HTLV-1 bZIP factor dysregulates the Wnt pathways to support proliferation and migration of adult T-cell leukemia cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ), the viral gene transcribed from the antisense strand, is consistently expressed in ATL cells and promotes their proliferation. In this study, we found that a Wnt pathway-related protein, disheveled-associating protein with a high frequency of leucine residues (DAPLE), interacts with both HTLV-1 Tax and HBZ. In the presence of DAPLE, Tax activated canonical Wnt signaling. Conversely, HBZ markedly suppressed canonical Wnt activation induced by either Tax/DAPLE or ß-catenin. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets lymphoid enhancer-binding factor 1, one of the key transcription factors of the pathway, and impairs its DNA-binding ability. We also observed that the canonical Wnt pathway was not activated in HTLV-1-infected cells, whereas the representative of noncanonical Wnt ligand, Wnt5a, which antagonizes canonical Wnt signaling, was overexpressed. HBZ was able to induce Wnt5a transcription by enhancing its promoter activity through the TGF-ß pathway. Importantly, knocking down of Wnt5a in ATL cells repressed cellular proliferation and migration. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways.

Lack of modulation of Ib inhibition during antagonist contraction in spasticity.

OBJECTIVE: To examine the modulation of non-reciprocal group I (Ib) inhibition during tonic contraction of antagonist muscles in patients with spasticity vs normal subjects. METHODS: The authors studied 10 patients with spastic paraplegia due to cervical compression myelopathy and 16 age-matched normal subjects. Ib inhibition to soleus motoneurons was recorded as the change in size of the H-reflex of the soleus, evoked by conditioning stimulus to the nerve innervating the medial gastrocnemius muscle. The extent of inhibition was studied at rest and during tonic contraction of the pretibial muscles of variable strength. RESULTS: In the resting state, the extent of inhibition in the patients did not differ from normal controls. During antagonist contraction, the extent of inhibition increased both in the normal subjects and patients. The increment was smaller in the patients, especially in those with severe spastic gait. The smaller increment in the inhibition was correlated with the time required to walk 10 m in the patients. CONCLUSION: The authors observed a lack of modulation of Ib inhibition during tonic antagonist contraction in patients with spasticity, especially those with gait disturbance. Disturbed central modulation of non-reciprocal (Ib) interneurons may be responsible for spasticity.

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Age and sex differences in the P-wave signal-averaged electrocardiogram in a Japanese study population.

Normal P wave signal-averaged electrocardiogram (SAE) values were determined in 120 healthy Japanese adults (56 men, 64 women), aged 44.5+/-10.2 years (mean+/-SD). The P wave trigger method was used with a Fukuda FDX6500 recorder. We used bipolar Frank leads (X,Y,Z), and recordings were made with forward and backward digital Butterworth filters [40 Hz (18 dB / oct) - 300 Hz (12 dB / oct)]. The recordings were taken for the following five parameters: forward and backward filtered P wave duration [fPd (F); tPd (B)]; bidirectionally corrected fPd [tPd (C)]; and 20 ms of the terminal portions of voltage at forward and backward filtering (RMS20). Overall, fPd (F) was 117.8-136.4 ms, fPd (B) 116.4-134.4 ms, fPd (C) 97.4-115.2 ms, RMS20 (F) 1.6-3.6 microV, and RMS20 (B) was 2.2-5.4 microV. Between the sexes, there were significant differences in fPd (F) (p<0.001) and fPd (B) (p<0.01) and in RMS20 (F) (p<0.05) and RMS20 (B) (p<0.05). Weak positive correlations were observed between fPd (F) and body surface area, fPd (F) and age, fPd (B) and body surface area, fPd (B) and age, fPd (C) and body surface area, and fPd (C) and age. There was no evident correlation, however, between either forward or backward RMS20 and body surface area or between forward or backward RMS20 and age. Differences in the normal P wave values between the sexes and age groups were evaluated in this study.

Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarization.

