Search

Objectives: To characterise cartilage intermediate layer protein (CILP)-induced arthropathy in mice. Methods: The first and second halves of the nucleotide triphosphate pyrophosphohydrolase (NTPPHase) non-homologous region of human CILP were prepared [...]

High-dose methotrexate (Hd-MTX) therapy has recently been applied to the treatment of adult acute lymphoblastic leukemia (ALL) based on pediatric protocols; however, its effectiveness for adult ALL has not yet been confirmed in a rigorous manner. We herein conducted a randomized phase III trial comparing Hd-MTX therapy with intermediate-dose (Id)-MTX therapy. This study was registered at UMIN-CTR (ID: C000000063). Philadelphia chromosome (Ph)-negative ALL patients aged between 25 and 64 years of age were enrolled. Patients who achieved complete remission (CR) were randomly assigned to receive therapy containing Hd-MTX (3?g/m2) or Id-MTX (0.5?g/m2). A total of 360 patients were enrolled. The CR rate was 86%. A total of 115 and 114 patients were assigned to the Hd-MTX and Id-MTX groups, respectively. The estimated 5-year disease-free survival rate of the Hd-MTX group was 58%, which was significantly better than that of the Id-MTX group at 32% (P=0.0218). The frequencies of severe adverse events were not significantly different. We herein demonstrated the effectiveness and safety of Hd-MTX therapy for adult Ph-negative ALL. Our results provide a strong rationale for protocols containing Hd-MTX therapy being applied to the treatment of adult ALL.

As leukemic transformation of myeloproliferative neoplasms (MPNs) worsens the clinical outcome, reducing the inherent risk of the critical event in MPN cases could be beneficial. Among genetic alterations concerning the transformation, the frequent one is TP53mutation. Here we show that retroviral overexpression of Jak2 V617Fmutant into wild-type p53murine bone marrow cells induced polycythemia vera (PV) in the recipient mice, whereas Jak2 V617F-transduced p53-null mice developed lethal leukemia after the preceding PV phase. The leukemic mice had severe anemia, hepatosplenomegaly, pulmonary hemorrhage and expansion of dysplastic erythroid progenitors. Primitive leukemia cells (c-kit+Sca1+Lin−(KSL) and CD34−CD16/32−c-kit+Sca1−Lin−(megakaryocyte-erythroid progenitor; MEP)) and erythroid progenitors (CD71+) from Jak2 V617F-transduced p53-null leukemic mice had leukemia-initiating capacity, however, myeloid differentiated populations (Mac-1+) could not recapitulate the disease. Interestingly, recipients transplanted with CD71+cells rapidly developed erythroid leukemia, which was in sharp contrast to leukemic KSL cells to cause lethal leukemia after the polycythemic state. The leukemic CD71+cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2 V617F-transduced p53-null erythroleukemia. Taken together, our results show that p53loss is sufficient for inducing leukemic transformation in Jak2 V617F-positive MPN.

This retrospective study was conducted in Japan to determine the incidence, risk factors and outcomes of sinusoidal obstruction syndrome (SOS) after allogeneic hematopoietic stem cell transplantation (HSCT). Among 4290 patients undergoing allogeneic HSCT between 1999 and 2010, 462 were diagnosed with SOS according to the Seattle criteria (cumulative incidence, 10.8%). The cumulative incidence of SOS diagnosed by the modified Seattle criteria was 9.3%. Of 462 patients, 107 met the Baltimore criteria and 168 had severe SOS with renal and/or respiratory failure. The median onset for SOS was 12 days after HSCT (range, −2–30). Overall survival at day 100 was 32% for SOS and 15% for severe SOS. Multivariate analyses showed that significant independent risk factors for SOS were the number of HSCTs, age, performance status, hepatitis C virus-seropositivity, advanced disease status and myeloablative regimen. SOS was highly associated with overall mortality (hazard ratio, 2.09; P<0.001). Our retrospective survey showed that the cumulative incidence of SOS in Japan was 10.8%, similar to that previously reported in Western countries, and that the overall survival of patients who developed SOS was low. Furthermore, several risk factors were identified. Preventive and therapeutic strategies for high-risk SOS patients must be established to improve overall survival.

