Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.

Bioinspired Total Synthesis of the Dimeric Indole Alkaloid (+)-Haplophytine by Direct Coupling and Late-Stage Oxidative Rearrangement.

A bioinspired convergent total synthesis of (+)-haplophytine, a dimeric indole alkaloid with diazabicyclo[3.3.1]nonane and hexacyclic aspidosperma segments, is described. This synthesis involves the direct coupling of the two segments in a AgNTf2 -mediated Friedel-Crafts reaction and construction of the diazabicyclo[3.3.1]nonane skeleton through late-stage chemoselective aerobic oxidation of the 1,2-diaminoethene moiety and a sequential semipinacol-type rearrangement.

Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction.

The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.

Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats.

Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

A rare polymorphic variant of NBS1 reduces DNA repair activity and elevates chromosomal instability.

Failure to expeditiously repair DNA at sites of double-strand breaks (DSB) ultimately is an important etiologic factor in cancer development. NBS1 plays an important role in the cellular response to DSB damage. A rare polymorphic variant of NBS1 that resulted in an isoleucine to valine substitution at amino acid position 171 (I171V) was first identified in childhood acute lymphoblastic leukemia. This polymorphic variant is located in the N-terminal region that interacts with other DNA repair factors. In earlier work, we had identified a remarkable number of structural chromosomal aberrations in a patient with pediatric aplastic anemia with a homozygous polymorphic variant of NBS1-I171V; however, it was unclear whether this variant affected DSB repair activity or chromosomal instability. In this report, we demonstrate that NBS1-I171V reduces DSB repair activity through a loss of association with the DNA repair factor MDC1. Furthermore, we found that heterozygosity in this polymorphic variant was associated with breast cancer risk. Finally, we showed that this variant exerted a dominant-negative effect on wild-type NBS1, attenuating DSB repair efficiency and elevating chromosomal instability. Our findings offer evidence that the failure of DNA repair leading to chromosomal instability has a causal impact on the risk of breast cancer development.

Antioxidative activity and chemical constituents of edible terrestrial alga Nostoc commune Vauch.

The extract of terrestrial alga Nostoc commune Vauch. has high antioxidative activity. Our study on N. commune Vauch. resulted in the isolation of two ß-ionone derivatives, nostocionone and 3-oxo-ß-ionone, together with four indole alkaloids, scytonemin, reduced scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyltryptamine. The structures of the isolated compounds were determined on the basis of 1D and 2D NMR and MS analyses. Among these isolates, nostocionone and reduced scytonemin demonstrated strong antioxidative activities which were assessed by using a ß-carotene oxidation assay.

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue.

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9) cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.

The novel and independent association between single-point SNP of NPHP4 gene and renal function in non-diabetic Japanese population: the Takahata study.

Nephronophthisis (NPHP) 4 gene coding nephrocystin-4 is involved in the development of renal tubules and its congenital mutations cause juvenile end-stage renal disease, NPHP. To investigate the association between single-point single-nucleotide polymorphism (SNP) of NPHP4 gene and renal function, we conducted a cross-sectional study in Japanese population. The subjects of this study were non-diabetic general population consisting of 2604 individuals >40 years in Takahata town, Japan. We genotyped 11 SNPs within NPHP4 gene that displayed frequent minor allele frequencies (>0.1) in Japanese general population. Among 11 SNPs in NPHP4 gene, only rs1287637 that induces amino acid substitution (A (Gln)/T (Leu)), located in the acceptor site of exon 21, showed a significant association with estimated glomerular filtration rate (eGFR; T/T: 81.3±15.6 (n=1886), A/T: 82.0±15.5 (n=652) and A/A: 87.4±21.4 ml min(-1) per 1.73m(2) (n=66); mean±s.d., P=0.006). This SNP was not in linkage disequilibrium with the surrounding SNPs. The multivariate analysis adjusted with possible confounders showed that the A/T+T/T genotype of rs1287637 was independently associated with reduced renal function (eGFR <90 ml min(-1) per 1.73m(2); odds ratio (OR) 1.75, 95% confidence interval (CI) 1.05-2.94, P=0.033). These results indicate the novel and independent association between single-point SNP rs1287637 in NPHP4 gene and renal function in non-diabetic Japanese population.

Histology of fibrovascular membranes of proliferative diabetic retinopathy after intravitreal injection of bevacizumab.

PURPOSE: The purpose was to study the histology of the fibrovascular membranes in proliferative diabetic retinopathy (PDR) with an intravitreal injection of bevacizumab. METHODS: Light and electron microscopic studies were performed on surgical specimens obtained during a pars plana vitrectomy from 6 PDR eyes after intravitreal injection of bevacizumab. The patients had preoperatively received no or scant retinal photocoagulations. The presence and distribution of CD34 was assessed as a marker of vascular endothelium using immunostaining. The presence of vascular endothelial growth factor was stained with a method of immunostaining. As controls, we examined 7 surgical specimens from 7 PDR eyes obtained during pars plana vitrectomy without bevacizumab therapy. All control patients had preoperatively received full or nearly full pan retinal photocoagulations. RESULTS: Light microscopy showed that the CD34-positive vascular endothelial cells formed capillarylike structures in the fibrovascular membranes of all 13 PDR eyes. Vascular endothelial growth factor was positively stained in the vascular endothelium of both groups; however, the number of vascular endothelial growth factor-positive vascular endothelial cells significantly decreased in the fibrovascular membranes with intravitreal injection of bevacizumab. Electron microscopy showed the newly formed vascular endothelial cells with junctional complex in both groups. CONCLUSION: The vascular endothelial cells with decreased expression of vascular endothelial growth factor are still present in the fibrovascular membranes of patients with PDR after intravitreal injection of bevacizumab.

