Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia.

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

The AAA+ ATPase RUVBL2 is a critical mediator of MLL-AF9 oncogenesis.

The most frequent chromosomal translocations in pediatric acute myeloid leukemia affect the 11q23 locus and give rise to mixed lineage leukemia (MLL) fusion genes, MLL-AF9 being the most prevalent. The MLL-AF9 fusion gene has been shown to induce leukemia in both mouse and human models. In this study, we demonstrate that leukemogenic activity of MLL-AF9 requires RUVBL2 (RuvB-like 2), an AAA+ ATPase family member that functions in a wide range of cellular processes, including chromatin remodeling and transcriptional regulation. Expression of RUVBL2 was dependent on MLL-AF9, as it increased upon immortalization of human cord blood-derived hematopoietic progenitor cells with the fusion gene and decreased following loss of fusion gene expression in conditionally immortalized mouse cells. Short hairpin RNA-mediated silencing experiments demonstrated that both the immortalized human cells and the MLL-AF9-expressing human leukemia cell line THP-1 required RUVBL2 expression for proliferation and survival. Furthermore, inhibition of RUVBL2 expression in THP-1 cells led to reduced telomerase activity and clonogenic potential. These data were confirmed with a dominant-negative Walker B-mutated RUVBL2 construct. Taken together, these data suggest the possibility of targeting RUVBL2 as a potential therapeutic strategy for MLL-AF9-associated leukemia.

Connectivity mapping identifies HDAC inhibitors for the treatment of t(4;11)-positive infant acute lymphoblastic leukemia.

MLL-rearranged infant acute lymphoblastic leukemia (ALL) is an aggressive type of leukemia characterized by a unique gene-expression profile. We uncovered that the activation of particular (proto-onco)genes is mediated by promoter hypomethylation. In search for therapeutic agents capable of targeting these potential cancer-promoting genes, we applied connectivity mapping on a gene expression signature based on the genes most significantly hypomethylated in t(4;11)-positive infant ALL as compared with healthy bone marrows. This analysis revealed histone deacetylase (HDAC) inhibitors as suitable candidates to reverse the unfavorable gene signature. We show that HDAC inhibitors effectively induce leukemic cell death in t(4;11)-positive primary infant ALL cells, accompanied by downregulation of MYC, SET, RUNX1, RAN as well as the MLL-AF4 fusion product. Furthermore, DNA methylation was restored after HDAC inhibitor exposure. Our data underlines the essential role for epigenetic de-regulation in MLL-rearranged ALL. Furthermore, we show, for the first time, that connectivity mapping can indirectly be applied on DNA methylation patterns, providing a rationale for HDAC inhibition in t(4;11)-positive leukemias. Given the presented potential of HDAC inhibitors to target important proto-oncogenes including the leukemia-specific MLL fusion in vitro, these agents should urgently be tested in in vivo models and subsequent clinical trials.

Complete nucleotide sequences of genome segments 1 and 3 of Rosellinia anti-rot virus in the family Reoviridae.

The nucleotide sequences of genome segments 1 and 3 of Rosellinia anti-rot virus (RArV) from a hypovirulent isolate, W370, of the plant pathogen Rosellinia necatrix were determined. The complete nucleotide sequence of the genome segment 1 encoded a putative RNA-dependent RNA polymerase (RDRP). The deduced amino acid sequence of RDRP of RArV showed 29% identity with RDRPs of Colorado tick fever virus (CTFV) and European Eyach virus (EYAV) in the genus Coltivirus, and identities of 23-21% with members of the genera Fijivirus and Cypovirus. Both RArV and the Coltivirus member might have originated from a common virus ancestor.

A Reovirus Causes Hypovirulence of Rosellinia necatrix.