Absolute total cross sections for electron scattering from Ar and Xe at electron energies ranging from 7 meV to 20 eV were obtained with the experimental technique employing the threshold-photoelectron source. The measured total cross sections are in good agreement with those obtained by other groups down to 100 meV, above which several experimental works have been reported. Scattering lengths for electron scattering from Ar, Kr, and Xe were determined from the present total cross sections and our recent results for Kr using the modified effective range theory. The values of the scattering length obtained in the present analysis differ from the values determined from the previous swarm experiments and beam experiments. The resonant structures in the total cross sections due to Feshbach resonances of Ar, Kr, and Xe with an improved energy resolution were also measured. Analyses of the resonant structure were carried out based on the spin-dependent resonant scattering theory in order to determine the values of the natural width of Feshbach resonances of Ar, Kr, and Xe precisely. [ABSTRACT FROM AUTHOR]

Abstract: Two-proton relative momentum distributions from the break-up channels 23Al→p+p+21Na and 22Mg→p+p+20Ne at an energy of 60–70 A MeV have been measured together with two-proton opening angles at the projectile fragment separator beamline (RIPS) in the RIKEN Ring Cyclotron Facility. The results demonstrate the existence of diproton emission component from single-step 2He for highly excited 23Al and 22Mg.

In order to explore the functions of the peripheral neurons in the enteric nervous system (ENS) of the gastropods, Aplysiaand Lymnaea, we investigated the correlation between peripheral neuronal activities and movements of the digestive tract. In Aplysia, movements of the gizzard were distinguished into two types of contraction: a large constriction of the whole gizzard following bursting activities of the neurons on the gizzard and EJP-like potentials in the musculature; and a small contraction of a restricted part of the gizzard following a slow muscle potential. When TTX was applied to isolated gizzard preparation, the bursting activities were blocked and the EJP-like potentials and the subsequent constriction disappeared, whereas the slow potentials in the musculature and partial contractions appeared to be unaffected. Therefore, it was suggested that the peripheral neurons on the gizzard were motor neurons for constriction, while the partial contraction was thought to be myogenic. In Lymnaea, we recorded periodic bursting activities in the enteric nervous system that were followed by EJP-like potentials and gastrointestinal movements. The results show that, in both species, there may exist motor neurons in the ENS that are responsible for neurogenic movements of the digestive tract.

Replica microchips for capillary array electrophoresis containing 10 separation channels 50 µm width, 50 µm depth and 100 µm pitch and a network of sacrificial channels 100 µm width and 50 µm depth were successfully fabricated on a polymethyl methacrylate PMMA substrate by injection molding. The strategy involved development of moving mask deep X-ray lithography to fabricate an array of channels with inclined channel sidewalls. A slight inclination of channel sidewalls, which can not be fabricated by conventional deep X-ray lithography, is highly required to ensure the release of replicated polymer chips from a mold. Moreover, the sealing of molded PMMA multichannel chips with a PMMA cover film was achieved by a novel bonding technique involving adhesive printing and a network of sacrificial channels. An adhesive printing process enables us to precisely control the thickness of an adhesive layer, and a network of sacrificial channels makes it possible to remove air bubbles and an excess adhesive, which are crucial to achieving perfect sealing of replica PMMA chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to simultaneously monitor electrophoretic separations in ten micro-channels with laser-induced fluorescence detection. High-speed and high-throughput separations of a 100 bp DNA ladder and X174 HaeIII DNA restriction fragments have been demonstrated using a 10-channel PMMA chip. The current work establishes the feasibility of mass production of PMMA multichannel chips at a cost-effective basis.

