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Biomedical subjects

K Taira

Publications and source records attributed to K Taira.

At least 181 records · Page 10Linked to original sources
PubMed1995

In vitro selection of RNA aptamers that can bind specifically to Tat protein of HIV-1.

The Tat protein of human immunodeficiency virus interacts specifically with its target RNA sequence, TAR, and activates viral gene expression at the early stage of infection. Here, we have used in vitro genetic selection strategy to determine what sequence or structural motifs might exist between RNA's that interact specifically with the Tat protein. Starting from a RNA pool that has a 120 base random sequence core, aptamers that bind specifically to the Tat protein were selected by repeating 11 rounds of selection and amplification. A comparative analysis of 64 aptamers that were isolated from the 11th generation revealed two main sequence classes. Interestingly, one of these two classes of aptamers had minimum of one U residues in bulge loop and 2 specific adjacent base pairs. This region is very much homologous to the core sequence of TAR RNA that is essential for the specific Tat-TAR interactions. Further analyses of the sequences from the 11th generation should reveal what kind of RNA structures are required in order to show a high affinity for the Tat protein.

Base Sequence↗
PubMed1995

Magnesium is essential for formation of an active complex of a hammerhead ribozyme with its substrate: an investigation by NMR spectroscopy.

The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high-resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson Crick three-base-paired duplex and a four-base-paired duplex that included a wobble base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate-recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex.

Base Sequence↗
PubMed1995

A novel screening system for construction and selection of active ribozymes using DHFR (dihydrofolate reductase) gene as selection marker.

Ribozyme is thought to be a powerful tool for suppression of specific gene expression, but there are many failure cases in their applications in vivo. One reason may be that there are many cellular proteins in vivo which may inhibit the catalytic activity of ribozymes. Such being the case, many researchers have made efforts to increase the activity of ribozymes. Here we have developed a new screening system for active ribozymes using DHFR (dihydrofolate reductase) gene as a selection marker. In our system, we set the ribozyme target site not to the DHFR gene itself but to the interspace between two ATG codons. One of which is the original initiation codon, ATG, for DHFR gene itself, and the second one locates upstream of the former. The upstream ATG has an ability to cause frame shift for DHFR gene. Since the upstream ATG is preferentially used during translation, unless it is removed, production of DHFR is negligible. By setting the ribozyme target site between the two ATGs, we can make this screening system as positive selection. To our best knowledge, this is the first successful screening system in vivo.

Base Sequence↗
PubMed1995

The synthesis and biological activities of oligodeoxynucleotides that are covalently linked to psoralen at their 5' ends.

Four oligodeoxynucleotides (15-mers), designated 13 through 16, and the corresponding conjugates, designated 17 through 20, in which psoralen was covalently linked to the 5' end of each oligonucleotide were synthesized. Compounds 16 and 20 contained a sequence that was complementary to part of the first four codons and the upstream sequence close to the ribosome-binding site of c-Ha-ras mRNA. Compounds 16 and 20 inhibited the growth of cells that had been transformed by the c-Ha-ras plasmid, with accompanying suppression of the expression of the activated c-Ha-ras oncogene. The antisense oligonucleotides 16 and 20 also appeared to cause partial reversion of the major phenotypic characteristics of transformed cells, which included inhibition of anchorage-independent growth. Compound 20, which contained psoralen, was a more efficient inhibitor of the growth of transformed cells than compound 16 without psoralen, a result that suggests psoralen might have increased the ability of the oligodeoxynucleotides to enter the cells.

Amino Acid Sequence↗
PubMed1994

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.

Base Sequence↗
PubMed1994

Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis.

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTP alpha S) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original self-cleavage activity whereas incorporation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme activity, and they are located at positions 0, 1, 4, 5, 21, 24, 25, 27, 28, 30-34, 40, 43 and 75. In particular, the pro-Rp oxygens at positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme.

Base Sequence↗
PubMed1994

A hammerhead ribozyme inhibits the proliferation of an RNA coliphage SP in Escherichia coli.