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP) [1-4]. Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair [5]. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh) [6-8]. Here, we visualized endogenous Dsh in the developing wing. A portion of Dsh colocalized with Fmi, and the distributions of both proteins were interdependent. Furthermore, Fz controlled the association of Dsh with cell boundaries, which association was correlated with the presence of hyperphosphorylated forms of Dsh. Our results, together with a recent study on Fz distribution [9], support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that its restricted localization directs cytoskeletal reorganization only at the distal cell edge.

Identification of Notch1 as a frequent target for provirus insertional mutagenesis in T-cell lymphomas induced by leukemogenic mutants of mouse mammary tumor virus.

In contrast to wild-type mouse mammary tumor virus (MMTV), the MMTV mutants with specific deletions in the U3 region of their long terminal repeats cause T-cell lymphomas. In 30% of T-cell lymphomas arising in BALB/c mice infected with MLA-MMTV, a leukemogenic MMTV mutant, we have found that MMTV proviruses were integrated into a short region of the Notch1 genome, so that truncated Notch1 transcripts encoding the transmembrane and the cytoplasmic domains of Notch1 protein could be expressed. Thus, Notch1 is a major target of provirus insertional mutagenesis in these T-cell lymphomas.

Biochemical characterization of the Drosophila axin protein.

In the Wnt/Wingless pathway, accumulation of beta-catenin/Armadillo protein is a key regulatory step. Vertebrate Axin is a negative regulator of Wnt signaling, promoting glycogen synthase kinase-3beta-mediated phosphorylation of beta-catenin and thereby destabilizing beta-catenin. Using Drosophila cell culture systems, we demonstrated that a Drosophila homolog of Axin (Daxin) inhibits Wingless-induced Armadillo accumulation and Drosophila T-cell factor-dependent transcription induced by Wingless, Dishevelled, and Armadillo. The carboxy-terminal portion of Daxin is not essential for the inhibitory activity, but a mutant only consisting of this portion behaves as a dominant-negative protein. Moreover, interactions between Daxin and Zeste-white 3, Armadillo, Dishevelled, protein phosphatase 2A and Daxin itself were shown, providing direct evidence that Daxin is a scaffold protein in the Wingless pathway.

Radiofrequency catheter ablation of ventricular tachycardia from the anterobasal left ventricle.

Ventricular tachycardia (VT) in coronary artery disease arises mostly from endocardial sites. However, little is known about the site of origin in other diseases. We report two patients who had VT originating from an anterior aspect of the left ventricle just below the mitral annulus, adjacent to the left ventricular outflow tract. The QRS configuration of VT showed an inferior axis and monophasic R waves in all the precordial leads. Radiofrequency current delivered to this site from the endocardial site successfully ablated the tachycardia in both.

High-dose-rate interstitial brachytherapy of Bartholin's gland: A case report.

OBJECTIVE: To preserve the organs and function of the anus and ureter, radiotherapy was chosen for an advanced vulvar tumor originating from Bartholin's gland. METHODS: The patient was a 74-year-old female with stage III (FIGO) vulvar cancer derived from the left Bartholin's gland. She received 63 Gy to the vulvar lesion with external beam irradiation followed by 30 Gy of high-dose-rate interstitial brachytherapy. RESULTS: One year after radiotherapy, simple vulvectomy and reconstructive surgery were performed. The anal and ureteral functions were preserved. Histological examination revealed no evident malignant cells. CONCLUSION: Owing to the technological development of radiotherapy and improved reconstructive surgery, radiotherapy in treatment of the vulvar area has become effective and safe and could be indicated more as a treatment option for the sake of organ preservation.

Murine leukemia provirus-mediated activation of the Notch1 gene leads to induction of HES-1 in a mouse T lymphoma cell line, DL-3.

Constitutive activation of Notch signaling is known to be associated with tumorigenesis. In a mouse T lymphoma cell line, DL-3, we found that a murine leukemia provirus was inserted in the Notch1 locus, which led to marked expression of a virus-Notch1 fusion mRNA encoding an intracellular portion of the Notch1 protein. Furthermore, expression and nuclear localization of this constitutively active form of Notch1 protein were confirmed. Corresponding to this finding, the transcription of the hairy/enhancer of split (HES-1) gene, a known target of Notch1 signaling, was elevated in this cell line. A potential role for overexpressed HES-1 in the development of the lymphoma was discussed.

