Biomedical subjects

Takashi Shimada

Publications and source records attributed to Takashi Shimada.

At least 19 recordsLinked to original sources

PubMed2007

AAV1 mediated co-expression of formylglycine-generating enzyme and arylsulfatase a efficiently corrects sulfatide storage in a mouse model of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.

Animals↗

PubMed2006

Comprehensive classification of the auditory sensory projections in the brain of the fruit fly Drosophila melanogaster.

We established a comprehensive projection map of the auditory receptor cells (Johnston's organ neurons: JONs) from the antennae to the primary auditory center of the Drosophila brain. We found 477 +/- 24 cell bodies of JONs, which are arranged like a "bottomless bowl" within the auditory organ. The target of the JONs in the brain comprises five spatially segregated zones, each of which is contributed by bundles of JON axons that gradually branch out from the antennal nerve. Four zones are confined in the antennal mechanosensory and motor center, whereas one zone further extends over parts of the ventrolateral protocerebrum and the subesophageal ganglion. Single-cell labeling with the FLP-out technique revealed that most JONs innervate only a single zone, indicating that JONs can be categorized into five groups according to their target zones. Within each zone, JONs innervate various combinations of subareas. We classified these five zones into 19 subareas according to the branching patterns and terminal distributions of single JON axons. The groups of JONs that innervate particular zones or subareas of the primary auditory center have their cell bodies in characteristic locations of the Johnston's organ in the antenna, e.g., in concentric rings or in paired clusters. Such structural organization suggests that each JON group, and hence each zone of the primary auditory center, might sense different aspects of sensory signals.

Animals↗

PubMed2006

Klotho converts canonical FGF receptor into a specific receptor for FGF23.

FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates kidney functions, whereas most other family members are thought to regulate various cell functions at a local level. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule, generates the FGF23 receptor. Using a renal homogenate, we found that Klotho binds to FGF23. Forced expression of Klotho enabled the high-affinity binding of FGF23 to the cell surface and restored the ability of a renal cell line to respond to FGF23 treatment. Moreover, FGF23 incompetence was induced by injecting wild-type mice with an anti-Klotho monoclonal antibody. Thus, Klotho is essential for endogenous FGF23 function. Because Klotho alone seemed to be incapable of intracellular signalling, we searched for other components of the FGF23 receptor and found FGFR1(IIIc), which was directly converted by Klotho into the FGF23 receptor. Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF23 receptor. These findings provide insights into the diversity and specificity of interactions between FGF and FGF receptors.

Animals↗

PubMed2006

Functional characterization of the N-terminal region of myosin-2.

All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release approximately 40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.

Actins↗

PubMed2006

Characterization of cyanobacteriochrome TePixJ from a thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1.

A putative photoreceptor gene, TepixJ, of a thermophilic cyanobacterium is homologous to SypixJ1 that mediates positive phototaxis in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. The putative chromophore-binding GAF domain of TePixJ protein was overexpressed as a fusion with a polyhistidine tag (His-TePixJ_GAF) in Synechocystis cells and isolated to homogeneity. The photoreversible conversion of His-TePixJ_GAF showed peaks at 531, 341 and 266 nm for the green light-absorbing form (Pg form), and peaks at 433 and 287 nm for the blue light-absorbing form (Pb form). At 77K, the Pg form fluoresced at 580 nm, while the Pb form did not emit any fluorescence. Mass spectrometry of the tryptic chromopeptide demonstrated that a phycocyanobilin isomer binds to the conserved cysteine at ring A via a thioether bond. It is established that TePixJ and SyPixJ1 are novel photoreceptors in cyanobacteria ('cyanobacteriochromes') that are similar, but distinct from the phytochromes and bacteriophytochromes.

Amino Acid Sequence↗

PubMed2006

Ex vivo cell-mediated gene therapy for metachromatic leukodystrophy using neurospheres.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophotometric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD.

Animals↗

PubMed2006

Adenoviral mediated MyoD gene transfer into fibroblasts: myogenic disease diagnosis.

