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T Ikuta

Publications and source records attributed to T Ikuta.

At least 37 records · Page 2Linked to original sources
PubMed1999

The nuclear localization signal of the human Ku70 is a variant bipartite type recognized by the two components of nuclear pore-targeting complex.

Ku protein is a complex of two subunits, Ku70 and Ku80. Ku is suspected to participate in both DNA double-strand break repair and transcription. Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins. In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70. Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells. Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells. The NLS of Ku70 was located at the region composed of 18 amino acid residues (positions 539 to 556). This region overlapped with the Ku80-independent DNA-binding domain reported previously. The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region. All the subregions were necessary for complete NLS activity. The amino acids in the nonbasic intervening region of Ku70 might be important for full NLS activity not only to provide sufficient length between the two separated clusters of basic amino acids but also to have an adequate amino acid sequence. All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70. The structure of the Ku70 NLS resembled the consensus of a bipartite-type NLS. The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97.

Amino Acid Sequence↗
PubMed1998

Alterations in protein-DNA interactions in the gamma-globin gene promoter in response to butyrate therapy.

The mechanisms by which pharmacologic agents stimulate gamma-globin gene expression in beta-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with beta-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of gamma-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in gamma-globin protein synthetic levels above baseline gamma globin ratios and a relative decrease in beta-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the gamma-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, alphaCP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which gamma-globin gene expression is being altered strongly suggest that nuclear protein binding, including alphaCP2, to the BRE-G1 region of the gamma-globin gene promoter mediates butyrate activity on gamma-globin gene expression.

Adolescent↗
PubMed1998

Structural analysis of mouse tenascin-X: evolutionary aspects of reduplication of FNIII repeats in the tenascin gene family.

Tenascin-X (TNX) is an extracellular matrix glycoprotein involved in both primary structural functions and modulating cellular activities in multicellular organisms. We determined the 67977bp nucleotide sequence of the entire mouse tenascin-X (Tnx) gene, which also includes the last exon of Creb-rp and Cyp21. We compared it with the orthologous human locus. Conservation of both position and orientation of the three functionally unrelated genes at this position was found. Comparison also revealed that introns 1, 4 and 6 of Tnx are highly conserved between species. The sequence showed that mouse Tnx contains 43 exons separated by 42 introns. The deduced amino-acid sequence (4114 residues) revealed that mouse Tnx has a primary structure characteristic of tenascins, which consists of a signal peptide and four heptad repeats followed by 18.5 epidermal growth factor-like (EGF) repeats, 31 fibronectin type III-like (FNIII) repeats, and a region homologous to fibrinogen. cDNA clones generated by alternative splicing of eight consecutive FNIII repeats (M15-M22) as well as a proximal FNIII repeat (M3) were also identified. The FNIII motifs that were subject to alternative splicing were assigned to the group of recently reduplicated FNIII repeats because they have a high level of amino-acid sequence similarity. We also analyzed the evolution of FNIII repeats in TNX.

Amino Acid Sequence↗
PubMed1998

Cellular and molecular effects of a pulse butyrate regimen and new inducers of globin gene expression and hematopoiesis.

Cooley's anemia is characterized by a deficiency of beta-globin chains, a relative excess of alpha-globin chains, and consequent accelerated programmed death of developing erythroid cells in the bone marrow. Increasing expression of the gamma-globin genes to adequately balance excess alpha-globin chains can ameliorate this disorder. Butyrates induce gamma-globin experimentally, but can also cause cell growth arrest with prolonged exposure or high concentrations, which in turn can accelerate apoptosis. To determine if these potentially opposing effects can be balanced to enhance therapeutic efficacy, an intermittent "pulsed" regimen of butyrate was evaluated. Following induction of gamma-globin mRNA and protein synthesis, total hemoglobin increased in beta-thalassemia patients by more than 2 g/dl above baseline, and Hb F increased above 20% in 5/8 sickle cell patients from baseline levels of 2% Hb F. Specific regulatory regions were identified in the gamma- and beta-globin gene promoters to which new binding of transcription factors, including alpha CP2 (an activator of gamma globin) occur during therapy solely in the butyrate-responsive patients. Other compounds which induce gamma globin, derivatives of acetic, phenoxyacetic, propionic, and cinnamic acids, and dimethylbutyrate, are under investigation. Some of these newer gamma-globin inducers (designed hemokines) provide better potential as therapeutics by also acting to increase hematopoietic cell viability and proliferation. Pharmacologic induction of expression of the endogenous gamma-globin genes is a realistic approach to therapy of the beta-globin disorders for many patients, with some effective agents available now and new therapeutics, with enhanced activities, under development.

