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H Sawa

Publications and source records attributed to H Sawa.

At least 37 records · Page 2Linked to original sources
PubMed2001

Distribution and function of JCV agnoprotein.

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), encodes six major proteins including agnoprotein, the function of which is unknown. To explore its function, we initially studied the expression and localization of agnoprotein in both cultured cells and PML brain using immunohistochemical methods. Employing a specific polyclonal antibody, agnoprotein was found mostly in the cytoplasm of persistently infected JCI cells and in the finely elaborated cytoplasmic processes of oligodendroglial cells in PML brain. The immunohistochemistry indicated that the cytoplasm of oligodendroglial cells was relatively well-preserved in the demyelinated foci. Agnoprotein coprecipitated with tubulin in immunoprecipitation assays and the colocalization of agnoprotein with cytoplasmic tubulin was verified by double immunostaining with confocal microscopy. Transfection of an agnogene deleted JCV Mad1 strain [Mad1(Delta agno)] into the susceptible cell line failed to produce not only agnoprotein but also VP1 and large T mRNAs, whereas the wild-type JCV Mad1 resulted in the expression of both large T and VP1 mRNAs. The cytoplasmic agnoprotein was phosphorylated and when coexpressed with GST-EGFP, was also localized in the cytoplasm. Inhibition of protein kinase A by its inhibitor H-89, however, reversed the cytoplasmic localization of agnoprotein to the nuclear compartment. Our results suggest that JCV agnoprotein may "shuttle" between the nucleus and cytoplasm in a phosphorylation-dependent manner during viral replication.

Cell Line↗
PubMed2001

Inhibitory effects of sodium butyrate on proliferation and invasiveness of human glioma cells.

OBJECTIVE: Sodium butyrate (SB), a differentiation-inducing agent, has been demonstrated to inhibit cellular proliferation in a number of human cell lines. Its precise mechanisms remain to be clarified, however. We investigated molecular mechanisms of SB-induced growth suppression as well as the effects of SB on the invasiveness of human glioma cells. METHODS: Human glioma U87MG and U251MG cells were treated with 1 or 2 mmol/L SB for 48 hours, and the inhibition of cell growth was assessed by spectrophotometric analysis. Cell cycle analysis was carried out by the 5-bromo-2'-deoxyuridine incorporation method, and expression of cell cycle-regulatory proteins was determined by immunoblotting. In addition, invasiveness was assessed using a Transwell chamber (Iwaki, Tokyo, Japan) with extracellular matrix substrate fibronectin or laminin (Iwaki). RESULTS: SB treatment resulted in significantly suppressed proliferation of both U87MG and U251MG cells in a dose-dependent manner. It inhibited the G1-S transition, which was associated with increased expression of p21 and cyclin D1 and reduced pRb phosphorylation. Treatment with antisense oligonucleotide for Rb abrogated SB-induced G1 arrest. p21 up-regulation was independent of the p53 status of the glioma cells. SB treatment also inhibited invasiveness on fibronectin and laminin. CONCLUSION: Our results indicate that SB may suppress the growth of human glioma cells through modulation of cell cycle progression and also may affect their invasiveness on extracellular matrix substrates, which suggests that SB may be a useful therapeutic agent in treating multiple aspects of malignant gliomas.

Brain Neoplasms↗
PubMed2001

Chemotherapy-induced unconjugated hyperbilirubinemia caused by a mutation of the bilirubin uridine-5'-diphosphate-glucuronosyltransferase gene.

Chemotherapy for malignant neoplasms sometimes causes unconjugated hyperbilirubinemia in the absence of liver dysfunction. We analyzed the association of chemotherapy-induced hyperbilirubinemia with mutations of the bilirubin uridine-5'-diphosphate (UDP)-glucuronosyltransferase gene (UGT1A1) from two leukemic patients in whom chemotherapy resulted in a hyperbilirubinemic response. We isolated genomic DNA from peripheral blood samples and amplified UGT1A1 by polymerase chain reaction. The amplified DNA fragments were analyzed by direct sequencing. The genes of the two patients revealed an identical heterozygous missense mutation in exon 1 (211G-->A: G71R). This UGT1A1 mutation may be the basis of chemotherapy-induced unconjugated hyperbilirubinemia.