Characterization of mouse dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway.

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse and Drosophila cell culture systems. We found that treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with our finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induced hyperphosphorylation of Dsh (Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995) Genes Dev. 9, 1087-1097). Furthermore, we showed that mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II was shown to coimmunoprecipitate with Dvl proteins, and Dvl proteins were phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway.

[Prognostic factors for oral tongue carcinoma treated with intra-oral cone electron beam irradiation].

Twenty-five patients with squamous cell carcinoma of the oral tongue were treated with intra-oral cone electron beam irradiation (IOC) during the period from 1985 to 1995. We analyzed the prognostic factors for local control and complications. IOC was applied for T1 (16 cases) and T2 (9 cases) tumors. Hypofractionation was used for IOC (20, 10, or 8 Gy/fr, 1f/wk). The total dose delivered ranged from 40 Gy to 78 Gy. Radiation dose homogenization was done through calculation of the normalized total dose (NTD) for alpha/beta = 10 (tumor) and alpha/beta = 3 (late normal tissue). The two-year local control rates for T1 and T2 were 80.4% and 77.8%, respectively. The two-year local control rates for patients whose overall treatment time (OTT) was < or = 28 days (n = 16) was 100% vs. 41.7% for patients whose OTT was > 28 days (n = 9) (p = 0.002). Multivariate analysis was applied to identify possible prognostic factors for local control. OTT (p = 0.02) was the only variable that significantly influenced local control. The incidence of radiation ulcer was 33.3% (7/21). Significant indicators of ulceration were fraction size (>> 10 Gy) and NTD (alpha/beta = 3) (>> 130 Gy) (p < 0.05). These results indicate that prolonged OTT was the major reason for the failure of IOC radiotherapy to control local disease and that the relatively high rate of ulceration was due to large fraction size and high NTD (alpha/beta = 3).

Intracisternal type A particle-mediated activation of the Notch4/int3 gene in a mouse mammary tumor: generation of truncated Notch4/int3 mRNAs by retroviral splicing events.

The int3 oncogene was discovered as a frequent target in mouse mammary tumor virus-induced mammary tumors and encodes the intracellular domain of a Notch4/int3 protein. In one spontaneous mammary tumor, no. 9, that developed in a BALB/c mouse, we have found an insertion of a 1.2-kb sequence, consisting of a 5' long terminal repeat and gag sequences of an intracisternal type A particle (IAP) as well as an extra copy of the Notch4/int3 genomic sequences containing exons 23 and 24, into the intron between exons 24 and 25 of the Notch4/int3 gene. In this tumor, unique splicing events between the IAP and the Notch4/int3 sequences generated two types of IAP-Notch4/int3 fusion transcripts encoding two different portions of the intracellular domain of Notch4/int3 proteins: one with a RAM domain and the other without. Interestingly, these two proteins showed different subcellular localizations in a mouse mammary epithelial cell line, HC-11.

Image storing system for radiation therapy (radiation oncology information system: ROIS) as a branch of diagnostic PACS; implementation and evaluation.

We have implemented the new system that is called as ROIS for storing medical images of the patients underwent radiotherapy as a branch of medium-scale diagnostic PACS. It was estimated that, for about nine years, we can store all medical images of the patients who received radiation therapy under the on-line storage condition. Further, we can retrieve all medical images of the radiotherapy patients on ROIS faster than conventional film library or on the diagnostic PACS, even if the patient had been treated many years ago. Implementation of ROIS provided us a great help in our clinical work.

Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling.

Wingless (Wg) treatment of Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling.

[Isoproterenol continuous nebulization for childhood status asthmaticus. I. Efficacy and side effects of high-dose method].

