Biomedical subjects

K Takai

Publications and source records attributed to K Takai.

At least 127 records · Page 7Linked to original sources

PubMed1997

Codon reading properties of tRNA variants substituted within the anticodon loop.

Effects of base substitution within tRNA anticodon loop on the codon reading activities were quantitatively analyzed with the use of a set of unmodified tRNA molecules with GGA anticodon. The first (position 32) and the last (position 38) nucleotides of the anticodon loop of the wild-type molecule was changed from C32A38 to U32A38, U32G38, and C32G38. The codon reading activities of these variants relative to that of the wild type molecule were measured in a cell-free translation system. The reading of both the UCU and UCC codons were lower in all the three variants than in the wild-type molecule.

Anticodon↗

PubMed1997

Codon recognition by artificial tRNA molecules with modified nucleosides in the anticodon.

Proteins with unnatural amino acids at specific positions can be produced through cell-free protein synthesis. The synthesis of such molecules can, in principle, be facilitated by improving the codon reading efficiency of the tRNA that inserts the unnatural amino acid. In the present study, we prepared tRNA molecules with 2'-O-methyl nucleosides at the second and third positions of the anticodon and measured their codon-reading efficiencies. The results indicated, contrary to our expectation, that the modification damaged the decoding function completely.

Base Composition↗

PubMed1997

Study on site specific cleavage of RNA.

The precursor of an RNA molecule from bacteriophage T4 infected Escherichia coli cell (p2Sp1 RNA) has the ability to cleave itself. It has been found that the site specific RNA cleavage reaction occurred at the pyridine-adenosine sequence in the presence of a monovalent cation and a non-ionic detergent. In order to investigate the mechanism of this cleavage reaction, we designed a RNA oligonucleotide (UUUAUU) and this RNA was cleavage activity at the U-A sequence.

Ammonium Chloride↗

PubMed1997

Synthesis and anti-influenza virus-A activity of circular dumbbell RNA DNA chimeric oligonucleotides.

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of circular dumbbell DNA/RNA chimeric oligonucleotides.

Antiviral Agents↗

PubMed1997

Inhibition of HIV-1 replication by foldback triple-helix forming oligonucleotides.

Replication of retroviral RNA into double-stranded DNA is catalyzed by reverse transcriptase (RT). The polypurine tract (PPT) serves as a primer for plus-strand DNA synthesis and is highly conserved among HIV-1. The PPT region is a possible target for triple-helix formation. Here, we show the effects of triple-helix formation by analyses of melting temperature and gel shift using a foldback triplex-forming-oligonucleotides (FTFOs). We found that the FTFOs containing phosphorothioate groups at the 3'- and 5'-ends, or inside the hairpin loop, exhibited greater exonuclease resistance than the unmodified FTFOs. Several triplex oligonucleotides have thermal stability. The abilities of the FTFOs (DsDG-37) containing the guanosine in place of the cytidine in the third Hoogsteen base-pairing strand to inhibit HIV-1 replications were examined. The FTFOs (DsDG-37) inhibit the replication of HIV-1 more efficiently than the FTFOs (DsD-37) indicating sequence-specific inhibition of HIV-1 replication.

Anti-HIV Agents↗

PubMed1997

Removing tRNA from a cell-free protein synthesis system for use in protein production.

The cell-free system for biosynthesis of proteins is becoming an important tool for protein engineering. In particular, introduction of the unnatural amino acids is achieved though cell-free protein synthesis with the use of chemically acylated tRNA that recognizes a specific codon. In the original method, however, it was difficult to control the system through changing tRNA composition, as the endogenous tRNAs are involved in the reaction. Thus, in the present study, we digested the tRNA within Escherichia coli S30 extract with resin-bound RNase A, and estimated the protein synthesis activity. It was revealed that this digestion process does not damage the activity, if a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), is present in the digestion reaction.

Cell-Free System↗

PubMed1996

Hairpin antisense oligonucleotides containing 2'-methoxynucleosides with base-pairing in the stem region at the 3'-end: penetration, localization, and Anti-HIV activity.