Adolescent↗
PubMed2001

Quantitation of varicella-zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete.

Varicella-zoster virus (VZV) reactivation causes facial nerve palsy in Ramsay Hunt syndrome (RHS) and zoster sine herpete (ZSH) with and without zoster rash, respectively. In the present study, we analyzed the VZV DNA copy number in saliva samples from 25 patients with RHS and 31 patients with ZSH using a TaqMan PCR assay to determine differences in the viral load between the two diseases. VZV copy number in saliva peaked near the day of the appearance of zoster in patients with RHS. Consequently, VZV DNA was less frequently detected in patients with RHS who exhibited facial palsy several days after the appearance of zoster. These findings suggest that the VZV load in saliva samples reflects the kinetics of viral reactivation in patients with RHS. In addition, VZV DNA was equally detected in saliva from patients with RHS and ZSH, and there was no significant difference in the highest viral copy number between patients with RHS and those with ZSH. The VZV load does not appear to reflect a major difference between RHS and ZSH.

DNA, Viral↗
PubMed2001

Critical role of Caenorhabditis elegans homologs of Cds1 (Chk2)-related kinases in meiotic recombination.

Although chromosomal segregation at meiosis I is the critical process for genetic reassortment and inheritance, little is known about molecules involved in this process in metazoa. Here we show by utilizing double-stranded RNA (dsRNA)-mediated genetic interference that novel protein kinases (Ce-CDS-1 and Ce-CDS-2) related to Cds1 (Chk2) play an essential role in meiotic recombination in Caenorhabditis elegans. Injection of dsRNA into adult animals resulted in the inhibition of meiotic crossing over and induced the loss of chiasmata at diakinesis in oocytes of F(1) animals. However, electron microscopic analysis revealed that synaptonemal complex formation in pachytene nuclei of the same progeny of injected animals appeared to be normal. Thus, Ce-CDS-1 and Ce-CDS-2 are the first example of Cds1-related kinases that are required for meiotic recombination in multicellular organisms.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix metalloproteinase and conditioned switching of its expression during the ovarian follicular development.

In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Deficiency of phospholipase C-gamma1 impairs renal development and hematopoiesis.

Phospholipase C-gamma1 (PLC-gamma1) is involved in a variety of intracellular signaling via many growth factor receptors and T-cell receptor. To explore the role of PLC-gamma1 in vivo, we generated the PLC-gamma1-deficient (plc-gamma1(-/-)) mice, which died of growth retardation at embryonic day 8.5-9.5 in utero. Therefore, we examined plc-gamma1(-/-) chimeric mice generated with plc-gamma1(-/-) embryonic stem (ES) cells for further study. Pathologically, plc-gamma1(-/-) chimeras showed multicystic kidney due to severe renal dysplasia and renal tube dilation. Flow cytometric analysis and glucose phosphate isomerase assay revealed very few hematopoietic cells derived from the plc-gamma1(-/-) ES cells in the mutant chimeras. However, differentiation of plc-gamma1(-/-) ES cells into erythrocytes and monocytes/macrophages in vitro was observed to a lesser extent compared with control wild-type ES cells. These data suggest that PLC-gamma1 plays an essential role in the renal development and hematopoiesis in vivo.

Animals↗
PubMed2001

Coronary reperfusion following ischemia: different expression of bcl-2 and bax proteins, and cardiomyocyte apoptosis.

