Hiroshi Harada

Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms

Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl-/- haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl+/+ HSPCs, and transduction of C-CBL mutants into c-Cbl-/- HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl+/+ background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.

Is eradication therapy useful as the first line of treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan.

A retrospective study was performed to determine the prevalence of Helicobacter pylori (H pylori) infection, the effect of H pylori eradication on platelet counts, and the characteristic clinical features of chronic immune or idiopathic thrombocytopenic purpura (ITP) with H pylori infection. H pylori infection was found in 300 patients, a group that was significantly older (P < .005) and had more cases of hyperplastic megakaryocytes in the bone marrow (P = .01) than patients without H pylori infection.H pylori eradication therapy was performed in 207 H pylori-positive ITP cases, and the platelet count response was observed in 63% of the successful eradication group and in 33% of the unsuccessful eradication group (P < .005). In the successful group, the complete remission and partial remission rates were 23% and 42%, respectively, 12 months after eradication. In the majority of responders, the platelet count response occurred 1 month after eradication therapy, and the increased platelet count continued without ITP treatment for more than 12 months. H pylori eradication therapy was effective even in refractory cases, which were unresponsive to splenectomy. In conclusion, H pylori infection was involved in most ITP patients older than 40 years in Japan, and eradication therapy should be the first line of treatment in H pylori-positive ITP patients.

Inhibition of influenza virus infection by pine cone antitumor substances

The anti-influenza virus activity of polysaccharides and other high molecular weight fractions from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. was investigated. None of the fractions affected the growth of MDCK cells. The acidic PCE substances markedly suppressed the growth of the influenza virus in MDCK cells. Significant inhibition of both the viral protein synthesis in infected cells and virion-associated RNA-dependent RNA polymerase activity was observed with these acidic fractions. Although amantadine inhibited virus plaque formation as effectively as PCE fractions, it was less effective in inhibiting the RNA polymerase activity. These results suggest that PCE, which has been shown to contain antitumor substance(s), also contains anti-influenza virus substance(s).

Possible involvement of lignin structure in anti-influenza virus activity

Commercial lignins suppressed the growth of influenza A virus infecting MDCK cells, and the RNA-dependent RNA synthesis, as efficiently as the high-molecular weight fractions extracted from pine cone of Pinus parviflora Sieb. et Zucc. The anti-influenza A virus activity of both pine cone extract and commercial alkali-lignin was considerably reduced by treatment with sodium chlorite, but was not affected by sulfuric acid or trifluoroacetic acid. The degraded components of lignin, various synthesized polyphenols unrelated to lignin, and natural and chemically modified glucans, were not appreciably inhibitory. The data suggest that the polymerized phenolic structure of lignified materials is responsible for the anti-influenza A virus activity.

AMPHIBIA: A Cognitive Virtualization Platform for End-to-End Slicing

To cope with the increasingly diversifying services, QoS, and network architectures, network virtualization is a promising technology that enables the concurrent deployment of multiple network technologies on a shared network. However, traditional research on network virtualization preliminarily focuses on a wired environment and network virtualization for a wireless environment is not well studied. Considering that in near-future, various wireless technologies will play the most important role in access networks and multi-mode wireless terminals will become more popular, it is crucial to extend the concept of network virtualization to wireless networks. We refer to building such extended virtual networks as end-to-end slicing. The key technical challenges for such extension are (1) abstraction of heterogeneous wireless access networks for maximizing radio frequency utilization and (2) isolation of wireless resources such as radio frequencies, throughput, or name spaces for accommodating multiple virtual networks on a single wireless access network. In this paper, we tackle the first challenge and propose a Cognitive Virtualization Platform, called AMPHIBIA, which enables end-to-end slicing over heterogeneous wired and wireless networks. AMPHIBIA is a platform to provide independent virtual networks each of which can be configured for the corresponding service and to coordinate the resource management in both sides of wired and wireless networks, exploiting the network virtualization and cognitive radio technology. AMPHIBIA is motivated by the shared property of reconfigurability of network virtualization and cognitive radio, and provides network operators with the capability of cooperative resource allocation over wired and wireless networks. In other words, AMPHIBIA virtualizes a cognitive base station to dynamically configure a wireless access network for each virtual network. In this paper, we first show the basic architecture of AMPHIBIA from the perspective of network virtualization. Then we show the hardware and software design of prototype system.

Enterolosaponins A and B, novel triterpene bisdesmosides from Enterolobium contortisiliquum, and evaluation for their macrophage-oriented cytotoxic activity.

Two novel triterpene bisdesmosides, designated as enterolosaponin A (1) and B (2), were isolated from Enterolobium contortisiliquum. The chemical structures of 1 and 2 were determined by analysis of their extensive spectroscopic data, as well as hydrolysis followed by chromatographic study. Enterolosaponins have a 2-amino-2-deoxy-D-glucosyl unit (D-glucosamine) as one of the monosaccharides constituting their oligosaccharide moieties, which have been rarely found in natural product research. Enterolosaponin A (1) exhibited a highly selective cytotoxicity against BAC1.2F5 mouse macrophages, and it should be notable that the macrophage death caused by 1 was shown to be neither necrotic nor due to induction of apoptosis from morphology of the died cells, whose cytosol occurred in vacuolation.