Hairpin antisense oligodeoxyribonucleotides containing 2'-methoxynucleosides were more active in the micromolar concentration range than linear and DNA hairpin phosphorothioate oligonucleotides with the same sequence. Furthermore, the abilities of hairpin antisense and random hairpin phosphorothioate oligonucleotides to inhibit HIV-1 replication were examined. Antisense oligonucleotides inhibit the replication and the expression of HIV-1 more efficiently than random-oligomers of the same length or with the same internucleotide modification. Four different target sites (gag, pol, rev, and tat) within the HIV-1 genome were studied with regard to the inhibition of HIV-1 replication by antisense oligonucleotides. Antisense oligomers complementary to the sites of the initiation sequences of gag were most effective. The [32P]-labeled hairpin phosphorothioate oligonucleotide was rapidly assimilated by MOLT-4 cells, whereas the [32P]-labeled hairpin phosphodiester oligonucleotide was not. In MOLT-4 cells treated with the FITC-hairpin phosphorothioate oligonucleotide containing 2'-methoxynucleosides by a confocal laser scanning microscope, diffuse fluorescence was observed in the cytoplasm. Interestingly, fluorescent signals accumulated in the nuclear region of chronically infected MOLT-4/HIV-1 cells after a 60 min incubation.

Antiviral Agents↗

PubMed1996

Purification and characterization of phosphoenolpyruvate carboxylase from the hyperthermophilic archaeon Methanothermus sociabilis.

Phosphoenolpyruvate carboxylase (PEPC) was purified for the first time from hyperthermophilic archaeon Methanothermus sociabilis, growing autotrophically with an optimum at 88 degrees C. The optimum temperature for enzyme activity was similar to that for growth and was 85 degrees C. The native enzyme was a homotetramer of 240 kDa molecular mass and the subunit displayed an apparent molecular mass of 60 kDa. The archaeal PEPC was insensitive to various metabolites which are known as allosteric effectors for most bacterial and eucaryal counterparts. The enzyme showed extreme thermostability such that there remained 80% of the enzyme activity after incubation for 2 h at 80 degrees C. These results implied that archaeal PEPC was significantly different from bacterial and eucaryal entities.

Archaea↗

PubMed1996

Hepatic extraction ratio of 5-fluorouracil in rats. Dose dependence and effect of uracil and interleukin-2.

The hepatic extraction ratios (EH) of 5-fluorouracil (FUra) in rats were studied to clarify the disposition of FUra in the liver. The EH of FUra in rats at infusion rates ranging from 0.375 to 3 mg/kg/min decreased from 0.750 to 0.225. The EH of values of tegafur, a pro-drug of FUra, were 0.076 to 0.103 over the range of infusion rates, 0.577 to 4.616 mg/kg/min, and were much lower than those of FUra. The EH of FUra at an infusion rate of 0.375 mg/kg/min combined with uracil (0.323 mg/kg/min) was 0.646, which was significantly lower than that of FUra alone, 0.750 (P < 0.001). The EH of FUra combined with interleukin-2 (IL-2) at an infusion rate of 7500 U/kg/min was significantly higher than that of FUra alone (P < 0.01). The dose dependence of the EH of FUra and the effects of uracil and IL-2 on the EH of FUra corresponded with clinical findings. These results suggest that this experimental model in rats may be useful for predicting the clinical pharmacokinetics and efficacy of FUra. We also studied the effect of IL-2 on the EH of mitomycin C (MMC). The EH of MMC combined with IL-2 was higher than that of MMC alone, but the difference was not significant.

Animals↗

PubMed1996

Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesis.

Unmodified tRNA molecules are useful for many purposes in cell-free protein biosynthesis, but there is little information about how the lack of tRNA post-transcriptional modifications affects the coding specificity for synonymous codons. In the present study, we prepared an unmodified form of Escherichia coli tRNA1Ser, which originally has the cmo5UGA anticodon (cmo5U = uridine 5-oxyacetic acid) and recognizes the UCU, UCA and UCG codons. The codon specificity of the unmodified tRNA was tested in a cell-free protein synthesis directed by designed mRNAs under competition conditions with the parent tRNA1Ser. It was found that the unmodified tRNA with the UGA anti-codon recognizes the UCA codon nearly as efficiently as the modified tRNA. The unmodified tRNA recognized the UCU codon with low, but detectable efficiency, whereas no recognition of the UCC and UCG codons was detected. Therefore, the absence of modifications makes this tRNA more specific to the UCA codon by remarkably reducing the efficiencies of wobble reading of other synonymous codons, without a significant decrease in the UCA reading efficiency.