The aim of this work was to examine factors that could be involved in the occurrence of apoptosis in rat hearts subjected to coronary occlusion followed by reperfusion. To this end, we studied the expression of the pro- and anti-apoptotic factors, bax and bcl-2, respectively, in reperfused ischemic hearts and in hearts injected with bFGF or saline. In anesthetized rats the left coronary artery was occluded for 45 min, the anesthesia withdrawn and the occlusion removed to allow reperfusion; in sham-operated rats the occlusion was omitted. After 4 hours the rats were decapitated and the heart excised. Sections from the left ventricle were stained with anti-bcl-2-antibody and anti-bax-antibody using the TUNEL method which detects apoptosis. Fragmentation of DNA isolated from reperfused ventricles was examined by agarose electrophoresis. In reperfused hearts no bcl-2 staining was observed in the discrete area in which many cardiomyocyte nuclei were stained by the TUNEL method; outside this area staining for bcl-2 was more marked than in sham-operated rats. Sections from reperfused hearts were stained for bax protein over a wide area including the apoptotic region; sham-operated hearts showed little reaction. Staining for bcl-2 was demonstrable in some nuclei in hearts from saline-injected rats; the numbers were unaffected by i. v. bFGF. Ischemia/reperfusion increases the overall expression of both bcl-2 and bax proteins, but bcl-2 is lost from the reperfused area as indicated by TUNEL staining. Accordingly, the ratio of bcl-2 to bax was reduced in the reperfused area, indicating a pro-apoptotic trend. The marked increase in bcl-2 outside the reperfused area could be a mechanism with which to salvage surviving cardiomyocytes.

Animals↗
PubMed2001

Metastatic bronchogenic carcinoma with human chorionic gonadotropin production manifesting as cerebellar hemorrhage--case report.

A 58-year-old male presented with a rare case of brain metastatic bronchogenic carcinoma with human chorionic gonadotropin (hCG) production causing cerebellar hemorrhage with symptoms of nausea, vomiting, and headache. Bronchogenic carcinoma manifesting as gynecomastia had been resected a few months previously. Neurological examination revealed left cerebellar ataxia. Neuroimaging showed multiple cerebellar metastases with cerebellar hemorrhage adjacent to the tentorium. Angiography demonstrated tumor staining fed by the hemispheric branch of the left posterior inferior cerebellar artery. Suboccipital craniectomy was performed. The left cerebellar hematoma was evacuated and the tumor was partially removed to prevent massive intraoperative hemorrhage and avoid brain stem injury. Histological examination showed the resected tumor was large cell carcinoma. hCG was detected in the cerebrospinal fluid and was identified by immunohistochemical staining in tumor cells. The primary lesion of bronchogenic carcinoma showed choriocarcinomatous change because the tumor could produce hCG. The choriocarcinomatous cells with higher metastatic potential formed lesions in the brain, and finally intratumoral hemorrhage occurred producing the rapid development of symptoms.

Carcinoma, Bronchogenic↗
PubMed2001

New rat model for attention deficit hyperactive disorder (ADHD).

PURPOSE: In a strain of the Long-Evans Cinnamon (LEC) rats, we found spontaneously hyperactive animals designated as "wiggling," and established a congenic wiggling (Wig) rat by transferring the gene from the LEC to the Wistar King-Aptekman/Hokkaido (WKAH) strain. We evaluated the feasibility of the Wig rat for an animal model of human attention deficit hyperactive disorder (ADHD). METHODS: Mode of inheritance was examined by use of linkage analyses. Motor activity, behavior, and working memory were assessed by use of electric digital counters, open field test, and Y-maze and water-maze tests. RESULTS: The abnormal behavior, including hyperactivity, was transmitted in autosomal recessive mode. Diurnal and nocturnal motor activity of 12- to 14-week-old Wig rats was markedly higher than that of controls, and this hyperactivity was more prominent during nighttime than daytime. Ambulation in the open-field test was significantly increased in Wig rats, but rearing was decreased in Wig rats, compared with controls. Results of the Y-maze tests indicated that spontaneous alternation behavior was significantly impaired in Wig rats, although there was no difference in the total arm entries. The water-maze test could not be performed because, when exposed to water, Wig rats panicked and almost drowned. CONCLUSIONS: Wig rats are hyperactive and have impaired working memory and impulsive behavior, as assessed by the motor activity and open-field tests and the Y-maze test, and these abnormalities are transmitted by a single gene with Mendelian pattern. Wig rats represent an excellent animal model of human ADHD.