Ribozymes are potentially powerful tools for the suppression of intracellular gene expression. However, the few reports that exist of their activities in bacteria have described mixed success. Chuat and Galibert (Chuat, J.-C., and Galibert, F. (1989) Biochem. Biophys. Res. Commun. 162, 1025-1029) failed to detect any trans-activities of hammerhead ribozymes in Escherichia coli, while Sioud and Drlica (Sioud, M., and Drlica, K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7303-7307) reported complete inhibition of expression of the gene for a nonbacterial protein, HIV-1 integrase, by trans-acting hammerhead ribozymes in E. coli. It is of interest to determine whether ribozymes can really be used in natural bacterial systems (Altman, S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10898-10900). We now report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmid in E. coli caused failure of the proliferation of progeny phage. Inactive ribozymes with altered catalytic sequences did not affect phage growth. These results indicate that it is mainly the catalytic activity of the ribozyme and not its function as an antisense molecule that is responsible for suppressing the proliferation of the RNA phage. Moreover, an analysis based on numbers of plaque-forming units and the function of the A2 protein indicated that antisense RNA may successfully compete with ribosomes in targeting mRNA while ribozymes in this study may not compete with ribosomes in naturally occurring bacterial transcription/translation-coupled systems.

Base Sequence↗
PubMed1994

Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis.

We have investigated in detail the higher order structure of the genomic hepatitis delta virus (HDV) ribozyme using various base-specific chemical probes under native, semi-denaturing, and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethyl sulfate [which reacts with A (at N1) and C (at N3)] and a carbodiimide [which reacts with U (at N3) and G (at N1)]. In addition, for probing G residues (at N7), RNA samples were treated with NaBH4 and aniline after modification by treatment with dimethyl sulfate. The sites of modified positions were identified by primer extension analysis with reverse transcriptase. In general, our results are consistent with the proposed pseudoknot model of secondary structure, a model that is based on data from ribonucleolytic cleavage experiments. Our results provide clues to the identification of interacting bases in the HDV ribozyme. Furthermore, using this method we identified local conformational changes in several stem variants.

Aniline Compounds↗
PubMed1994

[Clinical investigation of obstructive pneumonia with lung cancer].

Nineteen episodes (18 patients) of obstructive pneumonia occurring in 247 patients with lung cancer were clinically examined, and the following findings were obtained. 1) All 18 patients were male smokers, with an average of 64 years. 2) Body temperatures stood above 37 degrees C in 95% of the 19 episodes. Leukocytosis was detected in 41%, and neutrophilia was observed in 69%. CRP values were positive in all episodes. 3) The frequency of obstructive pneumonia according to the site of lung cancer was 13.2% in proximal tumors and 1.7% in peripheral tumors. Pathologically, the frequency was 5.2% in adenocarcinomas 7.6% in squamous cell carcinomas, 15.6% in small cell carcinomas and 9.1% in large cell carcinomas. Squamous cell carcinomas even in Stage I led to obstructive pneumonia at the rate of 13.0%. In contrast, higher frequency was observed in adenocarcinomas in Stage II or later and small cell carcinomas in Stage IIIB or later. 4) Obstructive pneumonia was detected at the time of discovery of lung cancer in 48% of the 18 patients, and during treatment and in the terminal stage in 26% respectively. 5) Chest X-ray examinations showed that 42% of the pneumonia cases were lobar pneumonia and 11% had pleural effusion. 6) In expectorated sputum, 50% of bacteria isolated were gram-negative, 10% were gram-positive and 40% were unknown. 7) Most of the bacteria isolated from expectorated sputum showed a good susceptibility to antimicrobial agents, but those proved clinically ineffective. 8) The prognosis of obstructive pneumonia was significantly affected by performance status and levels of total protein and choline-esterase.

Aged↗
PubMed1994

Trabecular meshworks in the sinusoidal endothelial cells of the golden hamster liver: a freeze-fracture study.

Trabecular meshworks (TMs) in the sinusoidal endothelial cells of fasted-refed golden hamster liver were studied in thin sections and in freeze-fracture replicas. TM was a plasmalemma-attached reticulum of anastomosing trabeculae composed of the plasmalemma and cytosol. The surface of TM appeared as a sieve on the plasmalemma. The floor of TM appeared as a depression with snapped trabeculae on the P-face plasmalemma and as a mound, or inverted depression, with pits containing snapped trabeculae on the E-face plasmalemma. Faint ridges were seen connecting some trabeculae on the P-face plasmalemma, whereas furrows lined with intramembranous particles (IMPs) were seen spanning some trabeculae on the E-face plasmalemma. Circles of lined IMPs (IMP-circles) were seen on the E-face plasmalemma, either alone or in association with TMs. Plasmalemmal invaginations were often centered in IMP-circles and were sometimes seen in a cluster. TMs were often blended with the sieve plates.