ABSTRACT White root rot, caused by Rosellinia necatrix, is a serious soilborne disease of fruit trees and other woody plants. R. necatrix isolate W370 contains 12 segments of double-stranded RNA (dsRNA) that is believed to represent a possible member of the family Reoviridae. W370 was weakly virulent and its hyphal-tip strains became dsRNA free and strongly virulent. The 12 segments of W370dsRNA were transmitted to hygromycin B-resistant strain RT37-1, derived from a dsRNA-free strain of W370 in all or none fashion through hyphal contact with W370. The W370dsRNA-transmitted strains were less virulent than their parent strain RT37-1 on apple seedlings, with mortality ranging between 0 to 16.7% in apple seedlings that were inoculated with the W370dsRNA-containing strains and 50 to 100% for seedlings inoculated with the dsRNA-free strains. Some W370dsRNA-containing strains killed greater than 16.7% of seedlings, but these were found to have lost the dsRNA in planta. These results indicate that W370dsRNA is a hypovirulence factor in R. necatrix. In addition, a strain lost one segment (S8) of W370dsRNA during subculture, and the S8-deficient mutant strain also exhibits hypovirulence in R. necatrix.

Human characteristics of pair assembly work in a microgravity environment.

This research focuses on analyzing the human characteristics of pair assembly work in a microgravity environment. To obtain the necessary data, two groups of test subjects participated in an experiment in a pseudomicrogravity environment, and how such factors as work allotment, sizes of assembled objects, and conditions of parts influence the work efficiency were studied. The results show when the assembled object is smaller that it is easier to change the object's position and that workers can move it to an appropriate site and work easily. If the assembly object is attached to an object with very large mass, the work allotment does not result in a difference in work time. As for positioning the objects, step-saving processes can shorten the work time. For instance, if a part can be put so that both ends automatically touch the proper spots, time can be shortened.

Nucleotide sequence of seed- and pollen-transmitted double-stranded RNA, which encodes a putative RNA-dependent RNA polymerase, detected from Japanese pear.

The nucleotide sequence of the largest double-stranded (ds) RNA (named dsRNA1) of three species of seed- and pollen-transmitted dsRNA species detected from Japanese pear was analyzed, and one strand was found to contain a single long open reading frame (ORF) of 1434 nucleotides that encoded a putative polypeptide containing 477 amino acid residues with a molecular mass of 54.9 kDa. This polypeptide contained amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Slow sedimentation of the dsRNA fraction suggests that the dsRNAs may be unencapsidated. The concentration of dsRNAs in the host, Japanese pear, was about 16 times higher than that from a cryptic virus, radish yellow edge virus (RYEV). These results suggest that the dsRNAs were not from cryptic viruses. Partial nucleotide sequences of the two smaller dsRNAs (named dsRNAs 2 and 3) and two other dsRNAs (named dsRNAs 4 and 5) detected from only the Japanese pear cultivar (cv.) Akita Tazawa 3 Gou were analyzed, and encoded nearly the same amino acid sequence encoded by dsRNA1.

TNF-alpha bifunctionally induces proliferation in primary hepatocytes: role of cell anchorage and spreading.

In the absence of a growth factor or an appropriate extracellular matrix (ECM), cells are arrested in the G0/G1 phase. In this report, we demonstrate the evidence that TNF-alpha induced DNA synthesis of primary mouse hepatocytes in vitro by activating two distinct pathways. TNF-alpha induced drastic spreading of hepatocytes on hydrophobic plastic, while the adhesion was not influenced. The effect was time and dose dependent. The cell spreading was accompanied by the phosphorylation of paxillin, indicating the stimulation of focal adhesion molecules. TNF-alpha-induced spreading of hepatocytes was not transient, and kinetic analysis and morphologic observation suggest that the effect was different from epidermal growth factor- or hepatocyte growth factor-induced transient hepatocyte spreading. TNF-alpha-induced hepatocyte spreading was blocked by cytochalasin D, Arg-Gly-Asp peptides, cycloheximide, or anti-integrin beta1 Ab. Results of competitive PCR for ECM proteins demonstrated that TNF-alpha increased the expression of laminin alpha3 and gamma1 chains in hepatocytes. These data suggested that TNF-alpha induced cell anchorage for hepatocytes by up-regulating ECM production. More importantly, TNF-alpha, but neither epidermal growth factor nor hepatocyte growth factor, induced DNA synthesis following the spreading in primary hepatocytes on hydrophobic plastic, while mere cell spreading on collagen did not induce DNA synthesis. The DNA synthesis was blocked by the inhibition of either cell spreading or DNA polymerase, demonstrating that TNF-alpha induced DNA synthesis in primary hepatocytes by activating two distinct pathways, i.e., forming the scaffold and inducing growth signals. Taken together, TNF-alpha bifunctionally regulates the proliferation of primary hepatocytes, serving as both an ECM inducer and a growth factor.

