal proteins by caspases. Thes...