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Biomedical subjects

K Takai

Publications and source records attributed to K Takai.

At least 19 recordsLinked to original sources
PubMed1999

In vitro codon-reading specificities of unmodified tRNA molecules with different anticodons on the sequence background of Escherichia coli tRNASer.

The codon-reading properties of wobble-position variants of the unmodified form of Escherichia coli tRNASer1 (the UGA anticodon) were measured in a cell-free translation system. Two variants, with the AGA and CGA anticodons, each exclusively read a single codon, UCU and UCG, respectively. The only case of efficient wobbling occurred with the variant with the GGA anticodon, which reads the UCU codon in addition to the UCC codon. Surprisingly, this wobble reading is more efficient than the Watson-Crick reading by the variant with the AGA anticodon. Furthermore, we prepared tRNA variants with AA, UC, and CU, instead of GA, in the second and third positions and measured their relative efficiencies in the reading of codons starting with UU, GA, and AG, respectively. The specificity concerning the wobble position is essentially the same as that in the case of the codons starting with UC.

Amino Acid Sequence↗
PubMed1999

A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell-free translation system.

5-Methoxyuridine was introduced into the first position of the anticodon of the unmodified form of tRNA(1Ser) from Escherichia coli. The codon reading efficiencies of this tRNA (tRNA(5-methoxyuridine UGA)) relative to those of the unmodified counterpart (tRNA(UGA)) were measured in a cell-free translation system. tRNA(5-methoxyuridine UGA) was more efficient than tRNA(UGA) in the reading of the UCU and UCG codons and was less efficient in the reading of the UCA codon. Thus, the single modification of U to 5-methoxyuridine can enhance the wobble readings.

Amino Acid Sequence↗
PubMed1999

Altered pharmacokinetics of a novel anticancer drug, UCN-01, caused by specific high affinity binding to alpha1-acid glycoprotein in humans.

The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human alpha1-acid glycoprotein (AGP) (Fuse et al, Cancer Res., 58: 3248-3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: (a) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was approximately 80%, although the values for other specimens, except monkey plasma (approximately 20%), were <1%, indicating that the dissociation from human AGP was specifically slower than from other proteins; and (b) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-O1 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 microM) completely inhibited the initial uptake of UCN-01 (1 microM) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 microM). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans.

Alkaloids↗
PubMed1999

Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli.

Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567-585]. In the present study, these two tRNA(Leu) species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA(Leu)5 was found to correspond to the tRNA gene reported as su degrees6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627-644]. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA(Leu)4 was identified as the minor leucine tRNA that has been sequenced previously (tRNA(Leu)UUR) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220-2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA(Leu)4. Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N was found to be a 2'-O-methyluridine derivative with a substituent having a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring. On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um). Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine. Because of the conformational rigidity of Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) species recognize the leucine codons UUA++ and UUG correctly, but never recognize the phenylalanine codons UUU and UUC.

Anticodon↗
PubMed1999

A modified immunoperoxidase assay for detection of antibody porcine reproductive and respiratory syndrome (PRRS) virus in swine sera.

MARC-145 cell monolayers infected with PRRS virus were fixed in 3% neutral buffered formalin and embedded in paraffin. The sections were stained by avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The positive reactions were detected by ABC method and indirect immunofluorescent assay (IFA). There was good correlation between ABC and IFA, and the titers in ABC were higher than those in IFA. The present results indicate that the immunohistochemical staining is a useful test for the detection and quantitation of PRRS virus antibody in swine sera as well as IFA.

Animals↗
PubMed1998

Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides.

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA(Asp) blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This 'knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.

Aspartic Acid↗
PubMed1998

Inhibition of influenza virus RNA polymerase by 5'-capped short RNA fragments.

