Biomedical subjects
Publications and source records attributed to T Imamura.
We report on a patient with pure transient global amnesia (TGA) whose magnetic resonance imaging (MRI) demonstrated a small region of increased signal intensity in the right hippocampus on diffusion-weighted imaging (DWI). DWI was sensitive and useful for evaluating the early stage of TGA and might help to explain the pathophysiology of TGA.
We report a 51-year-old male with adult T cell leukemia (ATL) who received a BMT from an HLA-identical unrelated donor. The ATL proved refractory to chemotherapy, and he underwent BMT conditioned with CY/TBI. Complications of encephalitis of unknown origin were successfully treated with steroid therapy and the patient has been in CR for 16 months after BMT. Human T cell leukemia virus type 1 proviral DNA loads were reduced to undetectable levels in PBMC sampled 12 months after BMT. This encouraging result suggests that BMT from an unrelated donor should be considered for ATL even if the disease is refractory to chemotherapy.
Infant acute lymphoblastic leukemia (ALL) is characterized by the presence of the proB phenotype (CD10(-)/CD19(+)), poor prognosis and frequent rearrangement of the mixed lineage leukemia (MLL) gene. The most frequent rearrangement is t(4;11)(q21;q23), the role of whose product, the MLL-AF4 fusion transcript, has been extensively studied in leukemogenesis. In a cell line of infant leukemia with MLL rearrangement denoted KP-L-RY, panhandle PCR amplification of cDNA revealed the presence of a fusion transcript, MLL-AF5q31, indicating that AF5q31 is also a partner gene of MLL. In this fusion transcript the MLL exon 6 is fused in frame to the 5' side of the putative transactivation domain of AF5q31. The AF5q31 protein is a member of the AF4/LAF4/FMR2-related family of proteins, which have been suggested to play a role in hematopoietic cell growth and differentiation. The MLL-AF5q31 fusion transcript, although probably rare, appears to be associated with the pathogenesis of infant ALL like MLL-AF4. Co-expression of HoxA9 and Meis1 genes in the KP-L-RY cell line indicated possible functional similarity between MLL-AF4 and MLL-AF5q31. Further understanding of the function of AF5q31 as well as the specific leukemogenic mechanism of MLL-AF5q31 awaits future studies.
The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas cichorii YN2 cultured with C6-C12 1-alkenes was studied. PHAs containing repeating units with terminal epoxide groups were obtained when C7-C12 1-alkenes were fed separately as the only carbon source, but no epoxidized unit was detected when 1-hexene was fed. The content of epoxidized units in the polymers was in the range of 4-20 mol%, which was not dependent on the C atom length of the 1-alkene used as a substrate. The polymers produced undergo a glass transition at around -40 degrees C, and number average molecular weights were in the range of 150000-200000 as determined by GPC relative to polystyrene, with M(w)/M(n) ratios of 1.9-2.5. As an intermediate, the corresponding 1,2-epoxyalkane was found in the culture medium. According to this result, the epoxidation of the 1-alkene is the initial step in the synthetic pathway of the epoxy unit in the polymer.
Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.
Insulin stimulates glucose transport by promoting translocation of the insulin-sensitive glucose transporter isoform 4 (GLUT4) from an intracellular compartment to the cell surface. This movement is accomplished by stimulation of GLUT4 exocytosis as well as inhibition of endocytosis. However, the molecular mechanisms for these effects remain unclear. In this study, we found that the GTP-binding protein Rab5 physically associated with the motor protein dynein in immunoprecipitants from both untransfected cells and cells transfected with GFP-Rab5 constructs. Microinjection of anti-Rab5 or anti-dynein antibody into 3T3-L1 adipocytes increased the basal level of surface GLUT4, did not change the insulin-stimulated surface GLUT4 level, and inhibited GLUT4 internalization after the removal of insulin. Photoaffinity labeling of Rab5 with [gamma-(32)P]GTP-azidoanilide showed that insulin inhibited Rab5-GTP loading. By using microtubule-capture assays, we found that insulin also caused a significant decrease in the binding of dynein to microtubules. Furthermore, pretreatment of cells with the PI3-kinase inhibitor LY294002 inhibited the effects of insulin on both Rab5-GTP loading and dynein binding to microtubules. In conclusion, these data indicate that insulin signaling inhibits Rab5 activity and the interaction of dynein with microtubules in a PI3-kinase-dependent manner, and that these effects may inhibit the rate of GLUT4 internalization. As such, our results present a previously uncharacterized insulin-signaling pathway involving Rab5, the motor protein dynein, and the cytoskeleton to regulate directional GLUT4 movement, facilitating GLUT4 distribution to the cell surface.
Both endothelin (ET) and adrenomedullin (AM), produced by cardiac myocytes, are thought to be locally-acting hormones in the heart. Recently, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have been shown to function together to serve as AM receptors stimulating cAMP production. In the present study, we examined the effects of ET on AM secretion, intracellular cAMP response to AM, and gene expressions of CRLR and RAMPs in cultured cardiac myocytes. Synthetic ET-1 dose-dependently increased AM secretion from the cardiomyocytes. AM increased the intracellular cAMP level in a dose-dependent manner and the cAMP accumulation by AM was significantly amplified by 24 h preincubation with ET-1. 10 nmol/L ET-1 significantly increased the CRLR mRNA level without any effect on RAMP1 mRNA. 1 micromol/L ET-1 significantly reduced the RAMP2 mRNA level, but ET-1 dose-dependently increased the RAMP3 mRNA level in the cardiac myocytes. These findings suggest that ET-1 not only stimulates AM secretion, but also modulates intracellular cAMP responses to AM probably by altering the expressions of CRLR and RAMPs in rat cardiomyocytes.