Animals↗
PubMed2000

CalDAG-GEFIII activation of Ras, R-ras, and Rap1.

We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, co-activation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.

Animals↗
PubMed2000

Transcriptional activation of JC virus by human T-lymphotropic virus type I Tax protein in human neuronal cell lines.

Polyomavirus JC (JCV) causes the human demyelinating disease, progressive multifocal leukoencephalopathy (PML). The recent demonstration of cases of PML in association with human T-lymphotropic virus type I (HTLV-I) infection prompted us to examine whether the HTLV-I-encoded regulatory protein Tax activates JCV transcription. By employing a dual luciferase assay, we initially found that the expression of Tax activated the transcriptional potential of both early and late promoters of JCV in human neuronal but not in non-neuronal cells. We subsequently analyzed the mechanism of Tax-induced activation of the JCV promoter in neuronal cells with the following results: 1) the JCV promoter that lacks the NF-kappaB-binding motif could not be activated by Tax; 2) the overexpression of IkappaBalpha abolished Tax-induced transcriptional activation of the JCV promoter; 3) a Tax mutant (M22) lacking the potential for activation via the NF-kappaB pathway did not activate the JCV promoter. Furthermore, Tax enhances the gene expression of JCV T antigen and VP1. We examined mechanisms of the cell-specific activation of the JCV promoter by Tax. Electrophoretic mobility shift assay demonstrated the presence of Tax-bound protein(s) that were specifically present in non-neuronal cells. This study is the first demonstration of the activation of JCV promoter by HTLV-I Tax in an NF-kappaB-dependent manner.

Base Sequence↗
PubMed2000

Membrane expression of annexin I is enhanced by calcium and TPA in cultured human keratinocytes.

Annexin I plays an important role in the process of keratinization as a component of the cornified envelope. To elucidate the function of annexin I in keratinization, we investigated the effects of calcium, epinephrine, hydrocortisone, and 12-O-tetradecanoyl phorbol 13-acetate (TPA) on the expression and localization of annexin I in cultured human keratinocytes. Normal human keratinocytes were cultured in serum-free culture medium (0.15 mM calcium) until 70% confluence. After incubation with a higher concentration of calcium (1.8 mM), TPA (100 nM), epinephrine (50 microM), or hydrocortisone (10 microM) for 24 h, the expression of annexin I protein and mRNA was examined using immunofluorescence, Western blot, and Northern blot techniques. Immunofluorescence microscopy showed increased membrane staining of annexin I by calcium, which was inhibited by the addition of epinephrine or hydrocortisone. Western blotting confirmed elevated annexin I on the cell membrane. It was increased in the cell membrane fraction, but not in the cytosol fraction. Interestingly, the mRNA level of annexin I was slightly reduced after incubation with calcium, whereas TPA upregulated both membrane expression and the mRNA level. Secretion of annexin I was increased by TPA but inhibited by calcium. Because calcium and TPA are known to promote keratinization, our data suggest that annexin I expression on the cell membrane is involved in the process of keratinization.

Annexin A1↗
PubMed2000

Components of the SWI/SNF complex are required for asymmetric cell division in C. elegans.

Asymmetric cell division is a fundamental process that produces cellular diversity during development. We have identified two mutants in C. elegans (psa-1 and psa-4) in which the asymmetry of T cell division is disrupted. psa-1 and psa-4 encode homologs of yeast SWI3 and SWI2/SNF2, respectively, which are components of the SWI/SNF complex. We show by RNA interference assay that homologs of other components of SWI/SNF are also involved in T cell division. psa-1 and psa-4 are likely to be required in the T cell during mitosis to cause asymmetric cell division. Because the SWI/SNF complex is required for asymmetric division in S. cerevisiae, these results demonstrate that at least some aspects of the mechanism of asymmetric cell division are conserved between yeast and a multicellular organism.