MyoD, a master regulatory gene for myogenesis, converts mesoderm derived cells to the skeletal muscle phenotype MyoD gene transfer into skin fibroblasts has been attempted in an effort to diagnose genetic muscle diseases. Although the gene transduction efficiency of adenoviral gene delivery systems is higher than that of various other systems, the rate of myo-conversion is insignificant. Since high adenovirus doses are cytotoxic and exogenous MyoD expression is insufficient for skin fibroblasts to re-differentiate into muscle cells, we constructed the novel adeno-MyoD vector, Ad.CAGMyoD using the recombinant CAG promoter. Even at a lower multiplicities of infection most skin fibroblasts infected with Ad.CAGMyoD could convert into myotubes without vector-induced cytotoxicity. The converted cells expressed muscle-specific desmin and full-length dystrophin, both of which were detected by Western blotting. Genetic and immunohistochemical analyses using skin fibroblasts and our vector system are reliable and useful for the clinical diagnosis of genetic muscle diseases.

Adenoviridae↗

PubMed2006

Reconstitution of blue-green reversible photoconversion of a cyanobacterial photoreceptor, PixJ1, in phycocyanobilin-producing Escherichia coli.

PixJ1, a photoreceptor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, mediates positive phototactic motility and contains two GAF domains, the latter of which binds a bilin chromophore. Full-length PixJ1 expressed and purified from Synechocystis showed unique reversible photoconversion between a blue light-absorbing (Pb) form and a green light-absorbing (Pg) form (1) in contrast to the reversible phototransformation between the red light-absorbing form and far-red light-absorbing form of the other GAF-containing photoreceptors such as plant or bacterial phytochromes. To clarify the origin of the blue-shifted photoconversion, we tried to reconstitute this blue-green reversible phototransformation by synthesizing the second GAF domain in Escherichia coli transformed with genes for biosynthesis of four different bilins, biliverdin (BV), bilirubin (BR), phycocyanobilin (PCB), and phycocyanorubin (PCR), as final products. The three expression systems, the BR system being the exception, produced a GAF polypeptide with a covalently bound bilin. The GAF polypeptide from the BV-synthesizing system exhibited an irreversible photoconversion, while that from the PCB-synthesizing system revealed photoconversion between Pb and Pg almost identical to that of the full-length PixJ1, indicating that PCB is responsible for the blue-green reversible photoconversion. Furthermore, the GAF polypeptide from the PCR-producing system exhibited almost the same reversible spectral change, possibly coming from the PCB accumulated in the PCR-biosynthetic pathway. Mass spectrometry (MS) of the main tryptic chromopeptide revealed that the chromophore binds to a 21-amino acid peptide that contains a cysteine-histidine motif for phytochrome chromophore binding and that an ion signal can be assigned to desorbed PCB. The absorption spectra of the denatured GAF polypeptide suggested that PCB is attached to the protein moiety in a twisted conformation that disrupts the pi-electron conjugation between the A and B rings, possibly being held in position through a second covalent linkage.

Bile Pigments↗

PubMed2006

Corrective effect on Fabry mice of yeast recombinant human alpha-galactosidase with N-linked sugar chains suitable for lysosomal delivery.

We have previously reported the production of a recombinant alpha-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant alpha-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant alpha-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.

Animals↗

PubMed2006

Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens.

Various genotypes of the hepatitis B virus (HBV) induce liver disease of distinct severity, but the underlying virological differences are not well defined. Huh7 cells were transfected with plasmids carrying 1.24-fold the HBV genome of different genotypes/subgenotypes (2 strains each for Aa/A1, Ae/A2, Ba/B2 and D; 3 each for Bj/B1 and C). HBV DNA levels in cell lysates, determined by Southern hybridization, were the highest for C followed by Bj/Ba and D/Ae (P < .01), and the lowest for Aa (P < .01), whereas in culture media, they were the highest for Bj, distantly followed by Ba/C/D and further by Ae/Aa (P < .01). The intracellular expression of core protein was more than 3-fold lower for Ae/Aa than the others. Hepatitis B e antigen (HBeAg) was excreted in a trend similar to that of HBV DNA with smaller differences. Secretion of hepatitis B surface antigen (HBsAg) was most abundant for Ae followed by Aa, Ba, Bj/C and remotely by D, which was consistent with mRNA levels. Cellular stress determined by the reporter assay for Grp78 promoter was higher for C and Ba than the other genotypes/subgenotypes (P < .01). Severe combined immunodeficiency mice transgenic for urokinase-type plasminogen activator (uPA/SCID), with the liver replaced for human hepatocytes, were inoculated with virions passed in mouse and recovered from culture supernatants. HBV DNA levels in their sera were higher for C than Ae by 2 logs during 4-7 weeks after inoculation. In conclusion, virological differences among HBV genotypes were demonstrated both in vitro and in vivo. These differences may influence HBV infections with distinct genotypes in clinical and epidemiological settings.