Transition of Polycythemia Vera to Chronic Neutrophilic Leukemia

Two cases of polycythemia vera (PV) had transition to a hematological condition compatible with chronic neutrophilic leukemia (CNL) 17 and 8 years after diagnosis, respectively. One patient was treated with carboquone followed by hydroxyurea (HU) and the other with HU during PV phase. On transition, both had neutrophilia with white blood cell count above 40,000/microl, elevated neutrophil alkaline phosphatase activity, splenomegaly, normal karyotype without bcr-abl rearrangement. Busulfan was temporally effective in controlling the neutrophil count. However, one patient progressed to the so-called spent phase and the other subsequently had multiple transitions between PV and CNL. These cases may represent a form of uncommon evolution of PV and support the contention that CNL is a type of myeloproliferative disorder and that at least some CNL cases have derangement at the hematopoietic stem cell level.

Flower-induction in excised shoot apices of Pharbitis and Chrysanthemum cultured in vitro

THE existence of flower-inducing substances has been demonstrated by many workers, although it has been done in a more or less indirect way. Efforts to extract and isolate these substances, however, have so far had but few encouraging results. One of the main reasons for the rather disappointing results could be attributed to the lack of a proper means of detecting the active substances. The use of the entire plant as a system for bioassay is relatively easy, but has many shortcomings1,2. The use of tissues such as the root3 and the stem4 of Cichorium intybus and the stem of Plumbago indica2 has opened up new possibilities in the search for flowering substances. While plant apices were long thought to be adequate material for this purpose, the results so far obtained with them have been rather discouraging5.

Cytogenetics, FISH and RT-PCR Analysis of Acute Promyelocytic Leukemia: Structure of the Fusion Point in a Case Lacking Classic t(15;17) Translocation

The chimeric gene product PML-RAR α, the result of a reciprocal t(15;17) translocation, plays an important role in the pathogenesis of acute promyelocytic leukemia (APL). In the present study on clinical effects of cytogenetics and molecular events in APL, we performed chromosome analysis, fluorescence in situ hybridization (FISH) using gene-specific probe, and reverse transcription-polymerase chain reaction (RT-PCR) analysis in 10 patients with APL. Patients were treated with all- trans retinoic acid (ATRA) and/or chemotherapy, and all achieved complete remission. Cytogenetic analysis revealed the classic translocation t(15;17) in nine of 10 patients, and a remaining patient had an apparently normal karyotype. Interphase FISH was performed in nine patients, and revealed the presence of PML-RAR α fusion gene in all patients. RT-PCR analysis in 10 patients showed that eight expressed long (L)-form type, one expressed a short (S)-form type, and the other expressed a variable (V)-form type. Metaphase FISH of the patient with normal karyotype revealed a juxtaposed PML-RAR α fusion signal on one chromosome 17 homologue, an RAR α signal on the other chromosome 17 homologue, and one PML signal on each chromosome 15 homologue. Moreover, V-form of the PML-RAR α transcript was detected, and a portion of RAR α intron 2 was found inserted in the breakpoint region. ATRA differentiation induction therapy was effective in treating this patient, a result infrequently reported in cytogenetic and molecular investigations of APL.

Antimicrobial activity induction by PSK subfractions: dependence on molecular weight.

A protein-bound polysaccharide, PSK, extracted from the mycelium of Coriolus versicolor (Fr.) Quel, has been 100recognized for its host-mediated antitumor activity [1] and the ability to induce antimicrobial activity in mice [2]. PSK has at least four different subfractions with distinct 80molecular weights [3], which we compared for their induction of antimicrobial activity. PSK [1] and its subfractions, obtained by successive filtra60tion through membrane filters [3], were provided by Ku~reha Chem. Ind. Co., Ltd., Tokyo, Japan. When female ICR mice (six to seven weeks old) were ino 40E oculated with about one minimum lethal dose (4 x l06 cells) of Escherichia coli, only 17% survived after five o days. However, if mice were pretreated intraperitoneally ~ 20with 0.4-40 mg/kg PSK two days before the E. coli chalE lenge, the survival rate increased dose dependently up to o_ o 80% (Figu re 1 ). The effect of PSK became detectable 4 h -- after treatment, and reached its maximum after one to o 57 three days (not shown). Antimicrobial activity induced by ~the highest molecular weight fraction (MW > 200 kD) exo ~o ceeded that of unfractionated PSK (Figure 1). Effects of e4 this subfraction were detected at 0.04 mg/kg, and reached ~ maximum at 0.4-40 mg/kg. The subfraction with MW 2 100-200 kD was less potent, and those with MW < 50 kD, ~ 3 and 50-100 kD induced no activity. Treatment of mice ~0 rwith any PSK subffactions for 24--48 h significantly enO hanced generation of luminol-dependent chemiluminescence (LDCL) by peritoneal exudate cells (Figure 2A), 2. but by 72 h, the LDCL generation, except in mice treated with the highest molecular weight subfraction, declined to the control level. Although LDCL generation by adher| ent macrophages in the treated mice was less than 10% of that of the total peritoneal exudate cells, stimulation of