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X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

The ice surface provides two-dimensional reaction fields for bimolecular collisions in interstellar space. As H2O molecules on the surface are typically exposed to cosmic rays, H2O in the excited state is easily dissociated into H + OH, where a H atom is released from the surface to the gas phase. In the present study, the reaction dynamics of small-sized water clusters on the triplet-state potential energy (T1) surface, following the vertical electronic excitation from the ground state (S0), were investigated using direct ab initio molecular dynamics to provide insights into the generation mechanism of H atoms from an irradiated ice surface. In all clusters, that is, (H2O)n(n= 2–6), the H atom was directly dissociated from one of the H2O molecules in the clusters (direct dissociation), whereas the OH radical remained in the cluster. In the branched form of H2O tetramer (n= 4) and the book form of H2O hexamer (n= 6), the dissociated hydrogen atom (H′) collided with the neighboring H2O molecule, and the exchange of H atoms occurred as H′ + H2O → H′-H2O (collision) → H′OH + H (hydrogen exchange). The translational energy of the exchanged H atom decreases significantly (by approximately 50%) because the kinetic energy of the H′ atom is efficiently transferred to the vibrational modes of the cluster during the H-exchange reaction. The mechanism of H atom dissociation is discussed based on theoretical results.

Background:

The electronic properties of nanocarbons can be tuned via doping. In this study, the interaction energies of Li with (n,n)-carbon belts (n= 5–20) are investigated using density functional theory to clarify the effect of curvature on doping. The magnitude of the interaction energies range from −20.6 to −14.8 kcal mol−1, with a maximum at n= 8. Essentially, two types of charge transfer occur, depending on the carbon belt diameter. For n≥ 8, the charge is widely distributed on the carbon belt, whereas for n< 8, back-donation charge transfer to Li occurs.

The activation energy of radical addition to polycyclic aromatic carbons consisting of only six-membered rings decreases with increasing curvature. In this study, the position of monohydrogenation in carbon nanotubes containing five- and six-membered carbon rings was investigated through density functional theory calculations. The activation energies of the monohydrogenation of carbon at the intersection of three six-membered rings were 2.0–2.8 kcal mol−1, and those of carbon-containing one five-membered ring were close to zero. Bonding sites involving a five-membered ring were found to have lower deformation energies for becoming sp3-like, and induced stronger interactions with hydrogen atoms.

Alkylation of the cylindrical part of single-walled carbon nanotubes can improve solubility and oriented aggregation. In this study, the activation energy of the monomethylation of carbon belt surfaces was investigated using density functional theory to clarify the effect of curvature on nucleophilic radical addition. The activation energy decreases with increasing curvature, and at κ= 0.290 Å−1, it is approximately half (7.1 kcal·mol−1) of that of the monomethylation of flat graphene flakes. The curvature significantly affects the repulsive energy between the CH3and carbon belt portions, as well as local deformation around the bonding site.

The hydration structure and electronic state of Li+doped complexes on the surface of C60were investigated by density functional theory calculations. This system is a simple model for the solvation of Li+at the anode of a lithium-ion battery. C60and Li+bind at approximately 35 kcal mol−1. The new band of C60formed the lowest excited state, 0.1 eV smaller after interaction with Li+. The water molecule preferentially interacted with the Li portion of the C60-Li+complex, and a micro-hydration structure was formed around Li+. When four or more water molecules were added to the system, Li+was removed from the C60surface by the water molecules, and a hydration shell was formed around both Li+and C60(separate hydration). The electronic interaction between C60and Li+was completely disrupted by the formation of a microscopic-hydrated shell. Herein, the mechanism is discussed based on the theoretical results.

The surface functionalization of single-walled carbon nanotubes (SWCNTs) by direct radical addition has received considerable attention. The introduction of substituents is useful for tuning the π-character, enhancing the substrate anchoring, and improving the solubility. In this study, we investigated the binding energies of mono-methylated carbon belts (short SWCNTs) using density functional theory to elucidate the effect of curvature. The binding energy decreased as the curvature κdecreased and was approximately 25 kcal mol−1less for κ= 0.166 Å−1than for κ= 0.364 Å−1. This is because a change in curvature significantly impacts the interaction energy between the CH3moiety and the carbon belt portion but not the deformation energy of the system. These results suggest that curvature can control the grafting onto the SWCNT surface.