Adult↗
PubMed1998

Nuclear localization and export signals of the human aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1beta). We found in this study that Ahr contains both nuclear localization and export signals in the NH2-terminal region. A fusion protein composed of beta-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a ligand-dependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligand- and signal-dependent nucleocytoplasmic shuttling protein.

Biological Transport↗
PubMed1998

Cholesterol sulfate activates transcription of transglutaminase 1 gene in normal human keratinocytes.

Cholesterol sulfate and transglutaminase 1 are essential for the process of keratinization. Cholesterol sulfate is formed during keratinization and activates the eta isoform of protein kinase C. Transglutaminase 1 is a key enzyme for formation of the cornified envelope in terminally differentiated keratinocytes. In this study, we demonstrated that cholesterol sulfate acts as a transcriptional activator of the transglutaminase 1 gene in normal human keratinocytes. Growth of normal human keratinocytes was inhibited by cholesterol sulfate, but not by its parental cholesterol. Treatment of normal human keratinocytes with cholesterol sulfate induced activity of transglutaminase 1 in a dose- and time-dependent manner. Activation of transcription of transglutaminase 1 by cholesterol sulfate was demonstrated by northern blotting analysis, whereas that by cholesterol was not. In order to identify a cholesterol sulfate responsive region in the transglutaminase 1 gene, plasmids were constructed containing a luciferase reporter gene ligated to deletion fragments of the 5' upstream region of the tranglutaminase 1 gene and were transfected into normal human keratinocytes. Transfected cells were treated with cholesterol sulfate, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and a high concentration of Ca2+. Our results indicate that the responsive element(s) for cholesterol sulfate and phorbol ester is located upstream of the human transglutaminase 1 gene at a position(s) between -819 and -549, whereas the responsive element for Ca2+ is located at a position between -79 and -49.

5' Untranslated Regions↗
PubMed1998

The effects of qi-gong and acupuncture on human cerebral evoked potentials and electroencephalogram.

Although a number of studies on traditional Chinese medicine, such as qi-gong (QG), acupuncture (AC), moxibustion and Chinese herbal drugs, have been reported in recent years, there are few reports on human cerebral evoked potentials (EPs), especially relating only to QG and AC. In the present study, we examined the changes in EPs and electroencephalogram (EEG) by QG, and by AC stimulation to the point called "Zusanli" on the left lower leg, with one healthy male adult. 1. With regard to the effect of QG, significant changes in EP-components originated from the cortex suggest both facilitating and inhibitory effects of QG on the cortex. However, no significant changes in EP-components originated from the subcortex and no significant changes in EEG power% suggest that QG does not affect the subcortex. 2. With regard to the effects of AC, significant changes in EP-components originated from the cortex suggest facilitating and inhibitory effects of AC stimulation on the cortex. Furthermore, it is suggested that AC stimulation has few effects on the somatosensory and the visual pathways up to the cortex, while it has complicated effects on the auditory pathway up to the cortex.

Acupuncture Therapy↗
PubMed1997

A nuclear localization signal of human aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor 1beta is a novel bipartite type recognized by the two components of nuclear pore-targeting complex.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a component of the transcription factors, aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1, which transactivate their target genes, such as CYP1A1 and erythropoietin, in response to xenobiotic aromatic hydrocarbons and to low O2 concentration, respectively. Since ARNT was isolated as a factor required for the nuclear translocation of AhR from the cytoplasm in response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with beta-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathione S-transferase/green fluorescent protein. We found a clear nuclear localization of ARNT in the absence of exogenous ligands to AhR, and identified the nuclear localization signal (NLS) of amino acid residues 39-61. The characterized NLS consists of 23 amino acids, and can be classified as a novel variant of the bipartite type on the basis of having two separate regions responsible for efficient nuclear translocation activity, but considerable deviation of the sequence from the consensus of the classical bipartite type NLSs. Like the well characterized NLS of the SV40 T-antigen, this variant bipartite type of ARNT NLS was also mediated by the two components of nuclear pore targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in an in vitro nuclear transport assay.

Amino Acid Sequence↗
PubMed1997

Abrogation of IL-3 requirements and stimulation of hematopoietic cell proliferation in vitro and in vivo by carboxylic acids.

Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the beta-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced gamma globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observed in vivo in mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce gamma globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.

Animals↗
PubMed1997

Visual evoked potential and electroencephalogram of healthy females during the menstrual cycle.