We have demonstrated that 5'-capped short RNA fragments inhibit the expression of chloramphenicol acetyltransferase (CAT) in the murine 76 cell line, derived which expresses the genes for the RNA polymerases (PB1, PB2, and PA) and the nucleoprotein (NP) of influenza virus in response to treatment with dexamethasone. We have synthesized 5'-capped short RNA fragments (8-13 ntds long) with a 5'-capped structure (m7GpppGm) using T7 RNA polymerase. The 5'-capped short RNA fragments (8-13 ntds long) were encapsulated in liposomes and were tested for their inhibitory effect by a CAT-ELISA assay using the clone 76 cells. The RNA fragments that were 9-12 ntds long showed inhibitory effects. In particular, the 9 ntds long RNA fragment, was highly inhibitory. On the other hand, the inhibitory effect of the 13 ntds long RNA fragment was considerably decreased in comparison with the other short RNA fragments. The minimal RNA chain length required for priming activity was found to be 12 ntds long. Furthermore, the 5'-capped RNA fragments exhibited higher inhibitory activities than the antisense phosphorothioate oligonucleotide (PB2-AUG-as, 20 ntds long) complementary to the site of the PB2-AUG initiation codon. Liposome encapsulation protected the RNA fragments in serum-containing medium and substantially improved their cellular accumulation.

DNA-Directed RNA Polymerases↗
PubMed1998

Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, 'dumbbell RNA/DNA chimeric oligonucleotides', consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the nicked (NDRDON) and circular (CDRDON) dumbbell RNA/ DNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA, which gave RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligonucleotide (anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON with four target sites (influenza virus A RNA polymerases (PB1, PB2, PA) and nucleoprotein (NP)) was synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The circular dumbbell DNA/ RNA chimeric oligonucleotide (CDRDON-PB2-as) containing an AUG initiation codon sequence as the target of PB2 showed highly inhibitory effects.

DNA↗
PubMed1998

Site-specific cleavage of tRNA by imidazole and/or primary amine groups bound at the 5'-end of oligodeoxyribonucleotides.

Sequence specific RNA cleaving molecules were synthesized by attaching novel polyamine derivatives bearing imidazole and/or primary amine groups to the 5'-end of DNA oligonucleotides as the sequence-recognizing moieties. The actions of the molecules on a half-tRNA(Asp) were investigated. The oligonucleotides directed the nuclease activity (the imidazole and the primary amine are the catalytic groups) of the enzyme to the nucleotides directly adjacent to the complementary target sequence on the substrate RNA. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0, indicating the participation of protonated and non-protonated imidazoles residues in the process. The specificity of these hybrid enzymes can be easily altered, and they should prove to be useful tools for probing RNA structures in solution and as potential reactive groups in antisense oligonucleotide derivatives. We also describe the site-specific cleavage of tRNA(Asp) by the cleaving reagents bearing imidazole and/or primary amine groups at the 5'-end of oligodeoxyribonucleotides.

Amines↗
PubMed1998

Properties of nicked and circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligodeoxynucleotides.

We have designed a new class of oligonucleotides, 'dumbbell RNA/DNA chimeric phosphodiester oligonucleotides', consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the Nicked (NDRDON) and Circular (CDRNON) dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 45 mer RNA, which gave 45 mer RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide (anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligodeoxynucleotide. The circularization, achieved by joining the 3' and the 5' ends of RNA/DNA chimeric oligonucleotides containing two hairpin loop structures, increases the oligonucleotide uptake into cells, as compared with the nicked dumbbell RNA/DNA chimeric oligonucleotide and the linear antisense phosphodiester oligodeoxynucleotides. When the circular dumbbell RNA/DNA chimeric oligonucleotide is directly delivered into retrovirus infected cells, its antisense phosphodiester oligodeoxynucleotide function appears.

DNA, Antisense↗
PubMed1998

Allogeneic peripheral stem cell transplantation using positively selected CD34+ cells from HLA-mismatched donors.

