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We investigated the effects of antipsychotics on human induced pluripotent stem cell (hiPSC)-derived neural stem cell (NSC) differentiation. Induction of NSCs from hiPSCs was performed using PSC neural induction medium. Induced NSCs were subsequently cultured in neural differentiation medium containing antipsychotics. Cultured cells were subjected to neural differentiation marker analysis. As previously shown in rodent cells, antipsychotics promoted neural differentiation compared with vehicle treatment. Atypical antipsychotics appear to possess more differentiation induction potential than typical ones. Most NSCs do not express dopamine D2 receptor; however, our in vitro study indicates the clinical potential of antipsychotics could include effects independent of monoamine receptor expression in NSCs. Our study shows NSCs derived from hiPSCs provide opportunity to investigate the underlying direct effect of antipsychotics treatment on NSCs. [ABSTRACT FROM AUTHOR]

Abstract: Clinical trials with type 1 and type 2 diabetes have identified a phenomenon known as “metabolic memory” in which previous periods of hyperglycemia result in the long-lasting deleterious impact on cardiovascular events. Emerging evidence shows that transient hyperglycemic exposure of human endothelial cells induces histone 3 lysine 4 mono-methylation (H3K4me1) on the promoter and persistent mRNA expression of RelA and IL-8 genes, suggesting that epigenetic histone modification and chromatin structure remodeling is a key event underlying metabolic memory. This burgeoning hypothesis, however, critically remains to be tested for relevance in the disease process of diabetes in vivo, and for broader applicability to an array of genes involved in endothelial dysfunction. To address this, we used type 1 diabetes mouse model induced by streptozocin to be hyperglycemic for 8weeks, and isolated endothelial cells that were used either freshly after isolation or after 2 to 3-week cell culture in normoglycemic conditions. mRNA expression profiling in diabetic mouse endothelial cells revealed significant and persistent up-regulation of Serpine1 encoding PAI-1, the hypo-fibrinolytic mediator leading to thrombotic diseases in diabetes, along with Rock2, Fn1 and Ccl2, whereas only Serpine 1 was persistently elevated in high glucose-treated mouse endothelial cells. Chromosome immunoprecipitation assay in type 1 diabetic mouse endothelial cells showed predominant enrichment of H3K4 tri-methylation on Serpine1 promoter, suggesting a unique epigenetic regulation in diabetic mice as opposed to high glucose-treated human ECs. Our study demonstrates the importance of combining in vivo models of diabetes with high glucose-treated cell culture to better assess the epigenetic mechanisms relevant to disease. [Copyright &y& Elsevier]

Epstein-Barr virus (EBV) is involved in various clinical disorders and many of the disease entities are lymphoid and epithelial malignancies. The exact mechanisms that determine the exact form of EBV-related disorder are not clear at present. Many of the clinical manifestations of these diseases are based on the biological characteristics of the target cells for EBV infection and the expression and function of EBV gene, which also perturb host immune functions. In this monograph, I propose a hypothesis regarding the mechanism involved in shaping the manifestation of EBV infection that genomic instability of EBV-infected cells and how a defective immune surveillance system against such cells plays a critical role in determining the clinical manifestation of EBV infection. Using EBV-infected B-cells from patients and carriers with ataxia telangiectasia as an example of EBV infection, I present and discuss evidence in support of the proposed hypothesis. [Copyright &y& Elsevier]

Background: Preventing infection and managing febrile neutropenia (FN) is mandatory for children with cancer undergoing chemotherapy. However, the current situation in Japan is unknown. Methods: We conducted a nationwide web-based questionnaire survey in 153 institutions treating childhood cancer in Japan. We asked about the type prophylaxis used to prevent infectious disease and manage FN. If patients with childhood cancer were managed by both pediatricians and surgeons at the same institution, we asked both to reply. Results: We received replies from 117 departments at 111 centers: of these, 108 were from pediatricians. Laminar air flow for neutropenic patients, and frequent hand sanitization with ethanol, were widespread. Twenty-eight percent and forty percent of departments performed active surveillance by taking cultures from patients and the environment, respectively, before initiation of chemotherapy. Forty-four percent of departments administered prophylactic intravenous antibiotics according to patient status. Many departments measured serum (1,3)-β-D glucan, procalcitonin, and aspergillus galactomannan at the onset of FN. Twenty-eight percent of departments used carbapenem as empirical therapy for FN. Some departments used prophylactic granulocyte-colony stimulating factor for acute leukemia. Seventy-two percent of departments used prophylactic immunoglobulin for hypogammaglobinemia caused by chemotherapy. Palivizumab was administered widely for respiratory syncytial virus prophylaxis in immunocompromised infants. Conclusion: As a whole, intensive care for infectious prophylaxis or FN is applied in Japan; however, the methods vary among centers, and some are excessive or inadequate. Therefore, it is desirable to conduct clinical trials and establish adequate care protocols for infection in children with cancer in Japan. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethyl-arginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares L-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors. [ABSTRACT FROM AUTHOR]