We investigated the efficacy and the side effects of "high-dose isoproterenol continuous nebulization" for childhood status asthmaticus. Subjects were 34 children who were hospitalized and underwent the nebulization therapy. The 50 ml solution of 0.5% dl-isoproterenol was diluted in 500 ml of normal saline and nebulized through an ultrasound nebulizer. The period of continuous nebulization was 25.5 +/- 16.0 hours. The Wood's clinical score clearly decreased in 32 cases, the average score changing from 7.7 +/- 0.8 to 2.9 +/- 1.3. Heart rate was elevated significantly during the first 3 hours (156 +/- 25/min at the start of the nebulization, 180 +/- 20/min at 1 hour, 171 +/- 23 at 3 hours), and then it decreased gradually to 122 +/- 25/min at the cessation of the nebulization. Serum GOT, LDH, CPK, and potassium were elevated after the nebulization compared with the values before the treatment, though the changes were not statistically significant. CPK-MB fraction after the nebulization was higher than normal range in 12 of 13 subjects. Of 34 subjects, 11 (32%) complained nausea or vomited, 2 showed arrhythmia on ECG (ventricular premature conduction), 1 developed myocardiac infarction, and 1 developed possible heart failure, some of which might be attributable to the pharmacological side effects of isoproterenol nebulization. We conclude that "high-dose isoproterenol continuous nebulization" is an effective method for childhood status asthmaticus, but there is some risk of serious side effects. This method was originally developed as a method indicated for the case of respiratory failure or threatened respiratory failure following status asthmaticus, and we should not extend the indication of this method thoughness.

Accumulation of Armadillo induced by Wingless, Dishevelled, and dominant-negative Zeste-White 3 leads to elevated DE-cadherin in Drosophila clone 8 wing disc cells.

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (ZW-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate zw-3, which in turn down-regulates arm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which bind to Arm, we overexpressed Dsh, ZW-3, and Arm, in the Drosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression led to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wild-type and dominant-negative forms of ZW-3 decreased and increased Arm levels, respectively, indicating that modulation in zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevated DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative ZW-3-, and truncated Arm-induced accumulation of DE-cadherin protein was accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevated Arm levels by stabilizing Arm at cell-cell junctions.

Possible origin of murine AIDS-inducing sequence.

The murine AIDS (MAIDS) virus has a unique sequence in its gag p12 region. A transcript which hybridizes with this sequence is expressed in normal C57BL/6 mice. This transcript has been proposed to be the origin of the MAIDS virus, since the virus was originally isolated from radiation-induced leukemic C57BL/6 mice. The transcript, designated Edv, was molecularly cloned and sequenced. Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic defective MAIDS virus has 16-bp deletions and a 1-bp insertion in the 5' and 3' regions of the gag p12 sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the gag p12 region of the MAIDS virus is less homologous to that of the helper virus and Edv transcript due to the frameshift mutations. This suggested that the MAIDS virus was generated by such frameshift mutations in the gag p12 region during recombination between the helper virus and the Edv or a related sequence.

Catheter ablation of canine ventricular myocardium. The use of repetitive short time constant capacitive shocks to increase lesion volume.

Arching and barotrauma, seen with high energy DC catheter ablation, are responsible for diffuse cardiac damage and coronary sinus rupture. In six anesthetized dogs, we investigated the effects of an increasing number of short time-constant capacitive shocks on the volume of myocardial damage. Each dog received capacitive shocks of 2 J/kg at 3 sites in the left ventricle. One shock was delivered in 2 dogs, 2 shocks were delivered in 2 dogs and 3 shocks were delivered in 2 dogs. Shock delivery was not accompanied by hemodynamic collapse, sustained ventricular tachycardia or ventricular fibrillation. The dogs were sacrificed at 60 minutes. Mean (SEM) lesion volumes were 195 (39) mm3, 480 (41) mm3, and 595 (110) mm3, respectively. Despite variability in individual volume of damage, there was a significant increase in lesion volume with an increasing number of shocks. There was no evidence of perforation or tamponade. Increasing myocardial damage can be produced using repetitive capacitive shocks. Delivery of 2 shocks produces clinically useful lesions without the adverse effects associated with single high energy shocks. Repetitive capacitive shocks offer a method of increasing lesion volume without increasing energy and thereby without compromising safety.