Mammalian oocytes undergo significant growth during oogenesis and experience extensive cytoplasmic and nuclear modifications immediately before ovulation in a process commonly referred to as oocyte maturation. These changes are intended to maximize the developmental success after fertilization. Entry of a spermatozoon into the oocyte, which occurs a few hours after ovulation, initiates long-lasting oscillations in the free intracellular calcium ([Ca(2+)](i)) that are responsible for all events of oocyte activation and the initiation of the developmental programme that often culminates in the birth of young. Nevertheless, the cellular and molecular changes that occur during maturation to optimize development are transient, and exhibit rapid deterioration. Moreover, fertilization of oocytes after an extended residence in the oviduct (or in culture) initiates a different developmental programme, one that is characterized by fragmentation, programmed cell death, and abnormal development. Inasmuch as [Ca(2+)](i) oscillations can trigger both developmental programmes in mammalian oocytes, this review addresses one of the mechanism(s) possibly used by spermatozoa to initiate these persistent [Ca(2+)](i) responses, and the cellular and molecular changes that may underlie the postovulatory cellular fragmentation of ageing mammalian oocytes.

The aim of the work is to improve the power conversion efficiency of a voltage-source three-phase inverter with a single active auxiliary resonant DC link snubber circuit (ARDCL). The operating principle and salient features of the ARDCL snubber circuit are described. An effective space voltage vector generation method is of proposed zero voltage vector of a three-phase soft-switching inverter, which can reduce power losses in the ARDCL snubber circuit. The zero voltage holding interval in the DC bus-line of the three-phase inverter is controlled due to the generation method of the zero voltage vector. The feasible experimental results confirm the relationship between the inverter power conversion efficiency and the proposed zero voltage vector generation method is concretely implemented and evaluated from a practical point of view.

The highly neutron-rich nucleus 34Mg has been studied via Coulomb excitation using a radioactive 34Mg beam at 44.9 MeV/nucleon with a Pb target. The B(E2;0+g.s.→2+1) value for the previously proposed 2+1state in 34Mg was determined for the first time to be 631(126) e2fm4. This value corresponds to the quadrupole deformation parameter β2of 0.58(6), implying an anomalously large deformation of 34Mg.

The Isobaric Analog State (IAS) of the neutron-dripline nucleus 14Be has been found for the first time in a charge-exchange reaction 14Be(p,n)14B∗in inverse kinematics at E(14Be)=74 AMeV. The excitation energy and width of the observed IAS were measured to be 17.06±0.03 MeV and 0.11±0.05 MeV (FWHM), respectively. The Coulomb displacement energy and the measured width of the IAS are discussed in connection with a possible neutron-halo structure of the 14Be nucleus.

A novel prototype of auxiliary resonant DC link (ARDCL) snubber is proposed. The paper also describes a three-phase space voltage vector modulated sinewave soft-switching inverter using the proposed ARDCL snubber circuit, which together with new voltage vector correction principle can produce high-performance output voltage waveforms. The output voltage waveforms obtained by the software processing of the inverter are able to achieve a high quality sinewave on the basis of the new correction principle. The effectiveness of the resonant DC link three-phase voltage source soft-switching inverter is proven from a practical point of view.

Abstract: Gill motor neuron L7-induced longitudinal shortening of the gill in Aplysia kurodai and A. juliana was suppressed when extracellular stimuli were applied to a restricted dorsal central region of the abdominal ganglion. We found a neuron there which antagonized the L7-driven contraction. Since the contraction was suppressed when the identified neuron was activated simultaneously with L7, we refer to the newly found neuron as “Anti-L7”. Anti-L7 did not change the L7 impulse generation in the abdominal ganglion. No direct synaptic connection from L7 to Anti-L7 was detected. A fluorescent dye injected into the soma of Anti-L7 revealed that the neuron sent axonal branches to the branchial nerve. These results may show that Anti-L7 antagonizes L7 at the periphery inside the gill, rather than in the abdominal ganglion. EJPs induced by L7 were unaffected by Anti-L7. Activation of Anti-L7 alone did not induce any change in tone or membrane potential of the gill musculature. The suppressive effect of Anti-L7 lasts many seconds after the cessation of a train of Anti-L7 impulses. The results may suggest that the suppression is mediated through an inhibitory neuromodulatory mechanism without inhibition of L7 itself.