Animals↗
PubMed1994

[Clinical effect of the combined therapy of arbekacin and imipenem/cilastatin against methicillin-resistant Staphylococcus aureus].

We evaluated the efficacy of combined therapy of arbekacin (ABK) and imipenem/cilastatin (IPM/CS) against infections by methicillin-resistant Staphylococcus aureus (MRSA). The MICs of ampicillin, cefmetazole, cefotiam, cefuzonam, flomoxef, fosfomycin, ofloxacin, minocycline, ABK and IPM/CS against clinically isolated strains of MRSA were examined. Almost all strains of MRSA were resistant to these antibiotics except ABK. Furthermore, combination of ABK and IPM/CS showed smaller MICs than that of ABK or IPM/CS alone. All fractional inhibitory concentration indices (FIC indices) of ABK plus IPM/CS were lower than 0.75. The efficacy rate of combined therapy of ABK and IPM/CS in 22 patients with MRSA infections (15 patients with pneumonia, 3 patients with chronic bronchitis, 2 patients with sepsis, a patient with subcutaneous abscess and a patient with DPB) was 68%. And no patients had adverse reactions. Six (27%) of 22 strains of MRSA were eradicated. Significant correlations were found between bacteriological effect and severity of disease, and between serum albumin level and clinical effect.

Aged↗
PubMed1994

[A case of endometrial tuberculosis diagnosed by polymerase chain reaction].

A 30-year-old female was admitted to our hospital for further investigation of infertility. The menstrual culture and polymerase chain reaction (PCR) of the endometrial tissue for detection of Mycobacterium tuberculosis were both positive. The pelvic CT scan and hysterosalpingography showed a slightly expanded uterus and irregularity of the endometrium. Barium enema and pyelography showed no abnormality. Since these data established a diagnosis of early primary endometrial tuberculosis, the combined therapy of three antituberculous agents was commenced. Menstrual smear, culture and PCR for M. tuberculosis were examined monthly throughout the therapy. The mycobacterial culture became negative soon after the start of therapy, followed by a negative PCR result 3 months later. We conclude that PCR is useful for the rapid detection of M. tuberculosis not only in pulmonary but also in endometrial tuberculosis.

Adult↗
PubMed1993

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.

Base Sequence↗
PubMed1993

High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Its substrate domain forms a double helical stem and a 17-base loop that includes the sequence GAGA, the second adenosine of which corresponds to A4324. Recently, studies of mutant RNAs have shown that the four-nucleotide loop, GAGA, can function as a substrate for ricin. To investigate the structure that is recognized by ricin, we studied the properties of a short synthetic substrate, the dodecaribonucleotide r-CUCAGAGAUGAG, which forms a RNA hairpin structure with a GABA loop and a stem of four base pairs. The results of NMR spectroscopy allowed us to construct the solution structure of this oligonucleotide by restrained molecular-dynamic calculations. We found that the stem region exists as an A-form duplex. 5G and 8A in the loop region form an unusual G:A base pair, and the phosphodiester backbone has a turn between 5G and 6A. This turn seems to help ricin to gain access to 6A which is the only site of depurination in the entire structure. The overall structure of the GAGA loop is similar to those of the GAAA and GCAA loops that have been described but that are not recognized by ricin. Therefore, in addition to the adenosine at the depurination site, the neighboring guanosine on the 3' side (7G) may also play a role in the recognition mechanism together with 5G and 8A.

Base Composition↗
PubMed1993

Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus.

The kinetic behavior of ribozymes derived from two types of multiple-ribozyme expression vector were examined. In some cases, multiple ribozymes were expressed as a single RNA molecule and all the ribozymes were simply connected in tandem (connected type). In other cases, multiple ribozymes were flanked by cis-acting ribozymes at both their 5' and 3' ends so that, upon transcription, multiple ribozymes were trimmed at both their 5' and 3' ends, with resultant liberation of multiple independent ribozymes (shotgun type). When levels of ribozyme expression were examined for the shotgun-type vector, the level of the ribozyme transcript was found to be proportional to the number of units (n) connected in tandem. Accordingly, the activities of the shotgun-type ribozymes, in terms of the cleavage of HIV-1 RNA in vitro, were also found to be proportional to the number of units connected in tandem (n). By contrast, the activities of the connected-type ribozymes reached plateau values at around n = 3. These results indicate that, when the shotgun-type expression system is used, it is theoretically possible to generate various independent ribozymes, each specific for a different target site, without sacrificing the activity of any individual ribozyme.

Antiviral Agents↗