The surgeon's technical skill in suturing: an analysis of the actual suture tracks.

Ideal suturing was defined as advancing a needle along its curvature (needle circle) to minimize tissue trauma, while placing the suture with its intended span and tissue bite in the expected place. Actual suture tracks were analyzed to find the keys to produce such suturing. Correspondence of those tracks to the ideal track was then determined by the span, the initial needle angle (IA) into the tissue, and the center of the needle circle. Eight surgeons with 4-7 years of experience produced 22 ideal sutures in two types of tissue simulants: The entrance and exit points of the needle were level in flat suturing, while the entrance point was slanted 45 degrees for slant saturing. The correspondence was better with slant suturing than flat suturing (P < 0.01). The IA in flat suturing was 49.0 +/- 2.0 (mean +/- SE) degrees versus 33.0 for ideal suturing (P < 0.01), while that in slant suturing was 35.5 +/- 1.9 (P: ns). In conclusion, the IA was the key to good results, and was optimized in slant suturing, which was instinctively utilized in practice by using forceps. The forceps avoided a derangement of suturing stemming from the configuration of the needle employed and from the range of motion of the surgeon's arm (human engineering), while satisfying the surgeons inclination to take a large IA.

Suturing techniques for a restricted operating space.

The movements of the needle holder and the needle do not correspond when the needle grip is modified for suturing in awkward sites. In this study the range of movement of the needle holder was determined for three modified grips and the optimum suturing methods were determined for horizontal and vertical suturing fields and for restricted and open operating spaces. Gripping a needle obliquely resulted in a 90 degree counterclockwise rotation of the range of movement of the needle holder. Gripping it after rotation deviated the needle holder without altering the range of movement. A combined grip achieved by rotating after shifting had an intermediate effect. The optimum suturing for a restricted horizontal operating space was to use a half-circle needle in the first half of its range of movement with the rotated grip. With the oblique grip the last half of the range should be used and in the middle the combined grip is used although gripping the needle is most stable with the oblique grip. For a restricted vertical operating space, the third quarter of the movement range was allotted to the rotated grip and the last quarter to the combined grip.

Heterogeneity of immune complex-derived anti-DNA antibodies associated with lupus nephritis.

The mechanisms responsible for the tissue injuries associated with lupus nephritis have not yet been well explained. We have investigated the characteristics of anti-DNA antibodies in circulating immune complexes (CIC) and in the deposits of renal glomeruli in patients with active lupus nephritis. The CIC-derived antibodies expressed anti-DNA idiotypes (Id) designated as 0-81 Id and NE-1 Id, and bound mainly to single-stranded DNA but never to glomerular basement membrane (GBM) antigens. On the other hand, the immunoglobulins (Ig) eluted from renal glomeruli of lupus patients reacted not only with DNA but also with GBM, proteoglycan, and heparan sulfate. The binding of glomeruli-deposited Ig was markedly low when GBM antigens were used after treatment with heparitinase, suggesting that some anti-DNA antibodies may bind directly to GBM antigens associated with heparan sulfate, and form in situ IC in renal glomeruli. It was also revealed that the renal eluates obtained after passing through GBM antigen-coupled Sepharose lost the binding ability with GBM but still retained DNA-binding and 0-81 Id activity, showing the participation of circulating IC-derived anti-DNA antibodies in the glomerular deposits. Theoretically there may be two mechanisms in the pathogenesis of lupus nephritis through the deposition of circulating IC and through in situ formation of anti-DNA IC in renal glomeruli. The diversity of histological features in lupus kidneys may be attributed to the heterogeneity of the mechanisms.

Selective absorption of anti-DNA antibodies and their idiotype-positive cells in vitro using an anti-idiotypic antibody-affinity column: possible application to plasma exchange.