We examined five children who underwent allogeneic peripheral stem cell transplantation (PSCT) using positively selected CD34+ cells from three or two loci-mismatched donors. CD34+ cells mobilized from peripheral blood were separated by immunomagnetic beads. CD34+ cells at 2.2-6.2 x 10(6)/kg were transplanted into three patients with refractory leukemia, a patient with relapsed medulloblastoma and a patient with Fanconi's anemia following a conditioning regimen which included irradiation, alkylating agents and antithymocyte globulin treatment. The number of infused CD3+ cells included in grafts was 2.3-22.7 x 10(4)/kg. Four patients achieved engraftment and hematopoietic reconstitution (> 5 x 10(8)/l of neutrophils on day 10 or 11). Graft rejection was observed in the patient with Fanconi's anemia, but a rapid engraftment was obtained after second PSCT. Although no prophylactic agents other than ATG (included in the conditioning regimen) were used, greater than grade I acute GVHD was not observed, but limited chronic GVHD was observed in two patients. The two patients with leukemia relapsed on days 103 and 210, respectively, and the patient with medulloblastoma died of disease on day 159. The patient with Fanconi's anemia died of fungal infection. CMV and HHV-6 diseases developed in four and two patients, respectively. Thus, although SCT using positively selected peripheral CD34+ cells may be an alternative approach for overcoming graft rejection and GVHD from HLA- mismatched donors, persistent immune deficiency attributing to extremely low numbers of T cells in grafts can potentially lead to reactivation of herpes viruses.

Adolescent↗
PubMed1998

Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors.

Major dose-limiting factors of high-dose thiotepa (TEPA) and melphalan are life-threatening mucositis and neurotoxicity. To administer a maximum dose of these drugs safely and to obtain a maximum anti-cancer effect, a double-conditioning regimen with a single grafting, two cycles of administration of a combination of TEPA (300-600 mg/m2) plus melphalan (70-150 mg/m2) with a 1-week interval was attempted in 20 patients with pediatric advanced or chemotherapy-resistant solid tumors (seven rhabdomyosarcoma, four hepatoblastoma, three neuroblastoma and four other malignancy). Combinations of TEPA plus melphalan/busulfan (Bu) (8-10 mg/kg) and TEPA plus Bu were given to four and two patients with brain tumors, respectively. In an additional two patients, three cycles of drug administration were performed. According to the results of the dose-escalating study, the maximum tolerable doses of TEPA and melphalan for children aged 2 years old or older were 1000 mg/m2 and 280 mg/m2, respectively. Mucositis was dose-limiting. Renal toxicity was also dose-limiting in young children (<2 years old). There were two treatment-related deaths (7%) (fungal pneumonia and renal tubular acidosis). Among 13 patients who received high-dose chemotherapy during CR, 10 are alive with no evidence of disease (15-59 months, median: 35 months) and in 13 evaluable patients without CR, six are alive without regrowth of the disease (14-59 months, median: 39 months). Thus, these novel conditioning regimens allowed us to increase the dose intensity to nearly the maximum for each drug and seemed to reduce adverse effects compared to previously reported regimens with these drugs. With regard to the effect on outcome, the results of this study seem to be encouraging, but a further study on a larger number of patients is required.

Adolescent↗
PubMed1998

Acquired thermotolerance and temperature-induced protein accumulation in the extremely thermophilic bacterium Rhodothermus obamensis.

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95 degrees C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.

Amino Acid Sequence↗
PubMed1998

Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by liposomally encapsulated antisense phosphorothioate oligonucleotides in MDCK cells.

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza A virus replication in MDCK cells. Liposomally encapsulated and free antisense S-ODNs with four target sites (PB1, PB2, PA and NP genes) were tested for their abilities to inhibit virus-induced cytopathogenic effects in a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the site around the PB2 AUG initiation codon showed highly inhibitory effects. In contrast, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with that directed to the PB2 target site. The liposomally encapsulated antisense S-ODNs exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas free antisense S-ODNs were observed to inhibit viral adsorption to MDCK cells. Liposomal preparations of oligonucleotides facilitated their release from endocytic vesicles, and thus cytoplasmic and nuclear localization was observed. The activities of the antisense S-ODNs were effectively enhanced by using the liposomal carrier. Interestingly, the liposomally encapsulated FITC-S-ODN-PB2-as accumulated in the nuclear region of MDCK cells. However, weak fluorescence was observed within the endosomes and the cytoplasm of MDCK cells treated with the free antisense S-ODNs. The cationic lipid particles may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in vitro or in vivo.

Antiviral Agents↗