The granitoids of Dehnow in NE Iran are part of a calc-alkaline plutonic series (diorite-to-nalite-granodiorite) that intruded the remnants of the Paleo-Tethys oceanic crust during the Triassic. New major and trace element data together with isotopic compositions elucidate their I-type nature and a deep magma origin. P-T calculations based on amphibole and plagioclase suggest crystallization stages in the upper lithosphere at an approximate pressure of 6.4 kbar and temperature of 708 °C. The Dehnow granitoids are characterized by high concentrations of LILE, LREE, HFSE and low concentrations of HREE, similar to some worldwide I-type granites, including examples from Harsit (along the Alpine-Himalayan suture zone), Iberia and the Martins Pereira plutons. The new geochemical data in combination with mineral parageneses and field observations suggest that the origin of the low temperature, Caledonian-type, arc-related granitoids of Dehnow resulted from the subduction of the Paleo-Tethys oceanic slab beneath the Turan block (along the Alpine-Himalayan suture zone) and involved the contribution of lower crust and mantle melts in this collisional setting.

Three submarine Diamante cross-chain volcanoes in the southern Mariana arc mark a magma-healed zone of along-arc (north–south) extension that allows either mafic mantle-derived basalts or felsic magmas from the middle of thickened arc crust to erupt. The largest volcano is East Diamante, with a well-developed (5×10 km) caldera that formed via violent felsic submarine eruptions beginning nearly 0.5 Ma. One or more of these eruptions also formed a giant submarine dune field extending 30 km to the NW of the volcano. Felsic igneous activity continues at least as recently as c.20 000 years ago, with emplacement of resurgent dacite domes, some hot enough to power the only black smoker hydrothermal system known in the Mariana arc. In contrast, felsic eruptions do not occur on the two volcanoes to the west, implying that the mid-crustal felsic zone does not underlie the thinner crust of the Mariana Trough back-arc basin. Diamante cross-chain lavas define a medium K suite; mafic lava phenocryst assemblages show arc-like associations of anorthite-rich plagioclase with Fe-rich olivine. Magmatic temperatures for a basaltic andesite and three dacites are c.1100 °C and c.800 °C, respectively, typical for cool, wet, subduction-related felsic magmas. Felsic magmas formed under low-P crustal conditions. The Diamante cross-chain is the southernmost of at least seven and perhaps eight Mariana arc volcanoes in a c.115 km long arc segment characterized by felsic eruptions. This is the ‘Anatahan Felsic Province’, which may have formed above a mid-crustal tonalite body that formed by fractionation or was re-melted when heated by c.1200 °C mafic, mantle-derived magmas. Across- and along-arc variations suggest that felsic eruptions and dome emplacement occurred when midcrustal tonalite was remobilized by intrusions of mafic magma, while north–south extension facilitated the development of conduits to the surface.Supplementary material:Detailed Hyperdolphin ROV dive tracks, Cook 7 dredge locations, 40Ar/39Ar analytical data, analytical methods, major and selected trace element analyses of whole rock samples, and compositional data for minerals are available at http://www.geolsoc.org.uk/SUP18611

The interaction of a hydroxyl OH radical with a graphene surface has been investigated by the density functional theory (DFT) method in order to elucidate the radical scavenge mechanism of the graphene surface. The DFT calculation showed that the OH radical binds directly to the carbon atom of the graphene surface and a strong C--O bond is formed. The binding energies were dependent on the cluster size and were distributed in the 4.1--9.5 kcal/mol range at the B3LYP/6-31G(d) level of theory. The potential energy curve plotted as a function of the distance of OH from the surface carbon showed that the OH radical can bind to the carbon atom with a low activation barrier: the barrier heights for $n = 7$ and 14 were calculated to be 3.9 and 1.9 kcal/mol, respectively. Also, it was found that the structural change from sp2to sp3-like hybridization occurs by the approach of the OH radical.

Dysregulated production of hepcidin is implicated in anemia of inflammation, whereas interleukin-6 (IL-6) is a major inducer of hepcidin production. Overproduction of IL-6 is responsible for pathogenesis of multicentric Castleman disease (MCD), a rare lymphoproliferative disorder accompanied by systemic inflammatory responses and anemia. In this study, we investigated the roles of hepcidin and IL-6 in anemia of inflammation and the long-term effects of anti–IL-6 receptor antibody (tocilizumab) treatment on serum hepcidin and iron-related parameters in MCD patients. We found that tocilizumab treatment resulted in a rapid reduction of serum hepcidin-25 in 5 of 6 MCD patients. Long-term reductions, accompanied by progressive normalization of iron-related parameters and symptom improvement, were observed in 9 of 9 cases 1.5, 3, 6, and 12 months after the start of tocilizumab treatment. In in vitro experiments, IL-6–induced up-regulation of hepcidin mRNA in hepatoma cell lines was completely inhibited by tocilizumab but increased in the presence of patients' sera. Our results suggest that, although multiple factors affect serum hepcidin levels, IL-6 plays an essential role in the induction of hepcidin in MCD. This accounts for the long-term ameliorative effect of IL-6 blockage with tocilizumab on anemia by inhibiting hepcidin production in MCD patients.