We examined the possibility of using affinity columns coupled with anti-idiotype (Id) antibodies to selectively remove nephritogenic anti-DNA antibodies in order to determine their possible application to therapeutic plasmapheresis. Monoclonal anti-Id antibodies termed D1E2 or 1F5 were directed to idotypes of human anti-single-stranded and anti-double-stranded DNA antibodies. The mixture of D1E2- and 1F5-coupled Sepharose absorbed 26 to 92% of human anti-DNA antibodies in sera. The affinity columns were also effective in removing anti-DNA idiotype-positive cells from the blood samples of patients, especially those with active lupus nephritis. Thus, an anti-idiotypic antibody-coupled affinity column could, in theory, serve as a tool for selective plasma exchange in the therapy of autoimmune disease.

Biological activity of obiopeptide-1, a synthetic peptide derived from the native immune-regulator obioactin.

Tachyzoites of Toxoplasma gondii were killed in mouse macrophage and human somatic cell monolayers by a novel synthetic peptide (Obiopeptide-1) which is a Glycil-penta-Glutaminate (GpG) derivative of native Obioactin. In view of the worldwide prevalence of this protozoan disease and the lack of effective treatments, Obiopeptide-1 may be a new and unique antimicrobial active substance of non-antibiotic chemotherapeutic agents for intracellular parasites, T. gondii and associated nonspecific hypoimmune responses that occur in infected hosts.

Therapeutic treatment of New Zealand mouse disease by a limited number of anti-idiotypic antibodies conjugated with neocarzinostatin.

-81 and NE-1 idiotypes (Id) of human nephritogenic anti-DNA antibodies are interspecies Id expressed also in NZB/W F1 mice. We tried to manipulate the synthesis of spontaneously occurring anti-DNA antibody using monoclonal anti-Id antibodies (D1E2 and 1F5) conjugated with a cytotoxic agent, neocarzinostatin (NCS). In vivo administration of anti-Id antibodies conjugated with NCS brought about an improvement in the survival rate of female NZB/W F1 mice. It also caused a retardation of development of lupus nephritis and decreased the numbers of anti-DNA-producing cells. The suppression of anti-DNA antibody synthesis was specific and Id-mediated. The results indicate that the use of a limited number of anti-Id antibodies in combination with a cytotoxic agent may be applicable therapeutically to autoimmune diseases.

Hand-Schüller-Christian disease with occult diabetes insipidus, cardiac failure and renal dysfunction.

A 60-year-old man was diagnosed as Hand-Schüller-Christian disease due to the triad of exophthalmus, decalcification of the bone, and diabetes insipidus. He had xanthogranuloma on the face and a nuchal region, and unusual complications of ADH-resistant diabetes insipidus due to renal dysfunction, and chronic cardiac failure. Urine osmolality was hypotonic, but urine volume was within the normal limit, despite the presence of central diabetes insipidus. Hypophyseal, adrenal and thyroid function were not remarkable. The skin biopsy showed the infiltration of eosinophilic granuloma cells. Treatment with vincristine was effective to regress the xanthogranuloma. Diabetes insipidus was not treated because of the absence of polyuria and polydipsia.

Inhibitory effect of Toxoplasma lysate antigen on the multiplication of transplanted tumor in mice.

Tachyzoites of the RH strain of Toxoplasma gondii were pretreated by a process of freezing and thawing followed by ultrasonication. After ultracentrifugation at 144,000 g for 120 minutes, the resulting supernatant contained a protein (TLA 144), which consisted mainly of a component of protozoan origin with a molecular weight of 10,000-20,000, and additional sugars, peptides, and amino acids. TLA 144 was soluble in water and of very low toxicity. Mice that had been inoculated with allogeneic (S-180) and syngeneic (Meth A) transplantable tumor cells, were injected intramuscularly with 100 micrograms of TLA 144 once a week for some time beginning one week after transplantation. It was found that after TLA treatment the multiplication of tumor cells was more intensely inhibited than following administration of OK-432, one of the biological response modifiers (BRMs).