OBJECTIVE: To explore mechanisms for hypertriglyceridemia in diabetic patients with microalbuminuria, we examined an association between heparin-releasable lipoprotein lipase (LPL) and the von Willebrand factor (vWF), based on the hypothesis that LPL bound to endothelium is decreased by generalized endothelial damage. RESEARCH DESIGN AND METHODS: A total of 37 NIDDM patients with microalbuminuria and 69 patients with normoalbuminuria were studied. Plasma LPL mass in post-heparin plasma and plasma vWF antigen were quantified by sandwich-enzyme immunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: The NIDDM patients with microalbuminuria had higher plasma triglyceride (TG) and lower HDL cholesterol concentrations compared with the patients with normoalbuminuria. Heparin-releasable LPL mass was significantly lower in the microalbuminuric than in the normoalbuminuric subjects. Plasma level of vWF, a marker for endothelial damage, was significantly increased in microalbuminuric subjects compared with their normoalbuminuric counterparts. The LPL mass was inversely correlated with plasma vWF level at a high correlation coefficient value. The LPL mass was inversely related to TG and positively to HDL cholesterol concentrations. CONCLUSIONS: These results suggest that widespread endothelial damage occurred in NIDDM patients with microalbuminuria, thereby LPL moiety bound to the endothelium is decreased, which results in an impaired catabolism of TG-rich lipoproteins.

A difference was observed in the intracellular distribution between type I and II hexokinases in Ehrlich-Lettre hyperdiploid ascites tumor cells (ELD cells). Experiment of the rebinding to the mitochondria for either each or mixture of the partially purified preparations of the two types of hexokinase indicated that the accepting site on the mitochondrial membrane was common for both types. Mild treatment of the two isoenzymes with chymotrypsin resulted in loss of the binding ability to mitochondria without change in the catalytic activity. It was deduced from these results that the essential region in the two types of hexokinase to interact with mitochondria, which was cleaved by chymotrypsin, was the same or near-similar.

Varicella-zoster virus glycoproteins were purified by using monoclonal antibodies and analyzed for their effects on cell-free virus infection. Preinfection treatment of cells with gH:gL reduced the infection efficiency and increased the number of unadsorbed virus. Postinfection treatment of cells with gB increased the infection efficiency, but that with gE:gI reduced it. Treatment of gE:gI and gH:gL with neuraminidase (NA) abolished their inhibitory activity and the plaque formation was enhanced by NA treatment of glycoproteins and cells. Glycoproteins exhibited their diverse activities despite their common role in viral penetration, and sialyl residues were responsible for their function in cell-free virus infection.

Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by alternative splicing. Both forms have a DNA binding domain but, unlike AML1b, AML1a lacks a putative transcriptional activation domain. Here we demonstrate that overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dcl3 murine myeloid cells treated with granulocyte colony‐stimulating factor. These effects of AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that (i) AML1a, which on its own has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and (ii) AML1a exhibits the higher affinity for DNA binding compared with AML1b. These antagonistic actions could be important in leukemogenesis and/or myeloid cell differentiation because more than half of myelogenous leukemia patients showed an increase in the relative amounts of AML1a.

Flow visualization results for secondary flow phenomena at the exit of 90° and 180° bends and helically coiled pipes (1, 2 and 5 turns), (radius of curvature R_c = 381 mm, inside pipe diameter d=37.5 mm, curvature ratio d/2R_c = 0.049) and in the downstream straight pipe ( l/d = 30 ) are presented to study the stabilizing (relaminarization) effect in curved pipes with fully developed entry turbulent air flow and the destabilizing (re-transition from laminar to turbulent flow) effect in the downstream straight region. The entry Reynolds numbers are Re = 2200, 3200, 4300 and 5300). The related measurement results using a hot-film anemometer are presented for developing profiles of the time-mean streamwise velocity distribution and the axial turbulence intensity field in the 180° return bend and in the downstream straight pipe for Reynolds numbers Re = 3200, 4300, 6300 and 8200. The time traces showing the output of the hot-film sensor are also presented for developing fluctuating velocity field in the 180° bend and in the downstream straight pipe for the same Reynolds number range. Some aspects of the relaminarization phenomena in curved pipes and the re-transition phenomena from laminar to turbulent flow in the downstream straight pipe are clarified by the present experimental investigation.