Background: Low birth weight (LBW) and fetal growth restriction are associated with the development of cardio-metabolic diseases later in life. A recent Mendelian randomization study concluded that the susceptibility of LBW infants to develop hypertension during adulthood is due to the inheritance of hypertension genes from the mother and not to an unfavorable intrauterine environment. Therein, a negative linear association has been assumed between genetically estimated maternal blood pressure (BP) and birth weight, while the observed relationship between maternal BP and birth weight is substantially different from that assumption. As many hypertension genes are likely involved in vasculature development and function, we hypothesized that BP-increasing genetic variants could affect birth weight by reducing the growth of the placenta, a highly vascular organ, without overtly elevating the maternal BP.Methods: Using a birth cohort in the Japanese population possessing time-series fetal growth velocity data as a target and a GWAS summary statistics of BioBank Japan as a base data, we performed polygenic score (PGS) analyses for systolic BP (SBP), diastolic BP, mean arterial pressure, and pulse pressure. A causal mediation analysis was performed to assess the meditation effect of placental weight on birth weight reduced by maternal BP-increasing PGS. Maternal genetic risk score constituted of only "vasculature-related" BP single nucleotide polymorphisms (SNPs) was constructed to examine the involvement of vascular genes in the mediation effect of placental weight. We identified gestational week in which maternal SBP-increasing PGS significantly decreased fetal growth velocity.Results: We observed that maternal SBP-increasing PGS was negatively associated with offspring birth weight. A causal mediation analysis revealed that a large proportion of the total maternal PGS effect on birth weight was mediated by placental weight. The placental mediation effect was remarkable when genetic risk score was constituted of "vasculature-related" BP SNPs. The inverse association between maternal SBP PGS and fetal growth velocity only became apparent in late gestation.Conclusions: Our study suggests that maternal hypertension genes are strongly associated with placental growth and that fetal growth inhibition is induced through the intrauterine environment established by the placenta. [ABSTRACT FROM AUTHOR]

The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30 – 40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to γ-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8 – 9% in LBC282 and 5 – 7% in LBC286, while the dose of γ-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell... [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 5 (SCA5), a dominant spinocerebellar ataxia is caused by spectrin beta nonerythrocytic 2 gene (SPTBN2) mutation. It typically consists of a slow progressive cerebellar ataxia with an onset principally in adulthood. Here, we report on the first Japanese patient with infantile-onset SCA5 associated with a novel heterozygous SPTBN2 mutation. The patient, a 6-year-old girl, developed delayed motor development and unsteady arm movement during infancy. She also showed gaze-evoked nystagmus, saccadic eye pursuit, dysarthria, dysmetria, intention tremor and mild intellectual disability. Brain MRI revealed moderate cerebellar atrophy and mild pontine atrophy. Comprehensive target capture sequencing to identify the causative gene identified a novel missense mutation in SPTBN2 (c.1309C

Summary: The prognosis of paediatric acute myeloid leukaemia (AML) with primary induction failure (PIF) is extremely poor, and effective treatment strategies have not been established. We investigated the clinical and biological features of paediatric AML patients with PIF registered to the Japanese Paediatric Leukaemia/Lymphoma Study Group AML‐05 study. The 3‐year overall survival rate of the 41 PIF patients was 19.0%. High leucocyte count, M7 morphology, and unfavourable genetic aberrations, such as FLT3‐internal tandem duplication, NUP98‐NSD1 and high MECOM or PRDM16 expression, were risk factors for PIF. More effective treatment strategies based on leukaemia biology need to be urgently explored. [ABSTRACT FROM AUTHOR]

Background The combination of fludarabine (Flu), high-dose cytarabine (Ara-C) and granulocyte colony-stimulating factor (G- CSF; FLAG), with anthracyclines has become standard chemotherapy for refractory acute myeloid leukemia ( AML) in European children and adults. To clarify the efficacy and the safety of FLAG-idarubicin ( IDA) for children prospectively, we planned a multicenter phase II study ( AML-R11) by the Japanese Pediatric Leukemia/Lymphoma Study Group. Methods Patients with AML aged between 2 and 20 years old, who had the first bone marrow ( BM) relapse or induction failure, were enrolled. The FLAG- IDA regimen consisted of Flu 30 mg/m2 for 5 days, Ara-C 2 g/m2 for 5 days, G- CSF (lenograstim) 5 μg/kg for 6 days and IDA 10 mg/m2 for 3 days. The primary endpoint was remission rate after therapy. Results Due to drug supply issues, the trial was suspended after the inclusion of seven eligible patients. There were six cases of early relapse within 1 year of the first remission. All seven patients completed the therapy and no early death was observed. Hematological toxicity was common, and one patient developed grade 4 non-hematological toxicity of bacterial meningitis. Although only one patient with late relapse achieved complete remission, minimal residual disease was positive on both flow cytometry and Wilms' tumor 1 mRNA. Two patients were alive in remission following hematopoietic stem cell transplantation, whereas the other five patients died of either the disease or treatment-related causes. Conclusion FLAG- IDA might be tolerable for children with refractory AML although the efficacy should be further investigated. [ABSTRACT FROM AUTHOR]

