In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNAbinding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wildtype and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation. (Blood. 2010;115:677-686)

Aim: Metabolic syndrome (MetS) is a cluster of metabolic abnormalities and a predictor of both type 2 diabetes mellitus and adverse cardiovascular events. Whether there are gender differences in the association between early atherosclerosis and MetS has not yet been thoroughly elucidated. Methods: The subjects consisted of 388 men aged 64 16 years and 480 women aged 70 13 years. Early atherosclerosis was assessed by carotid intima-media thickness (IMT) on B-mode ultrasonography. Results: Carotid IMT values were significantly greater in both male (P 0.007) and female (P 0.002) subjects with MetS. After adjusting for established risk factors, the difference persisted on a significant level in women (P 0.003), but was weak in men (P 0.013). Multiple regression analysis using IMT as an objective variable, with adjustment for various risk factors as explanatory variables, showed that MetS (P 0.016) was a significant independent contributing factor along with known risk factors only in women. Among the components of MetS, hypertension (P 0.036) and dyslipidemia (P 0.008) had a strong impact on carotid IMT in men, whereas hypertension (P 0.003) ranked first in women. Conclusion: The effect of MetS in early carotid atherosclerosis is more pronounced in women than in men, and the impact of MetS components on carotid IMT differs between men and women.

Background: S-adenosylmethionine-dependent methyltransferase inhibitor, DZNep, targets the degradation of histone methyltransferase EZH2 that catalyzes H3K27 trimethylation.Results: DZNep induced erythroid-related genes, which may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2.Conclusion: DZNep has the capacity to induce erythroid differentiation.Significance: Our data may be exploited for therapeutic applications for hematological diseases, including anemia.

BackgroundPrimary adrenal lymphoma (PAL) is an extremely rare subtype of extranodal non-Hodgkin’s lymphoma. Some researchers have reported some of the characteristics of PAL and its association with poor prognosis; however, the clinicopathological features of PAL remain to be elucidated.MethodsFrom 2008 to 2011 we experienced seven cases of PAL in our institutions. We retrospectively analyzed the clinical and pathological features of these patients.ResultsThe patients ranged in age from 50 to 85 years, with a median of 71 years. The overall male:female ratio was 6:1. All seven patients were diagnosed with diffuse large B-cell lymphoma (DLBCL) pathologically. Bilateral adrenal involvement was confirmed in five patients. The median largest tumor diameter at diagnosis was 58 mm. The Ki-67 index was generally high (>70%). All patients were treated with rituximab-containing chemotherapy, and central nervous system (CNS) prophylaxis was conducted for three patients. One patient with CNS involvement at the time of the diagnosis also received whole-brain radiation. The overall survival rate at two years was 57% (median follow-up; 24.8 months). It is noteworthy that the three patients who received a full course of the rituximab-containing regimen and CNS prophylaxis are currently alive without disease relapse, and that none of the seven patients died due to progression of lymphoma.ConclusionsPrimary adrenal DLBCL can be a clinically aggressive disease entity. Rituximab-containing chemotherapy combined with CNS prophylaxis could be a reasonable option for the treatment of PAL; however, analyses of more PAL cases are needed for the establishment of this strategy.

Aim: Carotid intima-media thickness (IMT) is a useful surrogate marker of cardiovascular disease and is associated with cardiac events. We investigated cross-sectionally the association between carotid intima-media thickness (IMT), confounding risk factors, and metabolic syndrome (MetS) using the modified Japanese criteria.