Dysregulated production of hepcidin is implicated in anemia of inflammation, whereas interleukin-6 (IL-6) is a major inducer of hepcidin production. Overproduction of IL-6 is responsible for pathogenesis of multicentric Castleman disease (MCD), a rare lymphoproliferative disorder accompanied by systemic inflammatory responses and anemia. In this study, we investigated the roles of hepcidin and IL-6 in anemia of inflammation and the long-term effects of anti–IL-6 receptor antibody (tocilizumab) treatment on serum hepcidin and iron-related parameters in MCD patients. We found that tocilizumab treatment resulted in a rapid reduction of serum hepcidin-25 in 5 of 6 MCD patients. Long-term reductions, accompanied by progressive normalization of iron-related parameters and symptom improvement, were observed in 9 of 9 cases 1.5, 3, 6, and 12 months after the start of tocilizumab treatment. In in vitro experiments, IL-6–induced up-regulation of hepcidin mRNA in hepatoma cell lines was completely inhibited by tocilizumab but increased in the presence of patients' sera. Our results suggest that, although multiple factors affect serum hepcidin levels, IL-6 plays an essential role in the induction of hepcidin in MCD. This accounts for the long-term ameliorative effect of IL-6 blockage with tocilizumab on anemia by inhibiting hepcidin production in MCD patients.

Electronic states of graphenes, whose carbon atoms are terminated by hydrogen atoms (hydrogenated graphene, denoted H-graphene) and defective graphene (one carbon atom was removed from H-graphene, denoted D-graphene) have been investigated by density functional theory. The sizes of graphenes examined in the present study were n= 7, 14, 19, 29, 37, 44, and 52; where nis the number of benzene rings in the graphene. The excitation energies of H-graphenes were gradually decreased as a function of the number of rings. In D-graphene, new energy levels for the first and second excited states appeared as low-lying excited states. It was found that the formation of defect sites in graphene produces large decreases in the excitation energies for third and higher excited states. The highest occupied molecular orbital and lowest unoccupied molecular orbital (LUMO) in H-graphene were widely delocalized over the graphene surface. On the other hand, LUMO in D-graphene was localized only in the defect sites. The effects of vacancy defects on both the ground and excited electronic states of graphene were discussed on the basis of theoretical results.

Electronic states of normal graphene, the defective graphene (one carbon atom is removed from the normal graphene), the defective graphene anion (defective graphene plus an excess electron), and the defective graphene cation (defective graphene plus one hole) have been investigated by means of density functional theory (DFT) and direct molecular orbital−molecular dynamics (MO-MD) methods in order to elucidate the effect of vacancy defect on the electronic states of graphene. The HOMO and LUMO of normal graphene were widely delocalized as π-conjugated orbitals over the graphene surface in the normal graphene. On the other hand, the excess electron in defective graphene anion was localized in the defect site, indicating that the excess electron on the graphene circuit is efficiently trapped and stabilized by the vacancy defect site of graphene. The direct MO-MD calculations showed that the trapped electron in the defect site is stable at low temperature. Around room temperature (300 K), the structural change of the graphene backbone was found and the vacancy defect was reconstructed by thermal activation. The excess electron escaped from the defect site of the reconstructed graphene, while the spin density delocalized the graphene.