Poly (ADP-ribose) polymerase (PARP) is an indispensable component of the DNA repair machinery. PARP inhibitors are used as cutting-edge treatments for patients with homologous recombination repair (HRR)-defective breast cancers harboring mutations in BRCA1 or BRCA2. Other tumors defective in HRR, including some hematological malignancies, are predicted to be good candidates for treatment with PARP inhibitors. Screening of leukemia-derived cell lines revealed that lymphoid lineage-derived leukemia cell lines, except for those derived from mature B cells and KMT2A (MLL)-rearranged B-cell precursors, were relatively sensitive to PARP inhibitors. By contrast, acute myelogenous leukemia cell lines, except for RUNX1-RUNXT1 (AML1-ETO)-positive lines, were relatively resistant. Intriguingly, TCF3 (E2A)-HLF-positive leukemia was sensitive to PARP inhibitors. TCF3-HLF expression suppressed HRR activity, suggesting that PARP inhibitor treatment induced synthetic lethality. Furthermore, TCF3-HLF expression decreased levels of MCPH1, which regulates the expression of BRCA1, resulting in attenuation of HRR activity. The PARP inhibitor olaparib was also effective in an in vivo xenograft model. Our results suggest a novel therapeutic approach for treating refractory leukemia, particularly the TCF3-HLF-positive subtype. [ABSTRACT FROM AUTHOR]

The article reports on a study that explored the origin and evolutionary process of the sequential relationship between Langerhans cell histiocytosis (LCH) and acute lymphoblastic leukemia (ALL). Topics discussed include the comprehensive genomic analysis of the dynamics of clonal architecture for one case and the genomic analysis performed for another 2 ALL cases that subsequently developed LCH. The derivation of LCH from a founding clone with genomic alterations common to ALL is mentioned.

Etoposide, a topoisomerase 2 (TOP2) inhibitor, is associated with the development of KMT2A (MLL)-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL) rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL)-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP) assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq) identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll)-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective. [ABSTRACT FROM AUTHOR]

The Y chromosome plays a critical role in spermatogenesis. Formerly, it had been difficult to generate knockout mice with specific Y chromosome mutations using conventional gene-targeting strategies. Recently, a transcription activator-like effector nuclease (TALEN) was successfully used for editing a mouse Y chromosome-linked gene. Here, we report the generation of a mouse model with a mutation in Eif2s3y, a Y chromosome-linked gene, and analysis of its phenotype. The mouse carrying a targeted mutation of Eif2s3y was infertile and had hypoplastic testes. Histological and electron microscopic analyses showed that differentiation of spermatogonia was arrested at the stage of spermatogonial stem cells (undifferentiated spermatogonia) and that the progression of spermatogenesis was interrupted, resulting in azoospermia. Using TALEN, we verified that EIF2S3Y performs a key function in differentiation of spermatogonial stem cells. [ABSTRACT FROM AUTHOR]

We encountered a case of neonatal acute megakaryoblastic leukemia not associated with Down syndrome ( DS). Molecular cytogenetic analysis of leukemic blast cells indicated that increased blast cell status was caused by transient abnormal myelopoiesis with trisomy 21 and GATA1 mutation. Based on these molecular cytogenetic data, intensive chemotherapy was avoided, and the patient was successfully cured with low-dose cytarabine. Morphologically, leukemic blast cells of acute megakaryoblastic leukemia in a non- DS neonate are indistinguishable from a blast cell of transient abnormal myelopoiesis. The possibility of transient abnormal myelopoiesis should be carefully considered before intensive chemotherapy is adopted. [ABSTRACT FROM AUTHOR]

We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification. [ABSTRACT FROM AUTHOR]

It has been unclear whether chromosomally integrated human herpesvirus 6 (ciHHV-6) can be activated with pathogenic effects on the human body. We present molecular and virological evidence of ciHHV-6A activation in a patient with X-linked severe combined immunodeficiency. These findings have significant implications for the management of patients with ciHHV-6. [ABSTRACT FROM PUBLISHER]

Context Classical 3β-hydroxysteroid dehydrogenase (3β- HSD) deficiency (3β- HSDD) is caused by loss-of-function mutations in the HSD3B2 gene encoding type II 3β- HSD, which has a key role in steroid biosynthesis, converting Δ5-steroids to Δ4-steroids in adrenal glands and gonads. Patient A patient (46, XX) was found to have elevated 17-hydroxyprogesterone (17- OHP) [203 nmol/l (normal range: 2·94 ± 0·9 nmol/l)] by newborn screening. Endocrinological examination revealed dramatically increased Δ5-steroids [e.g. 17- OH pregnenolone: 910 nmol/l (normal range: 12·6 ± 10·5 nmol/l)]. The patient had virilization of external genitalia with labial fusion, suggesting classical 3β- HSDD. Methods and Results Consistent with the endocrinological data, the patient was a compound heterozygote for two novel missense mutations (p.Y190C and p.S218P) that were identified in HSD3B2. Both Y190 and S218 are conserved among mammals. The mutant proteins had severely impaired residual enzymatic activity in vitro, although both mutants retained higher activity for 17- OH pregnenolone than for the other Δ5-steroids. In a three-dimensional model of the enzyme based on the known structures of similar proteins, both mutations were located extremely close to the predicted substrate-binding pocket. This suggests that the mutations can cause a local conformational change in the substrate-binding pocket, leading to alterations of the binding affinities for Δ5-steroids. Conclusions We identified two novel missense mutations of HSD3B2 that resulted in unbalanced residual enzymatic activities for Δ5-steroids. As a potential novel mechanism, we propose that the mutations, which differently affect the activity towards different substrates, the effects of these mutations provide novel insights into the pathophysiology of 3β- HSDD. [ABSTRACT FROM AUTHOR]

