The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

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The voltage-sensitive sodium (Na+) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.

Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor (VEGFR) tyrosine kinases. Therefore, VEGFRs are an attractive therapeutic target for cancer treatment. In the present study, we show that a quinoline-urea derivative, KRN951, is a novel tyrosine kinase inhibitor for VEGFRs with antitumor angiogenesis and antigrowth activities. KRN951 potently inhibited VEGF-induced VEGFR-2 phosphorylation in endothelial cells at in vitro subnanomolar IC 50 values (IC 50 = 0.16 nmol/L). It also inhibited ligand-induced phosphorylation of plateletderived growth factor receptor-B (PDGFR-B) and c-Kit (IC 50 = 1.72 and 1.63 nmol/L, respectively). KRN951 blocked VEGFdependent, but not VEGF-independent, activation of mitogenactivated protein kinases and proliferation of endothelial cells. In addition, it inhibited VEGF-mediated migration of human umbilical vein endothelial cells. Following p.o. administration to athymic rats, KRN951 decreased the microvessel density within tumor xenografts and attenuated VEGFR-2 phosphorylation levels in tumor endothelium. It also displayed antitumor activity against a wide variety of human tumor xenografts, including lung, breast, colon, ovarian, pancreas, and prostate cancer. Furthermore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis revealed that a significant reduction in tumor vascular hyperpermeability was closely associated with the antitumor activity of KRN951. These findings suggest that KRN951 is a highly potent, p.o. active antiangiogenesis and antitumor agent and that DCE-MRI would be useful in detecting early responses to KRN951 in a clinical setting. KRN951 is currently in phase I clinical development for the treatment of patients with advanced cancer. (Cancer Res 2006; 66(18): 9134-42)

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domaincontaining protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.translational control | ribosome | 2-oxoglutarate oxygenase | hypoxia T he human genome encodes ∼60 2-oxoglutarate (2OG)-dependent oxygenases that catalyze diverse biological oxidations including hydroxylation of small molecules and proteins, and demethylation of histones and DNA/RNA (1). The identification of two types of 2OG oxygenase that regulate the transcriptional response to hypoxia by prolyl and asparaginyl hydroxylations in hypoxia-inducible factor (HIF), has led to the proposal that the hydroxylation of intracellular proteins may be involved in other signaling mechanisms (2). We have recently assigned 2OG oxygenases related to the HIF asparaginyl hydroxylase, factor-inhibiting HIF, as histidinyl and argininyl hydroxylases that catalyze hydroxylation of eukaryotic and prokaryotic ribosomes, respectively (3).2OG and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is a highly conserved 2OG oxygenase in eukaryotes. In the fission yeast, Schizosaccharomyces pombe, the homolog of OGFOD1, Ofd1, mediates oxygen-sensitive degradation of the N-terminal region of the transcription factor Sre1 [a homolog of sterol-response element-binding protein (SREBP)] after cleavage from the endoplasmic reticulum membrane, so contributing to oxygen-dependent regulation of the sterol response (4). The oxygen-sensitive SREBP pathway is not conserved in Saccharomyces cerevisiae (5), despite a conserved homolog of OGFOD1 [termination and polyadenylation 1 (Tpa1p)] in this species, suggesting Tpa1p has other roles. Indeed, TPA1 was identified in a screen for genes promoting stop codon readthrough (6). Tpa1p activity is linked to termination efficiency, mRNA polyadenylation, and mRNA stability. Structures of Tpa1p reveal the 2OG oxygenase characteristic doublestranded β-helix (DSBH) fold domain with typical Fe(II) and 2OG binding residues, but also indicate, as predicted f...

SummaryEfficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

Autophagy is a cytoplasmic degradation system that is important for starvation adaptation and cellular quality control. Previously, we reported that Atg5-null mice are neonatal lethal; however, the exact cause of their death remains unknown. Here, we show that restoration of ATG5 in the brain is sufficient to rescue Atg5-null mice from neonatal lethality. This suggests that neuronal dysfunction, including suckling failure, is the primary cause of the death of Atg5-null neonates, which would further be accelerated by nutrient insufficiency due to a systemic failure in autophagy. The rescued Atg5-null mouse model, as a resource, allows us to investigate the physiological roles of autophagy in the whole body after the neonatal period. These rescued mice demonstrate previously unappreciated abnormalities such as hypogonadism and iron-deficiency anemia. These observations provide new insights into the physiological roles of the autophagy factor ATG5.

This paper discusses the mechanisms of three-dimensional flows and of the associated losses occurring near the tip endwall region of a linear turbine cascade with tip clearance. The clearance gap sizes and the cascade incidences were chosen as the most important variables affecting the mechanisms. Flows close to the endwall and inside the clearance were surveyed in great detail using a micro five-hole pitot tube of 0.6 mm head size. The results gave very detailed information on the mechanisms, such as leakage flow vectors and pressure distributions throughout the clearance. Interaction of leakage flow with the endwall flow and their associated separation lines, effects of gap size and inlet flow angle on loss generation, and skewness of the three-dimensional endwall flows are also discussed.