The Samoan mantle plume is thought to host three isotopically (radiogenic) distinct low‐3He/4He components including EM2 (enriched mantle 2), dilute HIMU (high μ= 238U/204Pb), and a depleted mantle (DM) component, which were sampled by shield‐stage lavas from the Malu, Vai, and Upo trend volcanoes, respectively. However, it is unclear whether the isotopically distinct components are present as different lithologies. Using new Fe–Sr–Nd–Pb isotope data for Tutuila basalts (Samoa), combined with literature data for other Samoan basalts, we attempt to infer the lithological structure of the Samoan plume. The results show that “Malu trend” basalts have heavier Fe isotopic compositions (δ57Fe = 0.15–0.24‰) than “Vai trend” and “Upo trend” basalts. The latter two groups have average δ57Fe of 0.14 ± 0.07‰ (2SD) and 0.11 ± 0.03‰ (2SD), respectively, similar to normal midocean ridge basalts (N‐MORBs, δ57Fe = 0.15 ± 0.05‰, 2SD). The fractional‐crystallization‐corrected δ57Fe values of all shield lavas are positively correlated with (Gd/Yb)N, Pb/Nd and 87Sr/86Sr ratios whereas negatively correlated with Nb/Th and εNdratios, which cannot be explained by partial melting of a single garnet peridotite but point to heterogeneous source lithologies. The EM2 lavas are characterized with high δ57Fe and (Gd/Yb)N, low Nb/Th, and enriched Sr–Nd isotopic ratios, requiring a pyroxenitic source component with imprints of both recycled terrigenous sediments and oceanic crust. The Vai‐ and Upo‐trend lavas with MORB‐like δ57Fe can be explained by partial melting of peridotitic sources, although different extents of refertilization by recycled crust are essential for generating their distinct radiogenic isotope signatures. These observations highlight the lithological heterogeneity of the Samoan plume and relates the EM2 component with a pyroxenitic lithology. Lavas erupted on Samoan islands/seamounts construct three parallel volcanic lineaments: Upo‐, Malu‐, and Vai‐trend. Basalt samples from each of the lineaments have distinctive chemical and isotopic compositions. They are thought to be the surface expressions of three isotopically distinct components in the Samoan mantle plume beneath the volcanic lineaments. The physical nature (lithological identity) of the three distinct mantle components is still unclear. Recent efforts have found that iron isotope ratios (e.g., the ratio of iron‐57 to iron‐54) are a new tracer of mantle source mineralogy of basaltic lavas. In this study, we use iron isotopic compositions of basalts from Tutuila Island, American Samoa, together with literature data for other Samoan basalts, to infer the lithological identity of the three distinct mantle components. We find that these lavas have variable iron isotopic compositions, which cannot be ascribed to fractional crystallization and partial melting process. The correlations between iron isotopes and radiogenic isotopes of these lavas indicate that their difference in iron isotopic compositions is most likely caused by difference in mantle source mineralogy. Specifically, a pyroxene‐dominated lithology (e.g., pyroxenite) is required to explain the heavy iron isotopic compositions (high ratio of iron‐57 to iron‐54) of lavas from the Malu trend, while the partial melting of peridotitic mantle sources can reproduce the iron isotopic signatures of lavas from the Vai‐ and Upo‐trend volcanoes. These observations highlight the heterogeneous lithological structure of the Samoan mantle plume. Samoan shield‐stage lavas show heterogeneous Fe isotopic compositionsThe EM2 (enriched mantle 2) component in the Samoan mantle plume is pyroxenitic with heavy Fe isotopic compositionsThe other components in this mantle plume are most likely to be peridotitic with primitive‐mantle‐like Fe isotopic compositions Samoan shield‐stage lavas show heterogeneous Fe isotopic compositions The EM2 (enriched mantle 2) component in the Samoan mantle plume is pyroxenitic with heavy Fe isotopic compositions The other components in this mantle plume are most likely to be peridotitic with primitive‐mantle‐like Fe isotopic compositions

Hybrid density functional theory (DFT) calculations have been carried out for the radical cation and anion of permethyloligosilane Sin(CH3)2n2±(n 2−6) to elucidate the electronic structures at ground and low-lying excited states, and the results were compared with the corresponding experimental values. In particular, the assignment of electronic transition appeared at near-IR and visible regions, which is strongly correlated to hole and electron conductivity, and was carried out on the basis of time-dependent DFT calculation. The structure of oligosilane was generated at 300 K by direct PM3 molecular dynamics calculations, and then the geometry was fully optimized at the DFT(B3LYP)/6-311G(d,p) level. It was found that the hole in the radical cation and the electron in the radical anion of oligosilane are delocalized over the Si skeleton. The proton-hyperfine coupling constants calculated were in good agreement with those obtained by an electron spin resonance experiment. It was also found that the g-anisotropy of the radical anion was significantly larger than that of the radical cation. The IR bands of radical ions were assigned on the basis of theoretical calculations.

Introduction: Myelodysplastic syndromes (MDS) is thought to develop and progress as a result of accumulation of genetic mutations. This multicenter, open-label, phase II study examined impact of gene mutations on the effect of darbepoetin alfa (DA) for anemia. Death within 1 year and progression to acute myeloid leukemia (AML) were also examined.

Hepcidin, a key regulator of iron metabolism, is expressed in the liver, distributed in blood, and excreted in urine. However, to date, no reliable and practical method for measuring the bioactive form of hepcidin in serum has been developed. Here, we used surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) to analyze the distinctive serum proteomic patterns of patients receiving hemodialysis. In the range of 1000 to 15 000 m/z, we found 3 peptides at 2192, 2789, and 2851 m/z that showed a significant correlation with the serum ferritin levels. The molecular sizes of peptides at 2192 and 2789 m/z matched with the reported sizes of hepcidin-20 and -25, respectively, and the serum peptide at 2789 m/z was identified as hepcidin-25 by collision-induced dissociation tandem MS. By using SELDI-TOF MS, we developed a semiquantitative assay for hepcidin-25. In this assay, the level of serum hepcidin-25 correlated well with levels of serum ferritin and serum interleukin-6. Hepcidin-25 was found to accumulate in the serum of patients receiving hemodialysis; this could contribute to the pathogenesis of renal anemia by decreasing the available iron for hematopoiesis. Thus, SELDI-TOF MS would be a clinically useful tool to detect and semiquantify bioactive hepcidin in serum.