X-linked severe combined immunodeficiency (SCID-X1) caused by mutations in interleukin 2 receptor gamma (IL2RG) gene threatens the survival of affected boys during the first year of life unless hematopoietic stem cell transplantation is provided. Although viral vector-mediated gene therapy has been successfully performed in patients with no HLA-matched donors, leukemia caused by vector-mediated insertional mutagenesis has been reported in some individuals. Transcription activator-like effector nuclease (TALEN) is an artificial sequence-specific endonuclease that is expected to revolutionize the precise correction of disease-causing mutations and eliminate the risk of insertional mutagenesis. Here, we report TALEN-mediated genome editing of the IL2RG locus. We transfected TALENs along with a targeting vector into Jurkat cells, and we confirmed the precise introduction of the exogenous gene into the IL2RG locus. In addition, we found that the length of homology arm in the targeting vector influenced the efficiency of TALEN-mediated homologous recombination. [ABSTRACT FROM AUTHOR]

Abstract: A number of diseases exhibit neurodegeneration with/without additional symptoms such as immunodeficiency, increased cancer risk, and microcephalus. Ataxia telangiectasia and Nijmegen breakage syndrome, for example, develop as a result of mutations in genes involved in the DNA damage response. However, such diseases can be difficult to diagnose as they are only rarely encountered by physicians. To overcome this challenge, nine patients with symptoms that resemble those of ataxia telangiectasia, including neurodegeneration, hypogammaglobulinemia, telangiectasia, and/or elevated serum α-fetoprotein, were subjected to whole-exome sequencing (WES) to identify the causative mutations. Molecular diagnosis was achieved in two patients: one displayed CD40 ligand (CD40LG) deficiency, while a second showed a homozygous SIL1 mutation, which has been linked to Marinesco-Sjögren syndrome (MSS). Typical features of CD40LG deficiency and MSS are distinct from the symptoms usually seen in ataxia telangiectasia. These dissociations between phenotype and genotype make it difficult to achieve molecular diagnosis of orphan diseases. Whole-exome sequencing analyses will assist in the molecular diagnosis of such cases and allow the identification of genotypes that would not be expected from the phenotype. [Copyright &y& Elsevier]

Aims Proliferation of pulmonary arterial smooth muscle cells (PASMCs) is one histological sign of pulmonary arterial hypertension (PAH). We hypothesized that a signalling cascade from fibroblast growth factor 2 (FGF2) to plasminogen activator inhibitor 1 (PAI-1) and monocyte chemotactic protein-1 (MCP-1) via nuclear transcription factor nuclear factor kappaB (NF-kB) play a critical role in progression of PAH, and tested this hypothesis both in vivo and in vitro using a synthetic selective NF-kB inhibitor, N-(3,5-Bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide (IMD-0354). Methods and results Monocrotaline (MCT) was injected into 75 Sprague–Dawley rats. Starting at day 14 after MCT injection, we administered IMD-0354 (MCT + IMD group) or vehicle (MCT group) daily. At day 32, 65% of the MCT + IMD group were alive compared with 0% of the MCT group. IMD-0354 prevented increase of right ventricular pressure, and suppressed proliferation and induced apoptosis of PASMCs. mRNA transcript levels of FGF2, PAI-1, and tissue plasminogen activator (t-PA) were lower in MCT + IMD compared with MCT. In in vitro experiments, IMD-0354 inhibited p65 translocation to the nucleus promoted by FGF2 in PASMCs. Furthermore, the time courses of extracellular signal-regulated kinase (Erk) 1/2, MCP-1, and PAI-1 stimulated with FGF2 were each markedly shortened by IMD-0354. Conclusions We speculate that the positive-feedback loop (Erk1/2–NF-kB–MCP-1–Erk1/2) is associated with progression of PAH by causing FGF2-induced inflammation in MCT rats. IMD-0354 has potential as a new therapeutic tool for PAH. [ABSTRACT FROM PUBLISHER]

Highlights: [•] DNA damage-response pathway is activated in bone marrow of chronic-phase CML. [•] Loss of one of the allele of Atm accelerates the blast crisis. [•] ATM haploinsufficiency accelerates tumor development when oncogenic event arises. [Copyright &y& Elsevier]