Correlation between T cell phenotypes, especially activated T cells expressing early (EA1) and late (HLA-DR) activation antigens and clinical features were investigated in 22 patients with systemic lupus erythematosus (SLE) of childhood onset. Percentages of T cells expressing EA1 and HLA-DR in 22 patients with SLE were significantly higher than those in controls. Comparison of T cell phenotypes between patients with active and inactive SLE showed that eight patients with active disease had significantly increased percentages of HLA-DR positive T cells than 14 with inactive disease (P<0.01). Serial examinations showed that the percentages of HLA-DR positive T cells were decreased after therapy in seven with active non-renal or active non-renal and renal diseases but not in one with only active renal disease. A possible significance of T cells expressing EA1 and HLA-DR in the management of patients with SLE is discussed.

Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. Klagsburn. 1987. Proc. Natl. Acad. Sci. USA. 84:2292-2296) and in basement membranes (Folkman, J., M. Klagsburn, J. Sasse, M. Wadzinski, D. Ingber, and I. Vlodavsky. 1988. Am. J. Pathol. 130:393-400). The actual requirement of ECM-associated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.

1. In the guinea‐pig pelvic plexus‐vas deferens preparation, stimulation of the parasympathetic pelvic nerves contracted the vas deferens then depressed the contractile responses to stimulation of the sympathetic hypogastric nerves. 2. The contraction caused by stimulation of the pelvic nerves was initially phasic then tonic. The contractions were almost abolished by application of hexamethonium to the plexus. The phasic contraction was abolished by alpha,beta‐methylene adenosine triphosphate applied to the vas deferens. 3. Conditioning stimulation of the pelvic nerves preferentially depressed the phasic component of test contractions evoked by hypogastric nerve stimulation but did not affect the compound action potentials in postganglionic nerves evoked by test stimulation. 4. When the pelvic plexus was divided into two parts, one with the pelvic nerves and the other with the hypogastric nerves, conditioning stimulation of the pelvic nerves still depressed test contractions evoked by hypogastric nerve stimulation. 5. In the de‐ganglionated vas deferens preparation, conditioning stimulation of some postganglionic nerves also depressed contractions evoked by test stimulation of the other postganglionic nerves. 6. 8‐Phenyltheophylline (5‐20 microM) applied to the vas deferens antagonized the conditioning stimulation‐induced depression in both the pelvic plexus‐vas deferens and the de‐ganglionated preparations. 7. N6‐Cyclohexyladenosine (CHA) and N6‐(L‐2‐phenylisopropyl)‐adenosine at 0.5 microM preferentially inhibited phasic contractions evoked by the postganglionic nerve stimulation. The effect of CHA was antagonized by 8‐phenyltheophylline (10 microM). 8. The results indicate that the mechanism underlying the conditioning stimulation‐induced depression of phasic contractions operates not in the ganglia, but through activation of adenosine receptors in the vas deferens.

1. In the pelvic plexus‐vas deferens preparation of the guinea‐pig, conditioning stimulation of the pelvic nerves depressed the phasic component of biphasic contractions evoked by test stimulation of the hypogastric nerves, but potentiated the tonic component. 2. Similarly, in the deganglionated vas deferens preparation, conditioning stimulation applied directly to postganglionic nerves issuing from the pelvic nerve side of the pelvic plexus depressed the phasic component of biphasic contractions evoked by test stimulation of the nerves issuing from the plexus on the side of the hypogastric nerve, but potentiated the tonic component. 3. In the deganglionated preparation in the presence of alpha,beta‐methylene adenosine 5'‐triphosphate (alpha,beta‐mATP) by which the phasic component was removed, test contractions were markedly reduced by phentolamine and prazosin, and potentiated in a manner dependent on the magnitude of conditioning stimulation. 4. In the deganglionated preparations that were persistently exposed to alpha,beta‐mATP (5‐10 microM), attempts were made to identify the substance(s) mediating the potentiation of the prazosin‐sensitive contractions and their sites of action. 5. Noradrenaline and neuropeptide Y depressed the test contractions. 6. ATP, adenosine 5'‐diphosphate, adenosine 5'‐monophosphate and adenosine potentiated the contractions. Inosine and adenine were without effect. The effect of ATP was antagonized by 8‐phenyltheophylline. N6‐Cyclohexyladenosine (CHA) potentiated the contractions in a dose‐dependent and 8‐phenyltheophylline‐sensitive manner. 7. The conditioning stimulation‐induced potentiation of test contractions was antagonized by 8‐phenyltheophylline, but was further increased by dipyridamole. 8. Methoxamine‐evoked contractions were potentiated by conditioning stimulation of the nerves in a manner antagonized by 8‐phenyltheophylline, and also by CHA. 9. Adenosine and CHA inhibited in an 8‐phenyltheophylline‐sensitive manner field stimulation‐induced release of 3H‐activity from deganglionated vas deferens preloaded with [3H]noradrenaline. 10. In the deganglionated preparation that was not exposed to alpha, beta‐mATP, ATP‐evoked contractions were potentiated by conditioning stimulation and by CHA. 11. It is indicated that conditioning stimulation and adenosine exert opposite actions on phasic and on tonic contractions in spite of the results showing that each of them postjunctionally potentiates the responses to both ATP and noradrenaline. 12. These results suggest that conditioning stimulation releases adenosine and that adenosine potentiates tonic contractions by increasing the responsiveness of the muscle to noradrenaline through the postjunctional adenosine receptors.

