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Biomedical subjects

K Tamayose

Publications and source records attributed to K Tamayose.

11 recordsLinked to original sources
PubMed2001

An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson's disease.

Adeno-associated virus (AAV) vector delivery of an Apaf-1-dominant negative inhibitor was tested for its antiapoptotic effect on degenerating nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. The wild-type caspase recruitment domain of Apaf-1 was used as a dominant negative inhibitor of Apaf-1 (rAAV-Apaf-1-DN-EGFP). An AAV virus vector was used to deliver it into the striatum of C57 black mice, and the animals were treated with MPTP. The number of tyrosine hydroxylase-positive neurons in the substantia nigra was not changed on the rAAV-Apaf-1-DN-EGFP injected side compared with the noninjected side. We also examined the effect of a caspase 1 C285G mutant as a dominant negative inhibitor of caspase 1 (rAAV-caspase-1-DN-EGFP) in the same model. However, there was no difference in the number of tyrosine hydroxylase-positive neurons between the rAAV-caspase-1-DN-EGFP injected side and the noninjected side. These results indicate that delivery of Apaf-1-DN by using an AAV vector system can prevent nigrostriatal degeneration in MPTP mice, suggesting that it could be a promising therapeutic strategy for patients with Parkinson's disease. The major mechanism of dopaminergic neuronal death triggered by MPTP seems to be the mitochondrial apoptotic pathway.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Activation of an ataxia telangiectasia mutation-dependent intra-S-phase checkpoint by anti-tumour drugs in HL-60 and human lymphoblastoid cells.

In yeast cells, the intra-S-phase checkpoint slows down the rate of DNA replication in response to DNA damage. Here we showed that a similar checkpoint mechanism is present and activated by anti-tumour drugs in HL-60 and Epstein-Barr virus (EBV)-transformed human lymphoblastoid cells. Using bromodeoxyuridine (BrdU) pulse labelling combined with two-dimensional flow cytometric analysis, we clearly visualized the cell-cycle progression of the BrdU-positive population (cells originally belonging to the S phase) and detected even subtle changes in S-phase progression induced by mild drug treatment conditions free of apoptosis. The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU-positive HL-60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti-metabolite, cytosine beta-D-arabinofuranose (Ara-C; 50 nmol/l), kept them in early-to-late S phase after 8 h of incubation. Because 10 micromol/l of caffeine added 2 h later attenuated the S-phase retardation by these drugs in HL-60 cells, slowing of the S-phase progression should be actively regulated. Furthermore, two ataxia telangiectasia (AT)-derived lymphoblastoid cell lines were impaired in the doxorubicin-induced S-phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product. The inhibitory mechanism on S-phase progression elicited by anti-tumour drugs in HL-60 and lymphoblastoid cells may therefore correspond to the intra-S-phase checkpoint of the yeast cells.

Antimetabolites↗
PubMed2000

Site-specific integration of an adeno-associated virus vector plasmid mediated by regulated expression of rep based on Cre-loxP recombination.

Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5' end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

Base Sequence↗
PubMed2000

[Idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia and pleural effusion].

A 56-year-old man was admitted for further evaluation of mediastinal lymphadenopathy and left pleural effusion. A blood test revealed polyclonal hypergammaglobulinemia with an elevated erythrocyte sedimentation rate and level of C-reactive protein. Chest computed tomography disclosed bilateral hilar and multiple mediastinal lymph-node swelling and bilateral pleural thickening accompanied by a moderate amount of left pleural effusion. Biopsy specimens from mediastinal lymph nodes and the right pleura were obtained under video-assisted thoracoscopy. Histology on microscopic examination demonstrated mature plasma cells and lymphocyte infiltration in the conserved lymph-node structure and the pleura, suggesting a diagnosis of idiopathic plasmacytic lymphadenopathy (IPL) with polyclonal hyperimmunoglobulinemia. Interestingly, IL-6 was elevated in the pleural effusion but normal in serum. This was a rare and instructive case of IPL accompanied by pleural effusion that was considered to be an important determinant of the clinical spectrum of the disease.