Correlation between release of reactive oxygen intermediates and inhibition of Toxoplasma multiplication in mouse peritoneal and alveolar macrophages and kidney cells after in vitro incubation with Obioactin, lonomycin A, muramyl dipeptide, lipopolysaccharide or Toxoplasma lysate antigen.

The inhibition of Toxoplasma multiplication inside cells does not correlate with an enhanced release of oxygen intermediates except in the case of peritoneal macrophages treated with Obioactin. The inhibition observed in alveolar macrophages treated with Obioactin, in kidney cells treated with Obioactin or lonomycin A and in peritoneal macrophages treated with lonomycin A was not accompanied by an increment of release of oxygen intermediates. Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) enhanced the release of toxic oxygen intermediates in peritoneal macrophages, but did not have any toxoplasmacidal effect. Adenosine triphosphate (ATP) content increased during Obioactin, MDP or Toxoplasma lysate antigen (TLA) treatment. The actual oxygen consumption of the peritoneal macrophages treated with Obioactin increased dose dependently, but that of TLA-, lonomycin A- or MDP-treated cells did not change. These results suggest that the relationship between the intracellular killing of Toxoplasma protozoa and the release of oxygen intermediates differs according to the cells and/or the stimuli, and that the cellular mechanism of Toxoplasma killing in the peritoneal macrophages treated with Obioactin involves an energy-dependent mechanism.

Modulator effect of Toxoplasma lysate antigen in mice experimentally infected with Plasmodium berghei.

Normal mice were pretreated twice at an interval of 2 weeks with an emulsion of TLA (Toxoplasma lysate antigen), PLA (Plasmodium lysate antigen) or both in LMO (light mineral oil) or with a combination of the emulsion and Obioactin or Tp-LKs (Toxoplasma lymphokines) as an immunopotentiator. They were then given Obioactin or Tp-LKs 3 and 25 days after the first treatment and were further given parasitized erythrocytes with 1 X 10(2)-10(4) P. berghei 2 weeks after the second treatment. Thirty (3/10, number of survival/number of examined) per cent of mice treated with TLA, 50 (5/10)% of those treated with a combination of TLA and Tp-LKs and 60 (6/10)% of those treated with a combination of TLA and Obioactin survived as long as 20 days postinfection while none of untreated controls survived more than 15 days postinfection. Only 18.2 (2/11)% of mice treated with PLA or TLA + PLA survived and 20 (2/10), 18.2 (2/11) and 60 (6/10)% of those treated with TLA + Obioactin, PLA + Obioactin or TLA + PLA + Obioactin survived throughout the experiment, respectively while none of controls survived more than 13 days postinfection. Five mice of each group were killed right before infection, and 5, 10 and 15 days postinfection. In mice treated with TLA + Obioactin, more macrophage phagocytosis and macrophage migration inhibition induced by sensitized T-cells were observed than in those treated otherwise. No appreciable differences were noted according to the method of treatment in blood examination values. Cross immunities between Toxoplasma and Plasmodium antigens were tested by counter-immunoelectrophoresis and indirect fluorescent antibody technique. By using counter-immunoelectrophoresis, a specific precipitin line was observed between TLA and anti-PLA which was absorbed by mouse erythrocytes, leucocytes and liver powder. By the indirect fluorescent antibody technique, anti-Plasmodium IgM and IgG titers were detected in sera from mice treated with TLA or TLA-Obioactin before infection.

In situ microfluorometry of kinetoplast and nuclear DNAs in Trypanosoma gambiense. Unusual repairment of DNA after treatment with bleomycin.

The blood stream form of Trypanosoma gambiense was smeared on a nonfluorescent slide glass with 1 microgram/ml of Hoechst 33258 in 1 mM Tris-HCl buffer (pH 7.2) containing 1% 2-mercaptoethanol and subjected to the in situ microfluorometry. Effects of bleomycin (BL) on the kinetoplast (K)-DNA and nuclear (N)-DNA of T. gambiense were examined in the time course to 6 h after injection of BL into the infected mice. An enhancement of fluorescence occurred 30 min after the injection and then slowed down. This enhancement was due to DNA synthesis both in the K-DNA and N-DNA. This suggests that the strong repairment occurs in both DNAs after treatment with BL.