Adolescent↗

PubMed2006

Effects of phosphates on the expression of tissue-nonspecific alkaline phosphatase gene and phosphate-regulating genes in short-term cultures of human osteosarcoma cell lines.

We studied the effects of phosphates on the expression of the human tissue-nonspecific alkaline phosphatase (TNSALP) gene and phosphate-regulating genes in short-term cultures of human osteoblastic osteosarcoma cell lines. When human osteosarcoma cell lines, SaOS-2, MG-63, and U(2)OS were cultured with 10 mM inorganic sodium dihydrogenphosphate, 10 mM beta-glycerophosphate, 250 microM pyridoxal phosphate, or 100 microM inorganic pyrophosphate, enzymatic activity of alkaline phosphatase began to increase at 72 h after addition of sodium dihydrogenphosphate and beta-glycerophosphate in SaOS-2 cells. Pyridoxal phosphate and pyrophosphate did not induce alkaline phosphatase activity. U(2)OS cells slightly reacted to beta-glycerophosphate, but MG-63 cells did not react on exposure to phosphates. In SaOS-2 cells, TNSALP mRNA measured by real-time RT-PCR reached a peak level at 72 h after the addition of beta-glycerophosphate. PHEX and MEPE mRNAs were also induced by beta-glycerophosphate. These results suggest that TNSALP, PHEX and MEPE were concordantly induced by beta-glycerophosphate on mineralisation.

Alkaline Phosphatase↗

PubMed2006

Effect of first-trimester ultrasound examination for chromosomal aberrations in women undergoing amniocentesis.

OBJECTIVE: The purpose of this study was to assess the effect of first-trimester ultrasound examination for chromosomal aberrations in women who underwent amniocentesis. METHODS: To evaluate trends in the indications of amniocentesis and the number of chromosomal aberrations, we reviewed all amniotic fluid samples from genetic amniocentesis processed by the Tama-Nagayama Hospital of Nippon Medical School from 1991 through 2005. The referral indications included first-trimester abnormal ultrasound finding. RESULTS: A total of 1,054 women underwent genetic aminiocentesis in the first- to early second-trimester, and 1,063 amniotic samples were processed. The overall rate of chromosomal aberrations was 3.3% (35 of 1,063 samples), and the rate of aberrations remained unchanged during the study period. The number of cases with abnormal ultrasound finding increased from 5 (1.1%) in the first 5-year period to 46 (19.4%) in the last 5-year period (p<0.01). In contrast, the number of amniotic fluid samples per year tended to decline during the study period. CONCLUSION: First-trimester ultrasound examination had a significant effect on our amniocentesis cases. The application of first-trimester ultrasound examination may be associated with a lower rate of invasive genetic testing.

Amniocentesis↗

PubMed2006

Transient cardiomyopathy in a patient with congenital contractural arachnodactyly (Beals syndrome).

We report on an infant with Beals syndrome (congenital contractural arachnodactyly [CCA], MIM 121050) with transient cardiomyopathy showing ballon-like dilatation of the left ventricle that was similar to noncompaction. The patients father and two of his brothers were also found to have CCA without cardiovascular complications. CCA, which is caused by a mutation of the gene for fibrillin 2 protein is similar to Marfan syndrome (MIM 154700), which is caused by a mutation of fibrillin 1 but produces a life-threatening cardiovascular complications. This is the first report of CCA with transient cardiomyopathy. We discuss the mechanism of the spontaneous improvement of cardiomyopathy in this case on the basis of expression of the responsible gene.

Cardiomyopathies↗

PubMed2006

Selective extraction of uranium from a mixture of metal or metal oxides by a tri-n-butylphosphate complex with HNO(3) and H(2)O in supercritical CO(2).