Flash visual evoked potential (VEP) and electroencephalogram (EEG) changes during the menstrual cycle were studied using healthy females having regular menstruation, with 21 at the follicular phase (FP) and 23 at the luteal phase (LP). The following results were obtained. (1) The waveforms of Group Mean VEPs of both groups had approximately similar triphasic contours, consisting of 16 components of P 1-N 8 up to 500 msec of latency. (2) Latencies tended to be longer in LP. (3) Interpeak amplitudes tended to be larger in LP, and one VEP interpeak amplitude (P 5-N 7) of long latency component was significantly larger at LP after eliminating the effect of body height by ANCOVA for 2 CH. (4) Quantitative analysis of EEGs between FP and LP resulted in a tendency for increased alpha, and decreased beta power % at LP. Since estrogen increases the VEP amplitude, and decreases the VEP latency and the alpha activity of EEGs, the large VEP amplitude, the tendency for prolonged VEP latency, and the tendency for increased alpha power % at LP observed in this study indicate that the VEP amplitude at LP reflects the effect of estrogen, and that the VEP latency and EEGs at LP reflect the effect of progesterone.

Adult↗
PubMed1997

[Regional difference in the etiology of liver cirrhosis in Iwate].

To assess regional differences in the etiology of liver cirrhosis in Iwate, we analyzed 324 patients with liver cirrhosis treated at various hospitals. The etiology was HCV 44.8%, HBV 11.1%, HBV + HCV 4.6%, alcohol 27.5% (including heavy drinkers 17.9%), PBC 1.5% and non-B non-C 10.5% in Iwate. The incidence of alcoholic cirrhosis was higher than that in other prefectures, while that of HCV was lower. Especially in the northern area of Iwate, the rate of alcoholic cirrhosis was very high (39.1%--including heavy drinkers 21.8%) while viral cirrhosis was relatively low. Although the alcohol consumption volume in Iwate was not very high, marked alcohol consumption, especially shochu, was observed in the northern area of Iwate. The volume and kind of alcohol consumed in each area differed, and the etiology of liver cirrhosis differed regionally in Iwate. Thus, we should consider these districts and levels of alcohol consumption when treating patients with liver cirrhosis.

Female↗
PubMed1996

Globin gene switching. In vivo protein-DNA interactions of the human beta-globin locus in erythroid cells expressing the fetal or the adult globin gene program.

To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene.

Adult↗
PubMed1996

Recovery of high speed memory scanning after transient global amnesia: a case report.

We described the case of a 59 year old female with transient global amnesia (TGA) who had been examined neuropsychologically using Sternberg's paradigm and a random number generation (RNG) task on the following day, 1 week and 4 weeks after a TGA episode. The slope value of the linear function, a measure of cognitive memory scanning speed, decreased with time and showed a marked decrease 1 week after TGA, suggesting that the stage of serial and exhaustive scanning recovered within 1 week. The zero-intercept value, on the other hand, increased during 1 week but decreased 4 weeks later and was not related directly to recovery from TGA. The performance in RNG task improved 1 week later, but deteriorated 4 weeks after the episode. Such a change in RNG was in accordance with that of the zero-intercept value, predicting a relationship. It is concluded that the subclinical memory deficit, detected with these tasks, persisted longer than clinical recovery from TGA.

Amnesia↗
PubMed1996

Reproducibility and performance in random number generation.

We investigated the reproducibility of random number generation task (RNG) in 18 healthy subjects. They were tested using RNG on separate days, and the degree of randomness were measured according to RNG Index, Null score and Phase. The correlation coefficient between two repeated scores (test and retest) was significantly high for all randomness measures, and RNG Index and Null score remained unchanged. Next, 10 of 18 subjects were tested again after they were provided feedback on one statistical analysis of randomness (RNG Index). The correlations between the three measures became insignificant, and both RNG Index and Null score decreased significantly after the feedback. In conclusion, RNG is a useful neuropsychological test to evaluate cognitive dysfunction which is prone to change, since we can employ it repeatedly at short intervals.

Feedback↗
PubMed1996

Sex differences in visual evoked potential and electroencephalogram of healthy adults.

Sex differences in flash visual evoked potentials (VEPs) and electroencephalograms (EEGs) were studied in 200 healthy adults (100 females and 100 males). Following results were obtained. (1) Waveforms of Group Mean VEPs of each sex had roughly triphasic contour, consisted of components P1 approximately N8, within 500 msec of latency. (2) Latencies of VEP components by t-test, (3) By analysis of covariance, sex differences in peak-to-peak amplitudes were still significant after eliminating effects of body height, body weight or age, but not those in latencies after eliminating the effect of body height. (4) Quantitative analysis of EEGs resulted in significantly smaller absolute amplitudes, and significantly larger theta, larger beta 1 and smaller alpha 2 power % in females. (5) Peak-to-peak amplitudes of later components in VEPs for 5CH (O1-->Cz) were significantly and positively correlated with EEG absolute amplitudes within each sex group. (6) There were few significant differences in VEP components between follicular phase and luteal phase in females. The sex differences in VEPs verified in this study may be attributed to genetically determined sex differences in neuroendocrinological systems.

Adult↗