The AML1 gene encodes DNA-binding proteins that contain the runt homology domain and is found at the breakpoints of t(8;21), t(3;21), and t(12;21) translocations associated with myelogenous leukemias. AML1 heterodimerizes with PEBP2beta/CBFbeta, resulting in the enhanced affinity with DNA. The runt homology domain is responsible for binding with DNA and heterodimerizing with PEBP2beta/CBFbeta. AML1 is suggested to perform a pivotal role in myeloid cell differentiation, whereas it can cause neoplastic transformation when overexpressed in fibroblasts. In this study, we demonstrated that the reducing reagent, dithiothreitol (DTT), markedly enhances the DNA binding of AML1 expressed in COS7 cells. Oxidation by diamide or modification by N-ethylmaleimide of the free sulfhydryl residues inhibited the interaction of AML1 with DNA. The diamide effect was reversible with excess of DTT, whereas DTT could not restore the DNA binding of AML1 treated with N-ethylmaleimide. Site-directed mutagenesis of the amino acid residue 72, a highly conserved cysteine in the runt homology domain of AML1, to serine almost completely abolished DNA binding without altering the interaction with PEBP2beta/CBFbeta. This substitution also impaired transactivation through the consensus DNA sequence and transformation of fibroblasts induced by AML1b. These data indicate an essential role of the conserved cysteine residue in DNA binding of AML1, and it is possible that the redox state of AML1 could contribute to the regulation of its function.

The t(3;21)(q26;q22) translocation, which is one of the consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia (CML), is thought to play an important role in the leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t(8;21)(q22;q22) translocation found in acute myelocytic leukemia, was also rearranged by the t(3;21)(q26;q22) translocation. Screening of a cDNA library of the t(3;21)‐carrying leukemic cell line cells (SKH1) resulted in the isolation of two potentially complete AML1‐EVI‐1 chimeric cDNAs of 6 kb. Two species of AML1‐EVI‐1 fusion transcripts of 8.2 and 7.0 kb were detected in SKH1 cells. These cells expressed the 180 kDa AML1‐EVI‐1 fusion protein containing an N‐terminal half of AML1 including a runt homology domain which is fused to the entire zinc finger EVI‐1 protein. The AML1‐EVI‐1 fusion transcript was consistent in all three cases of the t(3;21)‐carrying leukemia examined by RNA‐based PCR. These findings strongly suggest that the t(3;21) translocation results in the formation of a new class of chimeric transcription factor which could contribute to the leukemic progression of CML through interference with cell growth and differentiation.

The AML1gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1gene, three proteins, AML1a, AML1b and AML1c, are produced by alternative splicings. We previously showed that AML1 is potentially involved in myeloid cell differentiation. Here we analyzed the expression of AML1in myeloid cell lines. It was revealed that AML1b and AML1c are the major AML1 proteins in these cell lines and that prior to morphological and functional differentiation, their expressions increase in U937 cells when treated with all-transretinoic acid. These data suggest that the expression of AML1is associated with myeloid cell differentiation.