Amino Acid Sequence↗

PubMed1996

Inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by unmodified, phosphorothioated, and liposomally encapsulated oligonucleotides.

We have demonstrated that antisense phosphodiester (ODNs) and phosphorothioate oligonucleotides (S-ODNs) inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line, which is a derivative of the murine C127 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to treatment with dexamethasone. Phosphodiester, phosphorothioate, and liposomally encapsulated oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The ODNs and S-ODNs complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites. Liposome encapsulation afforded oligomer protection in serum-containing medium and substantially improved cellular accumulation. The liposomal encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. The activities of the unmodified oligonucleotides are effectively enhanced by using the liposomal carrier.

Animals↗

PubMed1996

A dual computed tomography linear accelerator unit for stereotactic radiation therapy: a new approach without cranially fixated stereotactic frames.

PURPOSE: To perform stereotactic radiation therapy (SRT) without cranially fixated stereotactic frames, we developed a dual computed tomography (CT) linear accelerator (linac) treatment unit. METHODS AND MATERIALS: This unit is composed of a linac, CT, and motorized table. The linac and CT are set up at opposite ends of the table, which is suitable for both machines. The gantry axis of the linac is coaxial with that of the CT scanner. Thus, the center of the target detected with the CT can be matched easily with the gantry axis of the linac by rotating the table. Positioning is confirmed with the CT for each treatment session. Positioning and treatment errors with this unit were examined by phantom studies. Between August and December 1994, 8 patients with 11 lesions of primary or metastatic brain tumors received SRT with this unit. All lesions were treated with 24 Gy in three fractions to 30 Gy in 10 fractions to the 80% isodose line, with or without conventional external beam radiation therapy. RESULTS: Phantom studies revealed that treatment errors with this unit were within 1 mm after careful positioning. The position was easily maintained using two tiny metallic balls as vertical and horizontal marks. Motion of patients was negligible using a conventional heat-flexible head mold and dental impression. The overall time for a multiple noncoplanar arcs treatment for a single isocenter was less than 1 h on the initial treatment day and usually less than 20 min on subsequent days. Treatment was outpatient-based and well tolerated with no acute toxicities. Satisfactory responses have been documented. CONCLUSION: Using this treatment unit, multiple fractionated SRT is performed easily and precisely without cranially fixated stereotactic frames.

Brain Neoplasms↗

PubMed1996

5'-linked lipid-oligodeoxyridonucleotide derivatives as inhibitors of human immunodeficiency virus replication.

The covalent attachment of a phospholipid moiety, bound to the 5'-ends of phosphodiester and phosphorothioate oligonucleotides (L-ODNs and LS-ODNs), was achieved using H-phosphonate chemistry, and the lipid-oligonucleotides were assayed for the inhibition of virus replication in HIV-1 infected MT-4 cells. In the anti-HIV activity test, lipid-phosphorothioate oligonucleotides showed higher anti-HIV activities than non-lipid-phosphorothioate oligonucleotides, at the low concentration of 0.04 microM. LS-ODNs can inhibit HIV-1 reverse transcriptase activity through interactions with the enzyme. We found that the covalent attachment of a phospholipid group to the 5'-end of the phosphorothioate oligonucleotide enhances its nonsequence specific anti-HIV activity.

Base Sequence↗

PubMed1996

Properties of triple helix formation with oligodeoxyribonucleotides containing 8-oxo-2'-deoxyadenosine and 2'-modified nucleoside derivatives.