Molecular cytogenetic analysis of feline leukemia virus insertions in cat lymphoid tumor cells.

This study was conducted to map the acquired proviral insertions in the chromosomal genome of feline lymphoid tumors induced by feline leukemia virus (FeLV). Chromosome specimens of the lymphoid tumor-derived cell lines and normal cat lymphocytes were subjected to fluorescence in situ hybridization and tyramide signal amplification, using an exogenous FeLV-A genome as a probe. Specific hybridization signals were detected only on the metaphase chromosomes of the tumor cells. Poisson's distribution-based statistics indicated that 6 chromosomal loci in each cell line showed FeLV integration. In the examination of metaphase chromosomes of FL-74, FT-1 and KO-1 cells, significant signals were detected on B2p15-p14, B2q11, D1p14, E1p14-p13, E1q12 and F2q16; A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13 and E2p13-p12; and A2p22, A3q22, B1p13, B1q13, D1p13 and D3p15-p14, respectively. Consistently, Southern blot hybridization using an FeLV LTR-U3 probe specific for exogenous FeLV revealed the presence of at least 6 copies of exogenous FeLV proviruses at different integration sites in each cell line. These results indicate that there may be common FeLV integration sites at least in A2p22 and B2p15-p14. The cytogenetic analysis used in this study can promptly screen FeLV insertions and provide tags for identifying the novel common integration site.

BLNK suppresses pre-B-cell leukemogenesis through inhibition of JAK3.

Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK(-/-) mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27(kip1) expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27(kip1) induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7-dependent proliferation and survival of pre-B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre-B-cell leukemia.

NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells.

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

Clinical characteristics and genotype-phenotype correlation of hearing loss patients with SLC26A4 mutations.

CONCLUSIONS: The present study confirmed the clinical characteristics of patients with SLC26A4 mutations: congenital, fluctuating, and progressive hearing loss usually associated with vertigo and/or goiter during long-term follow-up. This clarification should help to facilitate appropriate genetic counseling and proper medical management for patients with these mutations, but there was no particular genotype-phenotype correlation among them, suggesting that other factors may contribute to such variability. OBJECTIVES: Due to the wide range of phenotypes caused by SLC26A4 mutations, there is controversy with regard to genotype-phenotype correlation. The present study was performed: (1) to determine phenotypic range in patients with biallelic SLC26A4 mutations, and (2) to evaluate whether possible genotype-phenotype correlation exists. SUBJECTS AND METHODS: Phenotypes in 39 hearing loss patients with SLC26A4 mutations were summarized and genotype-phenotype correlation was analyzed. RESULTS: Hearing level varied in the individuals from mild to profound severity. Most of the patients had fluctuating and progressive hearing loss that may have been of prelingual onset. Twenty-four (70.6%) patients had episodes of vertigo, and 10 (27.8%) patients had goiter, which had appeared at age 12 or older. In contrast to such phenotypic variabilities, no apparent correlation was found between these phenotypes and their genotypes.

Human and mouse protein-noncoding snoRNA host genes with dissimilar nucleotide sequences show chromosomal synteny.

snoRNAs are small protein-noncoding RNAs essential for pre-rRNA processing and ribosome biogenesis, and are encoded intronically in host genes (HGs) that are either protein coding or noncoding. mRNAs of protein-noncoding HGs differ in their nucleotide sequences among species. Although the reason for such sequential divergence has not been well explained, we present evidence here that such structurally different HGs have evolved from a common ancestral gene. We first identified two novel protein-noncoding HGs (mU50HG-a and mU50HG-b) that intronically encode a mouse ortholog of a human snoRNA, hU50. The sequences of mU50HG mRNA differed from that of hU50HG. However, a chromosome mapping study revealed that mU50HG is located at 9E3-1, the murine segment syntenic to human 6q15, where hU50HG is located. Synteny is a phenomenon whereby gene orthologs are arranged in the same order at equivalent chromosomal loci in different species; synteny between two species means it is highly likely that the genes have evolved from a common ancestral gene. We then extended this mapping study to other protein-noncoding snoRNA-HGs, and found again that they are syntenic, implying that they have evolved from genes of common ancestral species. Furthermore, on these syntenic segments, exons of adjacent protein-coding genes were found to be far better conserved than those of noncoding HGs, suggesting that the exons of protein-noncoding snoRNA-HGs have been much more fragile during evolution.