Biomarkers↗
PubMed1999

Inhibition of p34cdc2 dephosphorylation in DNA damage- and topoisomerase II inactivation-induced G2 arrests in HL-60 cells.

Doxorubicin induces DNA breakage by stabilizing a cleavable topoisomerase II-DNA complex. In contrast, topoisomerase II catalytic inhibitor ICRF-193 and uncoupling inhibitor aclarubicin interfere with the cleavable complex formation. We analysed combination effects of these drugs using two-dimensional flow cytometry of DNA content and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling assay. Both ICRF-193 and aclarubicin attenuated the cytotoxic effect of doxorubicin on HL-60 cells (85% and 46% maximum reduction, respectively), which suggested that doxorubicin exerts its cytotoxic effect at least partially through the topoisomerase II-dependent DNA cleavage. Doxorubicin and ICRF-193 both induced G2 arrest in HL-60 cells, by which they may have reduced the cytotoxic effect of vincristine. Indeed, although ICRF-193 inhibited doxorubicin-induced apoptosis, ICRF-193 and doxorubicin cooperated in arresting HL-60 cells at G2 phase. These results indicated that G2 arrest was caused not only by DNA damage but also through a DNA damage-free, topoisomerase II inactivation-induced pathway. Western blot analysis showed that both types of G2 arrest were mediated by the inhibition of p34cdc2 dephosphorylation.

Antineoplastic Agents↗
PubMed1996

A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography.

The extensive testing of adeno-associated virus (AAV) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid DNAs and infection with adenovirus in permissive cells. In an effort to resolve this problem, HeLa cell-based packaging cell lines were established. These packaging cells carry multiple copies of the AAV genome lacking the inverted terminal repeat (ITR) sequences. The AAV genes were silent in these cells but inducibly expressed by adenovirus infection. When the AAV vector plasmid containing the neoR gene flanked by the ITRs was also integrated into these cells, efficient production of the recombinant AAV particles occurred after adenovirus infection. AAV vector particles in cell lysates could be concentrated by sulfonated cellulose column chromatography. Using the packaging cells and the column chromatography technique, it is possible to prepare AAV vectors with the titer of higher than 10(8) cfu/ml or 5 x 10(10) particles/ml. This new strategy should be useful for testing AAV vectors in vivo.

Cellulose↗
PubMed1993

Hemorrhagic cystitis associated with allogeneic and autologous bone marrow transplantation for malignant neoplasms in adults.

We investigated the occurrence of hemorrhagic cystitis in 30 consecutive adult patients undergoing bone marrow transplantation (BMT) for hematological and non-hematological malignancies. Ten patients with hematological malignancies received allogeneic BMT, and twenty patients, seven with hematological and 13 with non-hematological malignancies, received autologous BMT. All 30 patients received high-dose cyclophosphamide-containing regimens (120 mg/kg in 16, 200 mg/kg in one, 6000 mg/m2 in 13) as preparative therapies. They all received 2-mercaptoethane sulphonate sodium (mesna) combined with hyperhydration 3 liter/day as prophylaxis for hemorrhagic cystitis. Bladder irrigation was not performed. Overall, five patients (16.7%) developed hemorrhagic cystitis; early-onset (within 48 hours of the end of the high dose chemoradiotherapy) hemorrhagic cystitis occurred in one (3.3%; 95% confidence interval, 0.6-16.7%) and late-onset occurred in four (13.3%; 95% confidence interval, 5.3-29.7%). In three of the four late-onset cases, adenovirus was isolated from the urine specimens at the onset of the hemorrhagic cystitis. Among the 13 patients with non-hematological malignancies receiving autologous BMT, one with recurrent breast cancer developed late-onset hemorrhagic cystitis associated with adenovirus type 11. We conclude the prophylactic measure with mesna and hyperhydration to be effective enough to prevent cyclophosphamide-induced hemorrhagic cystitis of early onset. Some BMT recipients, however, even those with non-hematological malignancies undergoing autologous BMT, develop late-onset hemorrhagic cystitis in which adenovirus is considered a principal causative agent.

Adult↗