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Takashi Shimada

Publications and source records attributed to Takashi Shimada.

63 records · Page 4Linked to original sources
PubMed2002

Universal fluorescent labeling (UFL) method for automated microsatellite analysis.

We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.

DNA Primers↗
PubMed2002

Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer.

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, alpha-galactosidase A (alpha-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the alpha-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that alpha-gal A activity in plasma was increased to approximately 25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-alpha-gal A antibodies. The alpha-gal A activity in various organs of treated Fabry mice remained 5-20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease.

Animals↗
PubMed2002

Importance of deletion of T at nucleotide 1559 in the tissue-nonspecific alkaline phosphatase gene in Japanese patients with hypophosphatasia.

The tissue-nonspecific alkaline phosphatase (TNSALP) gene in four unrelated patients with hypophosphatasia was analyzed using polymerase chain reaction-single strand conformation polymorphism and the direct sequencing method. Of the participating patients, one had childhood-type and three had perinatal-type disease. All carried a deletion of T at cDNA number 1559, which causes a frameshift downstream from codon L503, as a heterozygote. In the childhood-type patient, an F310L mutation was detected in the opposite allele. Similarly, a perinatal-type patient carried a V3651 mutation in the opposite allele. Mutations in the opposite alleles were not detected in the other two patients with perinatal-type disease. In addition, although both parents carried the deletion as a heterozygote in two families with childhood-type and perinatal-type disease, patients from those families were not homozygous for the deletion. Several single-nucleotide polymorphisms (SNPs) were also detected, which were shown to be useful for haplotype analysis. Allele frequency of the deletion among Japanese patients was 36% (10 of 28 alleles) but none occurred in Caucasian patients. These findings indicate that regardless of clinical type, deletion in the TNSALP gene occurs frequently among Japanese patients. Furthermore, haplotype analysis using SNPs suggested that the deletion might have derived from more than a single founder.

Adult↗
PubMed2002

Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders.

Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of Parkinson's disease. Our results suggest that AAV-Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as Parkinson's disease.

Adenoviridae↗
PubMed2002

New strategy for in vivo transgene expression in corneal epithelial progenitor cells.

PURPOSE: Efficient in vivo gene transfer into corneal epithelial cells and stable transgene expression would have broad clinical applications. We therefore assessed the capacity of adenoviral, adeno-associated viral (AAV) and lentiviral vectors encoding enhanced green fluorescent protein (EGFP) to transduce rat corneal epithelial progenitor cells. METHODS: Superficial cells of the corneal epithelium were shaved, after which 20 microl of the respective vector solutions were inoculated onto the basal cell layer of the limbal epithelium for 30 min. Results. Three days after transduction, fluorescence microscopic examination revealed the presence of EGFP+ cells in all corneas, irrespective of the vector used; however, EGFP+ cells were undetectable in corneas transduced with adenoviral and AAV vectors after 2 and 4 weeks, respectively. By contrast, EGFP+ cells were still detected among cells transduced with lentiviral vector 6 weeks after transduction. DAPI (4', 6-diamidino-2-phenylindole) staining confirmed that it was the basal layer cells that continued to express EGFP throughout the 6-week period. CONCLUSIONS: The use of a lentiviral vector for in vivo transfer of foreign genes into corneal epithelial stem and transient amplifying cells may represent a new and effective approach to the treatment of corneal disease, as well as to the study of the biology of these stem cells.

Adenoviridae↗
PubMed2002

Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.