Hepcidin, a key regulator of iron metabolism, is expressed in the liver, distributed in blood, and excreted in urine. However, to date, no reliable and practical method for measuring the bioactive form of hepcidin in serum has been developed. Here, we used surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) to analyze the distinctive serum proteomic patterns of patients receiving hemodialysis. In the range of 1000 to 15 000 m/z, we found 3 peptides at 2192, 2789, and 2851 m/z that showed a significant correlation with the serum ferritin levels. The molecular sizes of peptides at 2192 and 2789 m/z matched with the reported sizes of hepcidin-20 and -25, respectively, and the serum peptide at 2789 m/z was identified as hepcidin-25 by collision-induced dissociation tandem MS. By using SELDI-TOF MS, we developed a semiquantitative assay for hepcidin-25. In this assay, the level of serum hepcidin-25 correlated well with levels of serum ferritin and serum interleukin-6. Hepcidin-25 was found to accumulate in the serum of patients receiving hemodialysis; this could contribute to the pathogenesis of renal anemia by decreasing the available iron for hematopoiesis. Thus, SELDI-TOF MS would be a clinically useful tool to detect and semiquantify bioactive hepcidin in serum.

Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. (Blood. 2005;105:376-381)

Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. (Blood. 2005;105:376-381)

Exiguobacteriumsp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced viaan inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coliby simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.

Structures and electronic states of a molecular complex formed between fluorenone and sodium atom (denoted by FL–Na) have been investigated by means of ab initio density functional theory (DFT). The most stable structure of FL–Na has a C_s symmetry where the sodium atom is located on the C_s plane perpendicular to the molecular plane of FL where all atoms are located. The sodium atom binds to the carbonyl oxygen of FL and the angle of Na–O–C is calculated to be 157° at the B3LYP/6-311+G(d) level. The charge on the Na atom shows a positive value (~+0.80), and unpaired electron is delocalized over the FL molecule, indicating that the FL–Na complex exists as an ion-pair state expressed by (C&z.dbd;O)δ−(Na)δ+ at the ground state (δ ~ +0.80). The proton hyperfine coupling constants and excitation energies calculated at the B3LYP/6-311+G(d) and B3LYP/6-31++G(d,p) levels are in good agreement with previous experiments. The electronic states at the ground and first excited states were discussed.

Structures and electronic states of a molecular complex formed between fluorenone and sodium atom denoted by FL–Na have been investigated by means of ab initiodensity functional theory DFT. The most stable structure of FL–Na has a Cssymmetry where the sodium atom is located on the Csplane perpendicular to the molecular plane of FL where all atoms are located. The sodium atom binds to the carbonyl oxygen of FL and the angle of Na–O–C is calculated to be 157° at the B3LYP6-311Gd level. The charge on the Na atom shows a positive value ∼0.80, and unpaired electron is delocalized over the FL molecule, indicating that the FL–Na complex exists as an ion-pair state expressed by COδ−Naδat the ground state δ ∼ 0.80. The proton hyperfine coupling constants and excitation energies calculated at the B3LYP6-311Gd and B3LYP6-31Gd,p levels are in good agreement with previous experiments. The electronic states at the ground and first excited states were discussed.

Ab-initioDFT and configuration interaction CI calculations have been carried out for indium–carbonyl compounds, such as acetone–In Ac–In, 1,8-naphthalic anhydride–In In2NA and 3,4,9,10-perylenetetracarboxylic dianhydride–indium In4PTCDA, in order to shed light on the mechanism of electronic conductivity in the molecular devices. It was found that the electronic state of these complexes at the ground state is composed of ion-pair states expressed approximately by Acδ−Inδ, In2δNAδ−and In4δPTCDAδ−, respectively. On the other hand, the interaction at the first excited state was changed to a van der Waals interaction. Namely, the electron returned again to the In atom by electronic excitation. The second excited state is attributed to internal excitation within the carbonyl anion, while the charge on the indium was close to neutral. The mechanism of the electronic conductivity was discussed on the basis of theoretical results.