Unscheduled overexpression of human WAPL promotes chromosomal instability.

Previously, we have isolated and characterized a novel human gene termed human WAPL that has the characteristics of an oncogene in uterine cervical cancer. WAPL is inducible by human papillomavirus (HPV) E6 and E7 oncoproteins. On the other hand, recent studies have revealed that WAPL regulates sister chromatid resolution by controlling the association of cohesin and chromatin. However, the effects of WAPL overexpression on cervical carcinogenesis are still unclear. Here, we show that WAPL overexpression induces generation of multinucleated cells. In addition, WAPL-overexpressing cells demonstrated increases in chromatid breaks in comparison with control cells. These results were obtained even in HPV-negative cell lines. High frequent premature sister separation by disregulation of cohesin may lead to these results. Thus, our study suggests that unscheduled overexpression of WAPL disturbs mitosis and cytokinesis, and contributes to tumor progression by induction of chromosomal instability (CIN).

Enhanced energy expenditure and fat oxidation in humans with high BMI scores by the ingestion of novel and non-pungent capsaicin analogues (capsinoids).

The biochemical and physiological indices were monitored in 44 subjects after 4-week capsinoids (capsaicin analogues with low pungency) intake. The subjects were randomly assigned to 3 groups: CSNs3 (3 mg/kg of capsinoids), CSNs10 (10 mg/kg of capsinoids) and the control (placebo). Measurements were performed in the morning on overnight-fasted subjects. The oxygen consumption (VO(2)), resting energy expenditure (REE) and fat oxidation increased slightly compared to pre-administration values without any adverse effects, although the increase was not significant. The increase in fat oxidation was positively and significantly correlated with the body mass index (BMI). A meta-analysis was therefore conducted on a subgroup consisting of subjects with BMI >or= 25 (n=28). As a result, not only VO(2) increased significantly (p<0.05) in the CSNs10 group, but also REE in the CSNs10 group and fat oxidation in the CSNs3 and CSNs10 groups tended to increase (p<0.1). Consequently, a capsinoids intake would be able to enhance the energy expenditure and fat burning in humans, particularly those with high BMI.

Long-term (11-year) statin therapy following percutaneous coronary intervention improves clinical outcome and is not associated with increased malignancy.

BACKGROUND: Statins have been proven to reduce cardiac events and mortality. However, there are few studies dealing with the long-term efficacy of statin therapy following percutaneous coronary intervention (PCI). METHODS: We collected data from 575 consecutive patients who underwent PCI between 1987 and 1992. The baseline data, mortality and incidence of cardiovascular events of patients given statins and those not given statins at the time of PCI were compared. RESULTS: There were 243 patients in the statin group and 332 patients in the non-statin group. During follow-up (11.0+/-3.0 years), 68 patients died. At about 10 years, statin use was significantly associated with lower all-cause mortality (8.2% versus 14.5%, P=0.023) and cardiac death (2.5% versus 6.9%, P=0.017). After adjusting for variables, statin use was found to be an independent predictor of death from all causes (hazard ratio [HR] 0.54, 95% confidence interval [CI] 0.29-0.99, P=0.048) and cardiac death (HR 0.24, 95% CI 0.07-0.80, P=0.02). CONCLUSION: Statin use at the time of PCI was associated with a significantly reduced risk of death from all causes and cardiac death. Furthermore, this study provides evidence of a clinical benefit at about 10 years of statin use in patients who underwent PCI.

Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes.

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.

Acute effect of poly-gamma-glutamic acid on calcium absorption in post-menopausal women.

OBJECTIVE: Poly-gamma-glutamic acid (PGA) increases calcium (Ca) solubility in vitro and in vivo, and is associated with reduced bone loss in post-menopausal Japanese women. This study is the first to examine the effect of PGA on Ca absorption in humans. METHODS: A single-blind, randomized, crossover study with a 3-4 week wash-out was performed to determine the effect of PGA (80.6% glutamic acids) on Ca absorption measured by the double stable isotope method. Twenty-four healthy, non-smoking, postmenopausal women (mean age: 56.4 +/- SE 0.9) were given 200 g of orange juice containing 200 mg Ca as Ca-44 enriched CaCO(3), with or without 60 mg of PGA, after an overnight fast. The two tests were separated by 3-4 weeks. An intravenous injection of Ca-42 (CaCl(2) solution) was given 30 min after consuming the drink and a complete urine collection carried out from 24-48 h post-dosing. Ca absorption was calculated from the Ca isotope ratios measured by thermal ionization quadrupole mass spectrometry (TIQMS). RESULTS: Mean Ca absorption with PGA was significantly higher (P < 0.01) than without PGA, 39.1 (SE 1.6) % and 34.6 (SE 1.9) %, respectively. The effect of PGA on increasing Ca absorption was more marked in a sub-group of subjects whose baseline Ca absorption (without PGA) was lower than the population mean value. CONCLUSION: Postmenopausal women who received a single dose of PGA increased their intestinal Ca absorption particularly those individuals with lower basal absorptive capacity.