Immune defect in ataxia telangiectasia patients has been attributed to either the failure of V(D)J recombination or class-switch recombination, and the chromosomal translocation in their lymphoma often involves the TCR gene. The ATM-deficient mouse exhibits fewer CD4 and CD8 single-positive T cells because of a failure to develop from the CD4+CD8+ double-positive phase to the single-positive phase. Although the occurrence of chromosome 14 translocations involving TCR-&dgr; gene in ATM-deficient lymphomas suggests that these are early events in T-cell development, a thorough analysis focusing on early T-cell development has never been performed. Here we demonstrate that ATM-deficient mouse thymocytes are perturbed in passing through the &bgr;- or &ggr;&dgr;-selection checkpoint, leading in part to the developmental failure of T cells. Detailed karyotype analysis using the in vitro thymocyte development system revealed that RAG-mediated TCR-&agr;/&dgr; locus breaks occur and are left unrepaired during the troublesome &bgr;- or &ggr;&dgr;-selection checkpoints. By getting through these selection checkpoints, some of the clones with random or nonrandom chromosomal translocations involving TCR-&agr;/&dgr; locus are selected and accumulate. Thus, our study visualized the first step of multi-step evolutions toward lymphomagenesis in ATM-deficient thymocytes associated with T-lymphopenia and immunodeficiency. [ABSTRACT FROM AUTHOR]

The function of the kinase Btk in neutrophil activation is largely unexplored. Here we found that Btk-deficient neutrophils had more production of reactive oxygen species (ROS) after engagement of Toll-like receptors (TLRs) or receptors for tumor-necrosis factor (TNF), which was associated with more apoptosis and was reversed by transduction of recombinant Btk. Btk-deficient neutrophils in the resting state showed hyperphosphorylation and activation of phosphatidylinositol-3-OH kinase (PI(3)K) and protein tyrosine kinases (PTKs) and were in a 'primed' state with plasma membrane-associated GTPase Rac2. In the absence of Btk, the adaptor Mal was associated with PI(3)K and PTKs at the plasma membrane, whereas in control resting neutrophils, Btk interacted with and confined Mal in the cytoplasm. Our data identify Btk as a critical gatekeeper of neutrophil responses. [ABSTRACT FROM AUTHOR]

Abstract: Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47phox and p67phox deficiency. The p47phox or p67phox protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product. [Copyright &y& Elsevier]

We report a case of anomalous origin of the left main coronary artery (LCA) from the noncoronary sinus of valsalva (LCANCS) in a young healthy patient who presented with syncope and cardiopulmonary arrest during exercise. The enhanced computed tomography showed acute angle take-off (AAT) of LCA, and the exercise stress thallium-201 myocardial scintigraphy demonstrated a large defect at the LCA perfusion region. We propose that the coexistence of AAT and resulting ischemia causes sudden cardiac death during exercise in the patients with LCANCS. [ABSTRACT FROM AUTHOR]

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition. [ABSTRACT FROM AUTHOR]

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting. [ABSTRACT FROM AUTHOR]

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots. [ABSTRACT FROM AUTHOR]

The article presents several case studies on delayed-onset circulatory collapse in two newborn infants. It states that the first case involves a female infant who has diagnosed with hypothyroidism and apnea-bradycardia events after L-T4 administration. It concludes that hydrocortisone (HDC) treatment enhanced the patient's respiratory condition. It notes that the second case features a 24-weeks-old infant with suspected chronic lung disease with hypotension and hyperkalemia after L-T4 treatment.

Ataxia-telangiectasia-like disorder (ATLD) is caused by mutations of the MRE11 gene and is characterized by cerebellar ataxia, increased frequency of chromosomal translocations and hypersensitivity to ionizing radiation. ATLD is a rare genetic disease and the associated pathological changes in the brain are unclear. Here, we report the neuropathological findings in the first cases of genetically confirmed ATLD in a pair of Japanese male siblings. Magnetic resonance imaging studies performed during infancy revealed that both subjects had cerebellar atrophy. They died of pulmonary cancer at 9 and 16 years. The siblings had the same compound heterozygous mutations of the MRE11 gene. Brain autopsy demonstrated mild and severe cerebellar atrophy in the vermis and medial part of the hemispheres, oral to the horizontal fissure, respectively. Nuclear immunoreactivity for MRE11 was absent in neurons of cerebellar cortex, cerebral cortex, basal ganglia and midbrain, whereas being widespread in normal control brains. Immunoreactivity for the DNA oxidative stress marker, 8-hydroxy-2′-deoxyguanosine, was identified in nuclei of granule cells and Bergmann glial cells. The combination of MRE11 deficiency and DNA oxidative injury might have led to selective cerebellar degeneration. [ABSTRACT FROM AUTHOR]

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress. [ABSTRACT FROM AUTHOR]

Rare cases of possible materno-fetal transmission of cancer have been recorded over the past 100 years but evidence for a shared cancer clone has been very limited. We provide genetic evidence for mother to offspring transmission, in utero. of a leukemic cell clone. Maternal and infant cancer clones shared the same unique BCR-ABL1 genomic fusion sequence, indicating a shared, single-cell origin. Microsatellite markers in the infant cancer were all of maternal origin. Additionally, the infant, maternally-derived cancer cells had a major deletion on one copy of chromosome 6p that included deletion of HLA alleles that were not inherited by the infant (i.e., foreign to the infant), suggesting a possible mechanism for immune evasion. [ABSTRACT FROM AUTHOR]