Ab-initio DFT and configuration interaction (CI) calculations have been carried out for indium–carbonyl compounds, such as acetone–In (Ac–In), 1,8-naphthalic anhydride–In (In₂NA) and 3,4,9,10-perylenetetracarboxylic dianhydride–indium (In₄PTCDA), in order to shed light on the mechanism of electronic conductivity in the molecular devices. It was found that the electronic state of these complexes at the ground state is composed of ion-pair states expressed approximately by (Acδ−)(Inδ+), (In₂)δ+(NA)δ− and (In₄)δ+(PTCDA)δ−, respectively. On the other hand, the interaction at the first excited state was changed to a van der Waals interaction. Namely, the electron returned again to the In atom by electronic excitation. The second excited state is attributed to internal excitation within the carbonyl anion, while the charge on the indium was close to neutral. The mechanism of the electronic conductivity was discussed on the basis of theoretical results.

Iron is essential for cell proliferation, heme synthesis, and a variety of cellular metabolic processes. In most cells, transferrin receptor–mediated endocytosis is a major pathway for cellular iron uptake. Recently, transferrin receptor 2 (TfR2), another receptor for transferrin, was cloned. High levels of expression of TfR2 messenger RNA (mRNA) occur in the liver, as well as in HepG2 (a hepatoma cell line) and K562 (an erythroid leukemia cell line). In this study, TfR2 mRNA expression was analyzed in hematological cell lines, normal erythroid cells at various stages of differentiation, and leukemia and preleukemia cells. High levels ofTfR2 expression occurred in all of the erythroid cell lines that were examined. Erythroid-specific expression of TfR2 protein in bone marrow cells was confirmed by immunohistochemical staining. Expression of TfR2 mRNA was high in normal CD34+ erythroid precursor cells, and levels decreased during erythroid differentiation in vitro. Levels of expression of TfR2-α mRNA were significantly higher in erythroleukemia (M6) marrow samples than in nonmalignant control marrow samples. In addition, relatively higher levels of TfR2-αmRNA expression occurred in some samples of myelodysplastic syndrome that had erythroid hyperplasia in bone marrow, acute myelogenous leukemia M1, M2, and chronic myelogenous leukemia. Expression profiles of normal members of the erythroid lineage suggest that TfR2-α may be a useful marker of early erythroid precursor cells. The clinical significance of TfR2-α expression in leukemia cells remains to be determined.

Iron is essential for cell proliferation, heme synthesis, and a variety of cellular metabolic processes. In most cells, transferrin receptor–mediated endocytosis is a major pathway for cellular iron uptake. Recently, transferrin receptor 2 (TfR2),another receptor for transferrin, was cloned. High levels of expression of TfR2messenger RNA (mRNA) occur in the liver, as well as in HepG2 (a hepatoma cell line) and K562 (an erythroid leukemia cell line). In this study, TfR2mRNA expression was analyzed in hematological cell lines, normal erythroid cells at various stages of differentiation, and leukemia and preleukemia cells. High levels ofTfR2expression occurred in all of the erythroid cell lines that were examined. Erythroid-specific expression of TfR2 protein in bone marrow cells was confirmed by immunohistochemical staining. Expression of TfR2mRNA was high in normal CD34+erythroid precursor cells, and levels decreased during erythroid differentiation in vitro. Levels of expression of TfR2-αmRNA were significantly higher in erythroleukemia (M6) marrow samples than in nonmalignant control marrow samples. In addition, relatively higher levels of TfR2-αmRNA expression occurred in some samples of myelodysplastic syndrome that had erythroid hyperplasia in bone marrow, acute myelogenous leukemia M1, M2, and chronic myelogenous leukemia. Expression profiles of normal members of the erythroid lineage suggest that TfR2-α may be a useful marker of early erythroid precursor cells. The clinical significance of TfR2-αexpression in leukemia cells remains to be determined.

Complementary and genomic DNA for the murine transferrin receptor 2 (TfR2) were cloned and mapped to chromosome 5. Northern blot analysis showed that high levels of expression of murine TfR2 occurred in the liver, whereas expression of TfR1 in the liver was relatively low. During liver development, TfR2 was up-regulated and TfR1 was down-regulated. During erythrocytic differentiation of murine erythroleukemia (MEL) cells induced by dimethylsulfoxide, expression of TfR1 increased, whereas TfR2 decreased. In MEL cells, expression of TfR1 was induced by desferrioxamine, an iron chelator, and it was reduced by ferric nitrate. In contrast, levels of TfR2 were not affected by the cellular iron status. Reporter assay showed that GATA-1, an erythroid-specific transcription factor essential for erythrocytic differentiation at relatively early stages, enhanced TfR2 promoter activity. Interestingly, FOG-1, a cofactor of GATA-1 required for erythrocyte maturation, repressed the enhancement of the activity by GATA-1. Also, CCAAT-enhancer binding protein, which is abundant in liver, enhanced the promoter activity. Thus, tissue distribution of TfR2 was consistent with the reporter assays. Expression profiles of TfR2 were different from those of TfR1, suggesting unique functions for TfR2, which may be involved in iron metabolism, hepatocyte function, and erythrocytic differentiation.