In order to study the decontamination nature of the reactive extraction of uranium in the presence of some metal chemicals using a single-phase mixture of HNO(3), H(2)O and tri-n-butylphosphate (TBP) in supercritical carbon dioxide (SC-CO(2)), we measured the decontamination factors (DFs) of Sr, Zr, Mo, Ru, Pd, Ce and Nd from their mixture with U. These elements were originally added to U(3)O(8) as SrO, ZrO(2), MoO(3), RuO(2), Pd, CeO(2) and Nd(2)O(3), and the extraction was performed at 18 MPa and 323 K with the single-phase mixture. The DFs for these elements were determined to be greater than 10(3) when the molecular ratio of U to TBP in the extracted complex was greater than 0.3. Dilution by SC-CO(2) effectively increased the DFs.

Journal Article↗

PubMed2006

Nutrition education in medical schools in Japan: results from a questionnaire survey.

Diet is known to play a critical role in the pathogenesis of major age-related chronic diseases, which are rapidly becoming more common in Japan and other industrialized countries. However, traditional medical education has not provided adequate knowledge of nutrition. To understand the current status of nutrition education in Japan, we sent a questionnaire to all Japanese medical schools to survey their nutrition curricula. The questionnaire was sent to 79 medical schools in Japan, which includes all medical schools except for that of the authors. We also used a questionnaire to survey second year medical students just after they received a short nutrition course at Nippon Medical School. Fifty-seven medical schools (72.2%) responded to the questionnaire: 12 (21.1% of the responding schools) offered a " nutrition" course and 3 (5.4%) offered a "clinical nutrition" course. Including "nutrition" and "clinical nutrition" courses, 14 of the responding medical schools (24. 6 %) offered stand-alone nutrition courses in their undergraduate education. Although a total of 48 of the responding medical schools (84.2%) offered some nutrition topics, only 8 of the responding schools (14.0%) may have offered substantial nutrition education. No special postgraduate course in medical or clinical nutrition was offered by any of the schools. Despite this, more than 80% of the students that appeared to be interested in a nutrition course recognized the importance of nutrition education in medical school. This survey showed that nutrition education in Japanese medical schools remains inadequate and changes are necessary.

Curriculum↗

PubMed2005

Mutations in p53 cDNA sequence introduced by retroviral vector.

The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors.

Animals↗

PubMed2005

Design, synthesis, and biological activity of novel factor Xa inhibitors: improving metabolic stability by S1 and S4 ligand modification.

Serine protease factor xa (fXa) inhibitor 1 showed good ex vivo anti-fXa activity upon oral administration in rats. However, it has been revealed that 1 had low metabolic stability against human liver microsomes. To improve the metabolic stability, we attempted to modify the S1 and S4 ligands of 1. These modifications resulted in compound 34b, which exhibited selective anti-fXa activity and excellent anti-coagulation activity.

Administration, Oral↗

PubMed2005

Role of bone marrow cells in the healing process of mouse experimental glomerulonephritis.

Recent studies have shown bone marrow (BM) cells to differentiate into a variety of cell types and to thereby participate in the reconstitution of damaged organs. In the present study, we examined the extent to which BM-derived cells are incorporated into glomeruli during recovery from experimentally induced nephritis. To investigate the localization of BM cells in glomeruli, chimeric mice were prepared by transplanting BM cells from green fluorescent protein (GFP) transgenic mice into wild-type mice. Five weeks later, glomerulonephritis was induced by intravenous injection of Habu snake venom. Groups of mice were then killed every few days for 42 d, and harvested kidney samples were subjected to immunohistochemical and immunoelectron microscopic analyses with the aim of detecting the presence of GFP(+) cells within glomeruli. Chimeric animals injected with Habu venom developed proliferative glomerulonephritis within 1-3 d. The lesion gradually subsided and the glomerular structure returned to normal within 42 d. Consistent with the disease course, large numbers of GFP(+) cells were present within glomeruli on d 1-3, but most had disappeared by d 7. Nevertheless, some GFP(+) cells did remain within glomeruli showing mesangial proliferative changes, and were found to express thrombomodulin (TM), a specific endothelial cell marker. These GFP-TM-double-positive cells accounted for a mean of 1.31-2.24% of the total glomerular nuclei from d 7 through d 42, levels that remained stable for at least 12 mo. It thus appears that BM cells can give rise to endothelial cells that participate in the remodeling of glomeruli.

Animals↗