Adenosine Triphosphatases↗
PubMed2000

Pars plana vitrectomy for regression of choroidal neovascularization with age-related macular degeneration.

OBJECTIVE: In age-related macular degeneration, the posterior vitreous surface often remains attached to a higher degree than expected for the patient's age, suggesting the involvement of the posterior vitreous membrane in the pathophysiology of choroidal neovascularization. Thus, we performed simple pars plana vitrectomy and artificial posterior vitreous detachment in order to induce regression of choroidal neovascularization. METHODS: Vitrectomy was performed in 12 eyes of 11 patients with age-related macular degeneration in whom the posterior vitreous surface remained attached, and in whom there was evidence of highly active choroidal neovascularization. The posterior vitreous membrane was artificially detached during surgery, but photocoagulation and/or a subretinal procedure for choroidal neovascularization was not performed. Patients underwent fluorescein and indocyanine green angiography both pre- and postoperatively to assess the status of choroidal neovascularization. RESULTS: Six months after surgery, 6 of the 12 eyes showed regression of choroidal neovascularization, and in 2 eyes it had disappeared completely. Subretinal exudative changes in 8 eyes had improved. Visual acuity testing showed improved vision in 4 eyes, unchanged vision in 4 eyes, and worsening of vision in 4 eyes. The final best corrected visual acuity was 10/20 or better in one eye, 10/ 20 to 20/200 in 7 eyes, and 20/200 or worse in 4 eyes. CONCLUSION: The results of the present study suggest the involvement of the posterior vitreous membrane in the pathophysiology of age-related macular degeneration. Furthermore, the minimally invasive pars plana vitrectomy may represent a new treatment for age-related macular degeneration.

Aged↗
PubMed2000

MSI-1, a neural RNA-binding protein, is involved in male mating behaviour in Caenorhabditis elegans.

BACKGROUND: Neural RNA-binding proteins are thought to play important roles in neural development and the functional regulation of postmitotic neurones by mediating post-transcriptional gene regulation. RNA-binding proteins belonging to the Musashi family are highly expressed in the nervous system; however, their roles are poorly understood. RESULTS: We identified a Caenorhabditis elegans Musashi homologue, MSI-1, whose RNA-recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. We isolated a msi-1 mutant and found males with this mutation to have a mating defect. C. elegans male mating behaviour includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi-1 is required for the turning and vulva location steps. Like other Musashi family members, MSI-1 is expressed specifically in neural cells, including male-specific neurones required for turning and vulva location. However, msi-1 was not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems. CONCLUSIONS: Our results suggest that msi-1 is expressed specifically in postmitotic neurones in C. elegans. msi-1 is required for full development of male mating behaviour, possibly through regulation of msi-1 expressing neurones.

Alleles↗
PubMed2000

Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy.

Infection of the cerebral cortical neurons with JC virus (JCV) with possible dysplastic ganglion-like alteration of the infected neurons found in a case of progressive multifocal leukoencephalopathy (PML) is described. The patient was a 21-year-old man with common variable immunodeficiency who died of PML after a 9-month clinical course. At autopsy, the white matter of the cerebrum, brainstem, cerebellum, and spinal cord exhibited extensive demyelination and necrosis. Numerous inclusion-bearing oligodendrocytes and bizarre astrocytes were found. In the occipital and temporal cortex, thick band-like aggregates of dysplastic ganglion-like cells (DGLCs) were found. These DGLCs showed immunohistochemical properties of neurons, and nuclei of some DGLCs were immunoreactive for large T antigen of SV40/JCV and p53, but not for capsid protein JCV VP1. In situ hybridization for mRNA of JCV large T antigen revealed positive signals in the nuclei of some DGLCs. These results indicate that JCV infected neurons and it is suggested that binding of the large T antigen with cellular proteins could have resulted in the dysplastic, ganglion cell-like change of the infected neurons, although the possibility that the aggregates of DGLCs represent a pre-existent malformative lesion of the cortex cannot be excluded completely.

Adult↗