The aim was to identify the electrophysiological and psychological signs at a very early stage in asymptomatic boys with childhood cerebral X-linked adrenoleukodystrophy. Flash visual evoked potentials, pattern reversal, and visual eventrelated potentials were recorded in 6 radiologically asymptomatic boys with adrenoleukodystrophy and 22 control boys. The latency and amplitude of P100 of visual evoked potentials and P1 of event-related potentials were evaluated. Though all patients had normal intelligence quotient, performance intelligence quotient was significantly lower than verbal intelligence quotient in 2 patients. Both P100 and P1 amplitudes were significantly greater in adrenoleukodystrophy than in controls. The difference between performance intelligence quotient and verbal intelligence quotient exhibited significant correlation with P100 amplitude. Enlargement of visual evoked potentials might be a sign of cerebral involvement preceding the appearance of abnormalities on magnetic resonance imaging. Follow-up of asymptomatic boys with both electrophysiological and neuropsychological tests may serve as an aid for deciding the timing of therapeutic intervention. [ABSTRACT FROM AUTHOR]

Ectopic bone formation after joint replacement or brain injury in humans is a serious complication that causesimmobility of joints and severe pain. However, mechanisms underlying such ectopic bone formation are not fully understood. Bone morphogenetic protein (BMPs) are defined as inducers of ectopic bone formation, and they are regulated by several types of inhibitors. ANA is an antiproilferative molecule that belongs to Tob/BTG family, but its activity in bone metabolism has not been known. Here, we examined the role of ANA on ectopic bone formation activity of BMP. In ANA-deficient and wild-type mice, BMP2 was implanted to induce ectopic bone formation in muscle. ANA deficiency increased mass of newly formed bone in vivo compared with wild-type based on 3D-μCT analyses. ANA mRNA was expressed in bone in vivo as well as in osteoblastic cells in vitro. Such ANA mRNA levels were increased by BMP2 treatment in MC3T3-E1 osteoblastic cells. Overexpression of ANA suppressed BMP-induced expression of luciferase reporter gene linked to BMP response elements in these cells. Conversely, ANA mRNA knockdown by small interference RNA enhanced the BMP-dependent BMP response element reporter expression. It also enhanced BMP-induced osteoblastic differentiation in muscle-derived C2C12 cells. Immunoprecipitation assay indicated that ANA interacts with Smad8. Thus, ANA is a suppressor of ectopic bone formation induced by BMP, and this inhibitory ANA activity is a part of the negative feedback regulation of BMP function. [ABSTRACT FROM AUTHOR]

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.Kidney International (2008) 74, 1262–1269; doi:10.1038/ki.2008.405; published online 3 September 2008 [ABSTRACT FROM AUTHOR]

Mucopolysaccharidoses (MPS) are inherited disorders caused by the deficiency of lysosomal enzymes. Sanfilippo syndrome (MPS III) and Hunter syndrome (MPS II) are characterized by severe and mild neurological disorders, respectively, in which the neurodegenerative mechanisms remain to be clarified. We immunohistochemically examined the involvement of tauopathy/synucleinopathy, cell death and oxidative damage in the brains of three cases each of MPS IIIB and MPS II and age-matched controls. In cases of MPS IIIB, the density of GABAergic interneurons in the cerebral cortex immunoreactive for calbindin-D28K and parvalbumin was markedly reduced when compared with age-matched controls. The swollen neurons showed immunoreactivity for phosphorylated α-synuclein but not for phosphorylated tau protein or β-amyloid protein; those in the cerebral cortex demonstrated nuclear immunoreactivity for TUNEL, single-stranded DNA and 8-OHdG. Neither lipid peroxidation nor protein glycation was marked in MPS cases. The expression levels of superoxide dismutases (Cu/ZnSOD and MnSOD) and glial glutamate transporters (EAAT1 and EAAT2) were reduced in two MPS II cases. The disturbance of GABAergic interneurons can be related to mental disturbance, while synucleinopathy and/or DNA impairment may be implicated in the neurodegeneration of swelling neurons due to storage materials in MPS IIIB cases. These findings suggest the possibility of neuroprotective therapies other than enzyme replacement in MPS patients. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the quantification of 17α-hydroxyprogesterone (17OHP) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a highly proton-affinitive reagent, 2-hydrazinopyridine, and subjected to LC–MS–MS. Quantification was based on the selected reaction monitoring, and deuterated 17OHP was used as the internal standard. This method allowed the reproducible and accurate quantification of the salivary 17OHP using a 200-μl sample, and the limit of quantitation was 5.0pg/ml. The developed method was applied to clinical studies. A linear relationship was found to be positive (r 2 =0.975) between the blood 17OHP level and the salivary 17OHP level measured using the proposed method. The result from the salivary 17OHP measurement in patients with congenital adrenal hyperplasia demonstrated that the proposed method is very useful for monitoring of the therapeutic efficacy during hormone replacement therapy. [Copyright &y& Elsevier]