The ability of homopyrimidine oligonucleotides containing 8-oxo-2'-deoxyadenosine (dAOH), 2'-methoxyuridine (Um), 2'-fluorouridine (Uf), 2'-methoxycytidine (Cm), and 2'-fluorocytidine (Cf) to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2'-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. In particular, the substitutions of three deoxycytidine residues with 8-oxo-2'-deoxyadenosine showed higher endonuclease inhibition than the substitution of either one or two deoxycytidine residues with 8-oxo-2'-deoxyadenosine. In contrast, the oligonucleotides containing 2'-modified nucleosides (Uf, Um, Uf-Cf, Um-Cm, dAOH-Uf, and dAOH-Um) bind in a pH-dependent manner to the target duplex.

Binding Sites↗

PubMed1996

Nonenzymatic sequence-specific cleavage of duplex DNA via triple-helix formation by homopyrimidine phosphorothioate oligonucleotides.

Phenanthroline was attached covalently to the 5'-terminus of the unmodified and modified (3'-terminal phosphorothioate) oligonucleotide sequences, TTTTTTCTTCTCTTTCC (OP-17 mer) and TTTTTTTCTTCTCTTTCsC (OPRp-17 mer or OPSp-17 mer) via a phosphoramidite bond. Simian virus 40 DNA contains a single target site for these oligonucleotides. In the presence of copper ions, the efficient double-stranded cleavage at 37 degrees C and pH 7.0 was observed by agarose gel electrophoresis. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove, even though the linker was located in the major groove. Of particular interest are the 3'-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp), which were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3'-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes and antisense nucleic acid based anti-viral therapies, which could be used as tools in cellular biology.

Animals↗

PubMed1996

Rhodothermus obamensis sp. nov., a modern lineage of extremely thermophilic marine bacteria.

A novel extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent environment (depth, 22 m) in Tachibana Bay, Nagasaki Prefecture, Japan. The cells of this organism were gram-negative rods. Growth occurred at temperatures between 50 and 85 degrees C (optimum temperature, 80 degrees C; doubling time at optimum temperature, 90 min), at pH 5.5 and 9.0 (optimum pH, 7.0), and in the presence of 1 and 5% NaCl (optimum NaCl concentration, 3%). The new isolate was an aerobic heterotroph which utilized the following compounds as sole energy and carbon sources: yeast extract, peptone, starch, casein, Casamino Acids, a variety of sugars, some carboxylic acids, and amino acids. As determined by a sequence analysis of the 16S rRNA, the new isolate belongs to the genus Rhodothermus and represents a modern lineage of extreme thermophiles within the domain Bacteria. On the basis of the physiological and molecular properties of the new isolate, we describe a new species, Rhodothermus obamensis. The type strain of R. obamensis is strain OKD7 (= JCM 9785).

Base Sequence↗

PubMed1996

An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter.

The plc gene, which encodes phospholipase C (alpha-toxin) of Clostridium perfringens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter. The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNA-containing sequence, depending on its proper linear and rotational orientation. The in vitro transcriptional activity of the plc gene was also stimulated by the upstream sequence. In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively. A similar temperature effect was also observed with the chromosomal plc gene. These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.

Base Sequence↗

PubMed1996

A case of traumatic neuroma of the gallbladder in the absence of previous surgery and cholelithiasis.

We experienced a patient with traumatic neuroma of the gallbladder with no history of gallbladder surgery or cholelithiasis. A 74-year-old man was referred to our department after a gallbladder tumor was incidentally discovered during a preoperative screening examination for prostate hypertrophy. Ultrasonography, MRI, CT and endoscopic retrograde cholangiography revealed a protuberant lesion of the gallbladder. Laparoscopic cholecystectomy was attempted but adhesion between the liver and duodenum forced us to convert to open laparotomy. Cholecystectomy and adjacent liver tissue resection was performed. Diagnosis was made by frozen histology during operation. It revealed no malignancy. Postoperative pathological examination revealed traumatic neuroma associated with inflammation. To our knowledge, this is the first reported case of gallbladder neuroma without a history of gallstones or surgery in the English and Japanese literature since 1980. This traumatic neuroma should be considered in a differential diagnosis in treating gallbladder neoplasm, even in the absence of an operative history or cholelithiasis.

Aged↗