Complementary and genomic DNA for the murine transferrin receptor 2 (TfR2) were cloned and mapped to chromosome 5. Northern blot analysis showed that high levels of expression of murine TfR2 occurred in the liver, whereas expression of TfR1 in the liver was relatively low. During liver development, TfR2 was up-regulated and TfR1 was down-regulated. During erythrocytic differentiation of murine erythroleukemia (MEL) cells induced by dimethylsulfoxide, expression of TfR1 increased, whereas TfR2 decreased. In MEL cells, expression of TfR1 was induced by desferrioxamine, an iron chelator, and it was reduced by ferric nitrate. In contrast, levels of TfR2 were not affected by the cellular iron status. Reporter assay showed that GATA-1, an erythroid-specific transcription factor essential for erythrocytic differentiation at relatively early stages, enhanced TfR2 promoter activity. Interestingly, FOG-1, a cofactor of GATA-1 required for erythrocyte maturation, repressed the enhancement of the activity by GATA-1. Also, CCAAT-enhancer binding protein, which is abundant in liver, enhanced the promoter activity. Thus, tissue distribution of TfR2 was consistent with the reporter assays. Expression profiles of TfR2 were different from those of TfR1, suggesting unique functions for TfR2, which may be involved in iron metabolism, hepatocyte function, and erythrocytic differentiation.

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem.274, 20826–20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1or TfR2-αcDNA. TfR2-α-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression ofTfR2mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2mRNA was regulated in the cell cycle with the highest expression in late G1phase and no expression in G0/G1. DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-α-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-α developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth bothin vitroand in vivo.

Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: α (∼2.9 kilobase pairs), and β (∼2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-α protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-β protein lacked the amino-terminal portion of the TfR2-α protein including the putative transmembrane domain. Northern blot analysis showed that the α transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-α. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tf-bound 55Fe uptake. Taken together, TfR2-α may be a second transferrin receptor that can mediate cellular iron transport.

A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliaeAKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced α-, β-keto esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.

A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg2+-dependent and DNase ll-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast a clear difference was shown in the circulating CD34+cells, in that only Mg2+-dependent, Ca2+-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg2+-dependent and acidic endonuclease concomitantly with the disappearance of the Mg2-dependent endonuclease when CD34+cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg2+-dependent nuclease activity. Our results suggest an association of the Mg2+-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation.

Two co-dimers of styrene and p-chlorostyrene having regulated monomer sequences were obtained by the reactions of 1-(4-chlorophenyl) ethyl diphenyl phosphate with styrene and of 1-phenylethyl diphenyl phosphate with p-chlorostyrene. The structures of the co-dimers were confirmed by GC-MS and analyses of the oxidative cleavage products.

Copolymerization of 2,4-bisphenyl-1,3,2,4-dioxadiphosphetane-2,4-dioxide (DPO) with oxetane, a cyclic ether, was found to take place at 90 and 120°C without initiator. Copolymers obtained have a structure of phosphonate-ether units (1), which was determined by 31P, 1H and 13C NMR, IR and elemental analysis. Oxetane was incorporated into 1 in a slight excess in all runs(m value in 1=1.5–2.1). A small amount(<15%) of a by-product (2) was always formed. Other cyclic ethers such as ethylene oxide and propylene oxide did not afford copolymers but gave 1:1 addition products, 10 and 11, quantitatively. The copolymerization is reasonably explained by a mechanism in which DPO behaves as an electrophilic monomer and oxetane as a nuicleophilic one. A pyrophosphonate species is considered to be a key intermediate to lead to copolymer 1 and the whole reaction course is discussed in detail.

Dimers were preferably obtained in the oligomerization of styrene derivatives by diphenyl hydrogen phosphate. The reaction was assumed to proceed through the formation of esters from the monomers and diphenyl hydrogen phosphate as intermediate. This assumption was confirmed by the fact that treatment of the esters with alternative styrene derivatives gave linear codimers.

The presence of at least two distinct Mg2+- or Mn2+-dependent, Ca2+-independent endonuclease activities was shown in the myeloid leukemia cell line P39. One of them was recovered from nuclear extracts and the other from a cytoplasmic fraction. The molecular size of the former was 30 kDa in both gel filtration and activity gel and that of the latter approximately 130-140 kDa in gel filtration and 65-70 kDa in activity gel. These two activities were almost completely inhibited by 0.1 mM ZnCl2or 0.1 mM aurintricarboxylic acid, common inhibitors of apoptosis. Both could produce nucleosomal-size DNA fragmentation when incubated with diethyl-pyrocarbonate-treated nuclei as substrates, and the pattern of cleavage was 3′-OH and 5′-P. Taken together, either or both of these activities may be associated with apoptosis of myeloid leukemia cells.