FGF-23 is involved in the pathogenesis of two similar hypophosphatemic diseases, autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and tumor-induced osteomalacia (TIO). We have shown that the overproduction of FGF-23 by tumors causes TIO. In contrast, ADHR derives from missense mutations in FGF-23 gene. However, it has been unclear how those mutations affect phosphate metabolism. Therefore, we produced mutant as well as wild-type FGF-23 proteins and examined their biological activity. Western blot analysis using site-specific antibodies showed that wild-type FGF-23 secreted into conditioned media was partially cleaved between Arg(179) and Ser(180). In addition, further processing of the cleaved N-terminal portion was observed. In constrast, mutant FGF-23 proteins found in ADHR were resistant to the cleavage. In order to clarify which molecule has the biological activity to induce hypophosphatemia, we separated full-length protein, the N-terminal and C-terminal fragments of wild-type FGF-23. When the activity of each fraction was examined in vivo, only the full-length FGF-23 decreased serum phosphate. Mutant FGF-23 protein that was resistant to the cleavage also retained the activity to induce hypophosphatemia. The extent of hypophosphatemia induced by the single administration of either wild-type or the mutant full-length FGF-23 protein was similar. In addition, implantation of CHO cells expressing the mutant FGF-23 protein caused hypophosphatemia and the decrease of bone mineral content. We conclude that ADHR is caused by hypophosphatemic action of mutant full-length FGF-23 proteins that are resistant to the cleavage between Arg(179) and Ser(180).

Amino Acid Sequence↗
PubMed2002

Loss of DCC gene expression is of prognostic importance in acute myelogenous leukemia.

PURPOSE: Expression of the deleted in colorectal carcinoma (DCC) gene has been found to be lost in some patients with acute myelogenous leukemia (AML). Although this finding is critical to leukemogenesis, its prognostic significance remains uncertain. To evaluate this, loss of DCC gene expression in AML patients and their prognostic significance were investigated. EXPERIMENTAL DESIGN: A group of 170 patients with AML was analyzed. DCC gene expression in AML cells was determined by a semiquantitative reverse transcriptase-PCR. Simultaneous mutation analyses of the p53, N-ras, and FLT3 genes were performed in all of the AML cells by single-strand conformation polymorphism and sequencing subsequent to PCR. The importance of loss of DCC expression was evaluated by Cox proportional analysis and the Kaplan-Meier method. RESULTS: Loss of DCC expression was detected in 47 patients (27.6%). The p53, N-ras, and FLT3 mutations were detected in 20 (11.7%), 42 (24.7%), and 26 (15.2%) patients, respectively. The durations of overall survival (OS) and complete remission (CR) of the 47 DCC-negative AML patients were significantly shorter than that of the 123 DCC-positive patients (P < 0.0045 and <0.0060, respectively). Univariate and multivariate analyses showed that loss of DCC expression was an unfavorable prognostic factor for both OS (P < 0.0053 and <0.0084, respectively) and CR duration (P < 0.0146 and <0.0371, respectively). The 64 DCC-positive patients with wild p53, N-ras, and FLT3 had statistically better CR attainment compared with the other 106 patients (P < 0.0001). CONCLUSIONS: Loss of DCC gene expression was shown to be an independent prognostic factor in AML patients. Thus, loss of DCC gene expression might serve as an important molecular marker for predicting the CR duration and OS of patients with AML.

Acute Disease↗
PubMed2001

Selective transduction of HIV-1-infected cells by the combination of HIV and MMLV vectors.

Human immunodeficiency virus 1 (HIV-1)-infected cells are important targets of gene therapy for acquired immune deficiency syndrome. We have developed a novel strategy for targeted gene transfer into HIV-1-infected cells based on 2-step gene transfer. The first step involves the stable introduction of the HIV vector containing the ecotropic Moloney murine leukemia virus (MMLV) receptor gene (EcoRec) into human CD4+ T cells as a molecular switch. Because the HIV-long terminal repeat (HIV-LTR) is Tat inducible, it is expected that EcoRec is expressed only after HIV-1 infection. Northern blot analysis and a retrovirus binding assay confirmed that the HIV-LTR of the integrated vector was silent in transduced cells but strongly transactivated in HIV-1 infection. High levels of EcoRec expression were observed only in HIV-1-infected cells. These cells became highly susceptible to ecotropic MMLV infection and, therefore, in the second step, HIV-1-infected cells were selectively transduced with ecotropic MMLV vectors. More than 70% of HIV-1-infected cells were transduced by this strategy. These findings indicate that this 2-step method can be used for selective and stable gene transfer into HIV-1-infected cells.

Genetic Vectors↗