Otx2 is a paired type homeobox gene that plays essential roles in each step and site of head development in vertebrates. In the mouse, Otx2 expression in the anterior neuroectoderm is regulated primarily by two distinct enhancers: anterior neuroectoderm (AN) and forebrain/midbrain (FM) enhancers at 92 kb and 75 kb 5 of the Otx2 locus, respectively. The AN enhancer has activity in the entire anterior neuroectoderm at headfold and early somite stages, whereas the FM enhancer is subsequently active in the future caudal forebrain and midbrain ectoderm. In tetrapods, both AN and FM enhancers are conserved, whereas the AN region is missing in teleosts, despite overt Otx2 expression in the anterior neuroectoderm. Here, we show that zebrafish and fugu FM regions drive expression not only in the forebrain and midbrain but also in the anterior neuroectoderm at headfold stage. The analysis of coelacanth and skate genomic Otx2 orthologues suggests that the utilization of the two enhancers, AN and FM, is an ancestral condition. In contrast, the AN enhancer has been specifically lost in the teleost lineage with a compensatory establishment of AN activity within the FM enhancer. Furthermore, the AN activity in the fish FM enhancer was established by recruiting upstream factors different from those that direct the tetrapod AN enhancer, yet zebrafish FM enhancer is active in both mouse and zebrafish anterior neuroectoderm at the headfold stage.anterior neuroectoderm ͉ coelacanth ͉ enhancer ͉ tetrapod ͉ chondrichthyes T he vertebrate head is an evolutionary novelty called ''new head'' by Gans and Northcutt (1) that is characterized by structures that derive from the anterior neuroectoderm cells, cephalic neural crest cells, and placode cells. It is also a structure that has most dramatically changed during vertebrate evolution. Diversity in the animal body plan might have been brought about by changes in expression of a relatively limited number of key developmental regulators such as Hox genes in the trunk (2, 3). The Otx family of genes plays essential roles in head development (4-9). Otx genes encode a paired-type of homeoprotein homologous to a Drosophila head gap gene, otd. Gnathostomes possess three paralogues, Otx1, Otx2, and Otx5, whereas teleosts that underwent genome duplication could possess extra copies. In mouse, Otx2 plays major roles in each site of head development (5-11): epiblast, anterior visceral endoderm, anterior mesendoderm, anterior neuroectoderm, forebrain/midbrain, and cephalic neural crest cells. Fugu has two Otx2, but zebrafish has only one (9).To elucidate the molecular mechanisms regulating Otx2 expression during mouse brain development, we have identified three enhancers: anterior neuroectoderm enhancer (AN), forebrain/midbrain enhancer (FM), and forebrain/midbrain enhancer 2 (FM2) at 90 kb and 75 kb upstream and 115 kb downstream of the Otx2 translational start site, respectively (8, 9). The AN enhancer is not active in the epiblast but becomes active at embryonic day (E) 7.0 in the entire anterior...

Methods Generation of bone marrow mesenchymal stem cellsTo generate mouse BM-MSC, bone marrow cells from GATA2 conditional knockout mice were cultured in MesenCult MSC Basal Medium supplemented with 20% MSC stimulatory supplements (Stem Cell Technologies). The BM-MSC were transfected with the retroviruses expressing iCre to delete the DNA binding domain of GATA2 by inducing the Cre-loxP system. 21,22 To generate human BM-MSC, bone marrow mononuclear cells from healthy donors were cultured with Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 20% fetal bovine serum (Life Technologies), 10 ng/mL basic fibroblast growth factor (PeproTech), 10 mM HEPES (Life Technologies), and 100 μg/mL penicillin/streptomycin (Invitrogen). [23][24][25] Established BM-MSC were used until the seventh generation.The study was approved by the ethical committee of Tohoku University Graduate School of Medicine. Clinical samples were collected after obtaining written informed consent. The ethics policies of the Declaration of Helsinki were followed. Characterization of bone marrow mesenchymal stem cellsBM-MSC immunophenotypes were determined using a FACSAria II (BD). To induce differentiation into adipocytes, human Mesenchymal Stem Cell Adipogenic Differentiation Medium (Lonza) was used. After 12-16 days, morphological changes were assessed using an inverted microscope. Typical adipocytes were stained with Oil Red O.2 The area of mature adipocytes was determined using HistoQuest software (Novel Science). Quantitative reverse transcriptase polymerase chain reaction analysis and transcription profilingQuantitative reverse transcriptase polymerase chain reaction analysis (RT-PCR) was performed as previously described.26 Primer sequences are available upon request.For transcription profiling, the Human Genome U133 Plus 2.0 Array was used (Affymetrix). Gene ontology analysis was conducted using the DAVID bioinformatics program (http://david.abcc.ncifcrf.gov/). Short interfering RNA-mediated knockdownAnti-GATA2 and control short interfering RNA (siRNA) 26 were transfected into human BM-MSC with Lipofectamine TM RNAiMAX reagent (Life Technologies). Cells were analyzed 48 h after transfection. Viral vectors and cell transductionRetroviral overexpression of GATA2 was performed using the MSCV retrovirus vector, which co-expresses green fluorescent protein (GFP) by internal ribosome entry sites (IRES), transfecting into Platinum Retroviral Packaging Cell Lines (PLAT-F) 27 with FuGENE HD (Roche). Human BM-MSC were pretreated with Retronectin (TAKARA BIO.), and GFP-positive cells were sorted using FACSAria II (BD Biosciences).Co-culture of CD34-positive-enriched cells with a mesenchymal stem cell feeder layer BM-MSC were transfected with control or GATA2-siRNA. On day 3, control and GATA2 knockdowned BM-MSC, respectively, were replaced with serum-free medium containing CD34-positiveenriched cells (RIKEN). Serum-free medium (StemPro-34 SFM: Life Technologies) contained 100 ng/mL stem cell factor, 100 ng/mL interleukin (...

A 27-year-old