Abstract: Dentatorubral-pallidoluysian atrophy (DRPLA) is one of the CAG-repeat diseases, and is classified into juvenile and early adult types showing progressive myoclonus epilepsy (PME) in addition to late adult type. We immunohistochemically examined accumulation of oxidative products and expression of superoxide dismutase (SOD) in autopsy cases of DRPLA. Oxidative products to nucleosides, 8-hydroxy-2′-deoxyguanosine and 8-hydroxyguanosine, were accumulated in the lenticulate nucleus predominantly in DRPLA cases having PME. Neuronal accumulation of 4-hydroxy nonenal, a reactive lipid aldehyde, was found in the hippocampus, globus pallidus and cerebellar dentate nucleus in adult DRPLA cases and controls. Cytoplasmic immunoreactivity for Cu/ZnSOD was reduced in the external segment of globus pallidus, dentate nucleus and cerebellar cortex in DRPLA cases. Mitochondrial immunoreactivity for MnSOD was reduced in the lenticulate nucleus and cerebellum in DRPLA cases having PME. Some DRPLA cases showed reduced immunoreactivity for MnSOD in the cerebral cortex. Coexistence of reduced SOD expression and polyglutamine was observed in a few cases. It has been discussed in Huntington''s disease that expanded polyglutamine can lead to oxidative neurodegeneration. It is likely that oxidative stress can be involved in DRPLA, although relationship with expanded polyglutamine remains to be elusive. [Copyright &y& Elsevier]

The human chromosome X is closely associated with congenital disorders and mental retardation (MR), because it contains a significantly higher number of genes than estimated from the proportion in the human genome. We constructed a high-density and high-resolution human chromosome X array (X-tiling array) for comparative genomic hybridization (CGH). The array contains a total of 1,001 bacterial artificial chromosome (BACs) throughout chromosome X except pseudoautosomal regions and two BACs specific for Y. In four hybridizations using DNA samples from healthy males, the ratio of each spotted DNA was scattered between −3SD and 3SD, corresponding to a log2 ratio of −0.35 and 0.35, respectively. Using DNA samples from patients with known congenital disorders, our X-tiling array was proven to discriminate one-copy losses and gains together with their physical sizes, and also to estimate the percentage of a mosaicism in a patient with mos 45,X[13]/46,X,r(X)[7]. Furthermore, array-CGH in a patient with atypical Schinzel-Giedion syndrome disclosed a 1.1-Mb duplication at Xq22.3 including a part of the IL1RAPL2 gene as a likely causative aberration. The results indicate our in-house X-tiling array to be useful for the identification of cryptic copy-number aberrations containing novel genes responsible for diseases such as congenital disorders and X-linked MR. [ABSTRACT FROM AUTHOR]

Human acute myeloid leukaemia (AML) involving a core-binding factor (CBF) transcription factor is called CBF leukaemia. In these leukaemias, AML1 (RUNX1, PEBP2αB, CBFα2)-MTG8 (ETO) and CBFβ (PEBP2β)- MYH11 chimaeric proteins are generated by t(8;21) and inv(16) respectively. We analysed gene expression profiles of leukaemic cells by microarray, and selected genes whose expression appeared to be modulated in association with t(8;21) and inv(16). In a pair-wise comparison, 15% of t(8;21)- associated transcripts exhibited high or low expression in inv(16)-AML, and 26% of inv( 16)-associated transcripts did so equivalently in t(8;21)-AML. These common elements in gene expression profiles between t(8;21)- and inv(16)-AML probably reflect the situation that AML1-MTG8 and CBFβ- MYH 11 chimaeric proteins affect a common set of target genes in CBF leukaemic cells. On the other hand, 38% of t(8;21)-associated and 24% of inv( 16)-associated transcripts were regulated in t(8;21)- and inv(16)-specific manners. These distinct features of t(8;21)- and inv(16)-associated genes correlate with the bimodular structures of the chimaeric proteins (CBF- related AML1 and CBFβ portions, and CBF-unrelated MTG8 and MYH11 portions). [ABSTRACT FROM AUTHOR]

Topoisomerase II (Topo II) inhibitors are cell cycle-specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated-deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor-induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control. [ABSTRACT FROM AUTHOR]

Omenn syndrome is a variant form of severe combined immunodeficiency. It is fatal unless treated by allogeneic stem cell transplantation (SCT), which is the only curative approach. However, both treatment-related complications and graft rejection are major obstacles to treatment success. This report describes a case with Omenn syndrome who developed mixed chimerism after unrelated cord blood transplantation (UCBT). This case was successfully treated by altering the patient's immunosuppression and donor lymphocyte infusion (DLI) with donor cord blood-derived activated CD4+ T cells ex vivo expanded from the cord blood cell residues in an infused bag. This novel development to expand CD4+ T-lymphocytes from the donor cord blood unit for the use of DLI would serve as a useful method to overcome the risk of graft rejection in SCT for primary immunodeficiency disorders with residual cell-mediated immunity without compounding graft-vs.-host disease, especially in the UCBT setting. [ABSTRACT FROM AUTHOR]