As a convenient method for modifying the hole-transport property of fullerene materials, hydrogenation of fullerene C70is considered theoretically. Firstly, for the analysis of the carrier-transfer mechanism between C70H2molecules, the geometrical difference between C70H2and C70H2+, the natural population analysis (NPA) charge, and the electron spin resonance (ESR) parameters (spin density) of C70H2+are calculated by density functional theory [B3LYP/6-311G(d,p)]. Secondly, the reorganization energies ($\lambda$) and electronic coupling elements ($H_{\text{AB}}$) of eight isomers of C70H2with a small heat of formation are calculated and compared with that of C70. It is shown that four isomers of C70H2have a smaller $\lambda$ than C70(120 meV) and that the magnitude of $\lambda$ of C70H2isomers is closely related to the geometrical difference between C70H2and C70H2+. Four isomers of C70H2have larger $H_{\text{AB}}$ than C70(24 meV). Isomers with delocalized highest occupied molecular orbital (HOMO) tend to have small $\lambda$ and large $H_{\text{AB}}$. At 300 K, the best isomer has hole-transfer rate constant ($k_{\text{ht}}$) which is over six times as large as that of C70.

The functionalization of graphene surfaces by covalent bonds has attracted much attention. Hydrogenated graphene exhibits ferromagnetism, and the degree of hydrogenation on the graphene surface allows for tuning the band gap. In this study, to elucidate the effect of defects in graphene hydrogenation, we investigated defective graphene wherein one carbon atom was removed from the central region of graphene nano-flake, by density functional theory. Hydrogenation of a graphene nano-flake by electrophilic addition in the gas phase is an exothermic reaction only when defects exist. The activation energy of defective graphene nano-flake hydrogenation is approximately 25 kcal mol[?]1 lower than that of the normal graphene one due to the involvement of the defect in surface distortion and transition state stabilization. The results obtained suggest that the hydrogenation of a graphene surface can be controlled by carbon defect.

The functionalization of fullerenes, which are excellent n-type materials, has received much attention. The substituents enable tuning of the electronic state and fixing to the substrate. In this study, to elucidate the hydrofluorination mechanism of C60 fullerene, reaction mechanisms involving one or two hydrogen fluoride (HF) molecules were investigated by means of density functional theory. The hydrofluorination of C60 in the gas phase is an electrophilic addition reaction with only the 1,2-addition being an exothermic. The activation energy of hydrofluorination by two molecules of HF is about 30 kcal mol[?]1 lower than that with one molecule of HF. This is because hydrogen bonds between HF molecules are involved in the stabilization of the reactant and the aromaticity improvement of the transition state. These results suggest that hydrofluorination can be controlled by the size of the HF cluster.

Gallium-doped zinc oxide films prepared by atomic layer deposition have received much attention. When triethylgallium is hydrolyzed on the surface of zinc hydroxide, a multi-layered metal oxide film containing a specific amount of gallium at a controlled position is formed. In this study, to elucidate the micro-hydrolysis mechanism of triethylgallium, reaction mechanisms involving one or two water molecules were investigated by molecular orbital calculations. The complete hydrolysis of triethylgallium occurs in three steps with the first partial hydrolysis being the rate limiting step. The activation energy of hydrolysis by two molecules of water is [?]10 kcal mol[?]1 lower than that with one molecule of water for all steps. The addition of further water molecules significantly decreases the activation energy because the transition state is stabilized by hydrogen bonding between the water molecules. These results suggest the hydrolysis reaction proceeds spontaneously under mild conditions by controlling the concentration of water molecules.

Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations.

Morimoto: Grant-in-Aid for JSPS Research Fellow: Research Funding. Takaori-Kondo: celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria.

Sideroblastic anemias consist of a heterogeneous group of inherited and acquired disorders. The most common hereditary type is X-linked sideroblastic anemia (XLSA), which is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Heme synthesis starts with the polymerization of glycine and succinyl CoA polymerization and synthesis of δ-aminolevulinic acid (ALA) in the mitochondria. ALAS2encodes the enzyme that catalyzes these first steps in the heme synthetic pathway in erythroid cells, steps that require pyridoxal 5'-phosphate (PLP) as a cofactor. It has been found that treatment with PLP is effective for a small fraction of XLSA patients, but there are no effective treatments for the other fraction. The aim of this study is to explore the molecular mechanisms of XLSA and to develop new effective therapies.