Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins, which tag hypoxia-inducible factor (HIF) ␣ subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-␣ stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1 ؊/؊ or Phd3 ؊/؊ mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2␣, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1␣ accumulated in both the kidney and liver. Elevated HIF-1␣ levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2␣/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway. (Blood. 2008; 111:3229-3235)

SUMMARYHypoxia-inducible factor (HIF) α, which has three isoforms, is central to the continuous balancing of the supply and demand of oxygen throughout the body. HIF-α is a transcription factor that modulates a wide range of processes, including erythropoiesis, angiogenesis, and cellular metabolism. We describe a family with erythrocytosis and a mutation in the HIF2A gene, which encodes the HIF-2α protein. Our functional studies indicate that this mutation leads to stabilization of the HIF-2α protein and suggest that wild-type HIF-2α regulates erythropoietin production in adults.A widely recognized example of an oxygen-regulated pathway is the erythropoietin system, in which the kidney senses decreased tissue oxygenation and, in turn, produces erythropoietin, thereby increasing the red-cell mass. 1 Studies of the regulation of the EPO gene led to the discovery of HIF, which consists of a labile α subunit and a constitutively expressed β subunit. 2 Under normoxic conditions, the α subunit, which consists of three isoforms -HIF-1α, HIF-2α, and HIF-3α -is hydroxylated on two specific prolyl residues. 3 This hydroxylation targets HIF-α for degradation by the von Hippel-Lindau (VHL) tumor-suppressor protein. 4 Under hypoxic conditions, the hydroxylation is inhibited, thereby maintaining a stable HIF-α protein, which activates not only the EPO gene but also a broad range of other genes that orchestrate adaptation to hypoxia. 2,5Familial erythrocytosis provides an opportunity to study the oxygen-sensing mechanism. 6 This genetic disorder can be caused by a mutation in one of the proteins that hydroxylates HIF-α (prolyl hydroxylase domain protein 2 [PHD2]) 7,8 or by a mutation in the VHL protein. 9,10 These findings raise the question of whether there are mutations in HIF-α itself.The particular HIF isoform involved in erythropoietin regulation has been the subject of intensive investigation. Several results point to HIF-1α, 11-13 but mouse Hif-2α (also known as endothelial PAS domain protein 1 [Epas1] or Hif-1α-like factor [Hlf]) has been implicated as the principal regulator of erythropoietin in postembryonic mice. 14-17 We investigated the HIF2A gene in a family with erythrocytosis and found a missense mutation that impairs hydroxylation of the HIF-2α protein, thereby allowing both for maintenance of its stable conformation and for its induction of erythrocytosis.

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The facile fabrication of thin flexible electromagnetic interference (EMI) shielding materials with fast heat dissipation for adaptable tuning in both civil and military applications is in urgent demand. In our work, the flexible poly(vinylidene fluoride) (PVDF)/carbon nanotube (CNT) composite films decorated with anisotropy-shaped Co in flowers or chains were prepared and studied. The results showed that by increasing the Co filler contents, the EC (electrical conductivity), TC (thermal conductivity), and EMI shielding properties of such PVDF/CNT/Co (flowers or chains) flexible films were significantly improved. In contrast, the PVDF/CNT/Co-chain flexible films exhibit higher performance with respect to the EC, TC, and EMI shielding properties. Total shielding of 35.3 and 32.2 dB were, respectively, obtained by the PVDF/CNT/6 wt % Cochain with an EC of 2.28 S/cm and the PVDF/CNT/6 wt % Co-flower with an EC of 1.94 S/cm at a film thickness of 0.3 mm. Possibly owing to the conductive dissipation, interfacial polarization, magnetic loss, multiple reflections, and scattering of EM waves, such flexible composite films possessed a remarkable absorption-dominated EMI shielding behavior. These new composite films with enhanced TC are easily able to transform microwave energy into Joule heating systems, making themselves greatly potential for effective EMI shielding as well as rapid heat dissipation.

DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Here, a novel fluorescent nanosensor based on phosphorylation-specific exonuclease reaction and efficient fluorescence quenching of single-stranded DNA (ssDNA) by a WS2 nanosheet has been developed for monitoring the activity of PNK using T4 polynucleotide kinase (T4 PNK) as a model target. The fluorescent dye-labeled double-stranded DNA (dsDNA) remains highly fluorescent when mixed with WS2 nanosheets because of the weak adsorption of dsDNA on WS2 nanosheets. While dsDNA is phosphorylated by T4 PNK, it can be specifically degraded by λ exonuclease, producing ssDNA strongly adsorbed on WS2 nanosheets with greatly quenched fluorescence. Because of the high quenching efficiency of WS2 nanosheets, the developed platform presents excellent performance with a wide linear range, low detection limit and high signal-to-background ratio. Additionally, inhibition effects from adenosine diphosphate, ammonium sulfate, and sodium chloride have been investigated. The method may provide a universal platform for PNK activity monitoring and inhibitor screening in drug discovery and clinic diagnostics.

Abstract. ß-elemene has recently raised interest in P.R. China as a novel antitumor plant drug isolated from the Chinese medicinal herb Zedoary. To explore potentially useful combinations of ß-elemene with taxanes in the clinic, we characterized the effects of ß-elemene combined with taxanes in human lung cancer cells using a median effect analysis, micronucleus assay, apoptotic detection, and determination of gene expression in the signaling pathways of apoptosis. The synergistic analysis indicated that the interactions of ß-elemene with paclitaxel or docetaxel ranged from slight synergism to synergism. Combinations of ß-elemene with docetaxel induced much stronger synergistic interactions in p53 mutant H23 cells and p53 null H358 cells than in p53 wild-type H460 and A549 cells. Similar synergistic interactions were observed by micronucleus assay, apoptotic detection, and determination of apoptotic gene expression. Our findings indicate that the synergistic effects achieved with combinations of ß-elemene and taxanes are related to the augmented cytotoxic efficacy of taxanes owing to the action of ß-elemene. In H460 and A549 cells, dose-dependent upregulation of p53 protein expression was observed in cultures treated with docetaxel alone and with docetaxel plus ß-elemene, whereas no significant change in p53 expression was observed in any of the treatment groups in H23 cells. Fas revealed no alteration of expression with any of the treatments in this study. However, the combination treatments induced increased cytochrome c release from mitochondria, significant caspase-8 and -3 cleavage, and downregulation of Bcl-2 and Bcl-X L expression. These results suggest that, although p53 plays an important role in taxaneinduced cell death, apoptosis induced by ß-elemene or in combination with docetaxel thereof seems to be initiated through a p53-and Fas-independent pathway via mitochondria in our lung cancer cells. The suppression of specific 'survival' gene expression appears to be the key action leading to the synergistic effect of combination treatments with ß-elemene and taxanes. Finally, the ß-elemene-induced alteration of cell membrane permeability, which has potential to result in enhanced cellular uptake of taxanes, may also contribute to the synergistic interactions of the combination treatments.

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