Abstract: A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (γ-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-γ-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia–telangiectasia cells lacking ATM kinase activity, γ-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity. [Copyright &y& Elsevier]

Abstract: Autosomal recessive form of hyper-IgM syndrome type 2 (AR-HIGM2) is secondary to mutations affecting both alleles of AICDA gene encoding activation-induced cytidine deaminase, characterized by defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in most of the patients. We herein report the immunological phenotype of seven patients carrying a single heterozygous R190X mutation in AICDA. Variable defect in in vivo CSR inherited as an autosomal dominant (AD) trait strongly suggests that this heterozygous AICDA mutation causes HIGM (AD-HIGM2). In AD-HIGM2 B cells, CSR was consistently found impaired in vitro. However, in contrast to AR-HIGM2, the CSR-induced double-stranded DNA breaks in the switch region of IgM heavy chain gene were detected. The SHM frequency in V regions of IgM heavy chain gene in B cells was normal in all (but one patient). The characteristics of the AD-HIGM2 phenotype indicate that the AID C-terminal region may be involved in DNA repair machinery required for CSR. [Copyright &y& Elsevier]

Nagasawa M, Zhu Y, Isoda T, Tomizawa D, Itoh S, Kajiwara M, Morio T, Nonoyama S, Shimizu N, Mizutani S. Analysis of serum soluble CD40 ligand (sCD40L) in the patients undergoing allogeneic stem cell transplantation: platelet is a major source of serum sCD40L.Eur J Haematol 2005: 74: 54–60.© Blackwell Munksgaard 2005.CD40 ligand (CD40L) is expressed not only on activated T cells but also on activated platelets. A soluble CD40 ligand (sCD40L) is released from the activated T cells and platelets by ill-defined proteolytic processin vitro. It has been reported that sCD40L is elevated in the serum of patients with systemic lupus erythematosus, unstable angina, essential thrombocythemia, and autoimmune thrombocytopenic purupura. However, source of sCD40Lin vivoremains to be elucidated. We investigated the serial sCD40L in the serum in patients undergoing allogeneic stem cell transplantation and compared with the platelets number and soluble IL2R, which is a marker of activated T cells. The value of sCD40L was well correlated with platelet number or thrombopoiesis. In cases of severe graft vs. host disease with markedly increased sIL2R, sCD40L was not increasedin vivo. These results indicate that sCD40Lin vivois released mainly from the platelets or in the process of platelet production but not from the activated T cells. [ABSTRACT FROM AUTHOR]

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1-/-) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1+/+) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1-/- embryonic stem cell (ES) clones was also higher than that in Parp-1+/+ ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and 32P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB. [Copyright &y& Elsevier]

Monosomy 7 syndrome in infant is considered as pre-leukemic condition of poor prognosis. However, it seems controversial recently, because some cases of monosomy 7 syndrome showed spontaneous remission. We report 2-year-old girl with severe pancytopenia, who presented with monosomy 7. Morphologically, there was little dysplasia in the trilineage hematopoiesis. Monosomy 7 clone of CD34 positive cells, bone marrow mononuclear cells (BMMNC), and peripheral nuclear cells was 4.0, 40, and 3.8%, respectively. Immunosuppressive therapy was effective along with the disappearance of monosomy 7 clone. WT1 mRNA expression was not increased in monosomy 7 clone. Pathogenesis of monosomy 7 and its relation to aplastic anemia is discussed. [Copyright &y& Elsevier]

The ATM-dependent accumulation of p53 and induction of p21waf1 are key events for G1 cell-cycle checkpoint arrest following DNA damage. In ATM-null AT cells, even though the p53 and p21waf1 responses are kinetically delayed and quantitatively reduced, the G1 checkpoint is virtually disrupted, suggesting that these proteins arrive too late in G1 to enforce the arrest. As the precise mechanism remains unclear, we examined the response to DNA double-strand breaks generated by ?-radiation (IR), to determine if ATM deficiency affects the cell-cycle phase regulation of these molecules. We find that, after irradiation, whereas normal LCL-N cells markedly increase their levels of p53 in all phases of the cell cycle, AT cells fail to show any p53 increase in the G1?phase. In addition, whereas in LCL-N p21waf1 is induced in G1 and G2-M, in AT cells this induction is partly seen in G2-M, but not in G1, indicating a different cell-cycle phase regulation of p53 and p21waf1 as a result of ATM deficiency. The levels and catalytic activity of the p53-targeting kinases ATR and DNA-PK in LCL-N and AT cells are very similar throughout the cell cycle, both before and after IR, thus excluding a phase-specific activity for these kinases. Collectively, our findings demonstrate that, in ATM-deficient cells, the p53-dependent p21waf1 response to DNA damage is not only quantitatively reduced, but also specifically suppressed in the G1?phase, thus providing a mechanistic explanation for the severe disruption of the G1 checkpoint in AT cells.Oncogene (2003) 22, 7866-7869. doi:10.1038/sj.onc.1207086 [ABSTRACT FROM AUTHOR]