The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.
Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA.
BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.
Proadrenomedullin N-terminal 20 peptide (PAMP) is generated from post-transcriptional enzymatic processing of a 185-amino acid precursor for adrenomedullin (AM), a potent vasodilator peptide. We have reported that AM is secreted from cultured neonatal rat cardiac myocytes and fibroblasts, and that secreted AM modulates the growth of these cells; however, it is unknown whether or not the cardiac cells produce PAMP. In this study, we examined the production of PAMP in cultured neonatal rat cardiac myocytes and fibroblasts. Both the cardiac myocytes and fibroblasts cultured with serum-free media secreted PAMP time-dependently at rates of 5.7+/-0.9 fmol/10(5) cells/40 h and 8.4+/-0.7 fmol/5x10(4) cells/48 h (mean+/-SD), respectively. Reverse-phase high performance liquid chromatography showed that immunoreactive PAMP secreted from these cells was identical to PAMP[1-20], a whole active molecule. PAMP and AM secretions were significantly (P<0.01) stimulated by 10(-6) mol/L angiotensin II (Ang II) and 10% fetal bovine serum (FBS) in myocytes and fibroblasts, whereas the ratio of PAMP to AM secretion in the myocytes was smaller than that of the fibroblasts. These results suggest that PAMP is secreted along with AM from rat cardiac myocytes and fibroblasts, and the secretion is augmented by the growth-promoting stimuli of Ang II and FBS for these cells.
The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that alpha-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k(cat)/K(m) = 1.2 x 10(6) m(-)1 s(-)1) was 100-fold higher than that of RgpB (k(cat)/K(m) = 1.2 x 10(4) m(-)1 s(-)1). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534-S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis.
When released into the third ventricle of the brain, fibroblast growth factor (FGF)-1 acts as a feeding suppressor. To identify the cells that respond to FGF-1 in this process, we investigated the effects of intracerebroventricular administration of FGF-1 on Fos protein expression in the rat hypothalamus. Two hours after infusion of FGF-1, significantly more Fos signals were observed in the periventricular zone of the third ventricle and its surroundings. Double immunohistochemical studies using antibodies against Fos, glial fibrillary acidic protein, and neuronal nuclei revealed that the FGF-1-induced Fos signals in the periventricular zone were in astrocytes, not in neurons. FGF-1-induced Fos expression was not found in neurons in such hypothalamic nuclei as the lateral hypothalamic area, paraventricular nucleus, ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, or arcuate nucleus. These results suggest a possible involvement of periventricular astrocytes in the early stages of FGF-1-induced feeding suppression.
We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.
The nitrogen K-shell X-ray absorption spectra of the purine bases present in nucleic acids, adenine and guanine, were analyzed by using ab initio Hartree-Fock static exchange and restricted-active-space self-consistent-field calculations. A variety of derivative molecules were calculated to investigate the energetic shifts due to environmental effects on the nitrogen atoms. Shake-up excitations were also addressed.
Galactokinase (GALK) deficiency is an autosomal recessive disorder characterized by hypergalactosemia and cataract formation. Through mass screening of newborn infants, we identified a novel and prevalent GALK variant (designated here as the "Osaka" variant) associated with an A198V mutation in three infants with mild GALK deficiency. GALK activity and the amount of immunoreactive protein in the mutant were both 20% of normal construct in expression analysis. The K(m) values for galactose and ATP-Mg(2+) in erythrocytes with homozygous A198V were similar to those of the healthy adult control subjects. A population study for A198V revealed prevalences of 4.1% in Japanese and 2.8% in Koreans, lower incidence in Taiwanese and Chinese, no incidence in blacks and whites from the United States, and a significantly high frequency (7.8%; P < .023) in Japanese individuals with bilateral cataract. This variant probably originated in Japanese and Korean ancestors and is one of the genetic factors that causes cataract in elderly individuals.
The nonhistone chromosomal protein, high mobility group 1 (HMG1), which is ubiquitously expressed in higher eukaryotic cells, preferentially binds to cisplatin-modified DNA. The observation that HMG1 is overexpressed in cisplatin-resistant human cancer cells suggests that cisplatin resistance may be closely associated with HMG1. To decipher the mechanism of HMG1 overexpression in cisplatin-resistant cells, we isolated two overlapping genomic DNA clones containing the entire human HMG1 gene. These clones, which span approximately 15 kb of contiguous DNA, include 5 kb of the 5' flanking region as well as the entire coding sequence. We sequenced 1500 bp upstream of the first exon. The segment proximal to the transcription initiation site did not contain a TATA box but did possess an activating transcription factor site, an activator protein-2 site, one CCAAT box, and two CCAAT-binding transcription factor/nuclear factor-1 (CTF/NF-1) sites. HMG1 promoter activity was 3-10-fold higher in cisplatin-resistant KB-CP20 cells than in parental KB cells. An in vivo footprint experiment showed several differences of dimethyl sulfate modifications between KB and KB-CP20 cells in the area around the CTF/NF-1 sites. In addition, electrophoretic gel mobility shift assays showed that binding of a nuclear factor from cisplatin-resistant cells to the CTF/NF-1 site was significantly higher than the binding of the same factor from parental cells. Semiquantitative reverse transcription-PCR and Western blot analysis also showed that expression of CTF/NF-1 was 3-20-fold higher in the resistant cell line than in its parental counterpart. These findings suggest that, in cisplatin-resistant cells, the expression of HMG1 gene product is enhanced at the transcriptional level and that this probably occurs through the enhanced expression of the CCAAT binding factor, CTF/NF-1.
Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.