Protective effects of lidocaine in hepatic ischemia/reperfusion injury in vitro.
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Animals↗
Biomedical subjects
K Taira
Publications and source records attributed to K Taira.
A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo.
We have constructed an allosterically controllable novel enzyme (designated maxizyme) that can be transcribed in vivo under the control of a human tRNA(Val) promoter. The maxizyme has sensor arms that can recognize target sequences, and in the presence of such a target sequence only, it can form a cavity that can capture catalytically indispensable Mg2+ ions. As a target for a demonstration of the potential utility of the maxizyme, we chose BCR-ABL mRNA, the translated products of which cause chronic myelogenous leukemia. Only the maxizyme (but not conventional ribozymes) had extremely high specificity and high-level activity, not only in vitro but also in cultured cells including BV173 cells derived from a patient with a Philadelphia chromosome. The maxizyme induced apoptosis only in leukemic cells with this chromosome.
Allosteric Regulation↗
Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.
A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.
Base Sequence↗
Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli.
If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our first attempt, a sequence encoding either an active or an inactive ribozyme was connected upstream of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, there is the danger of selecting sequences that are not related to the activity of ribozymes. Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the resistance of Escherichia coli to TMP. Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected and 3'-connected ribozymes were compared. We found that the cleavage efficiencies in vivo were nearly identical for the two types of ribozyme, 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indicating that polysomes did not seem to inhibit the action of the 3'-connected ribozyme. In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP.
Base Sequence↗
Prevalence of use of medically prescribed hypnotics among adult Japanese women in urban residential areas.
Based on a population survey on insomnia among 3600 adult Japanese women living in urban areas, the prevalence of use of medically prescribed hypnotics is determined. The prevalence of use of medically prescribed hypnotics increases with an increase in age (<1.0% for those aged 49 or younger, while 14.3% for those aged 70 or older), in agreement with the results reported in many Western nations. The current sleep disturbance is mild in nearly half of these hypnotics users. More than one-third of the hypnotic users are receiving health care not for sleep problems but for depression, anxiety, or other reasons. More than one-third of the hypnotic users are found to be receiving hypnotics from non-psychiatrists. The percentage of this group is particularly high among those aged 60 or over, probably reflecting the fact that they are often consulting physicians for physical reasons. On the other hand, more than 80% of insomniacs are suggested to be untreated. Future public health research should focus on the quality of life and health care behaviors of untreated insomniacs and hypnotic users, especially among the elderly people, in order to assess the need for primary health care to prevent accidents, mortality, and psychiatric disorders related to sleep problems.
Adult↗
Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5'-oxy and 5'-thio substrates by a hammerhead ribozyme.
In a previous examination using natural all-RNA substrates that contained either a 5'-oxy or 5'-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2'-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5'-thio group (R11S) and (ii) the departure of the 5' leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5'-oxygen, because a switch in metal ion specificity was not observed with the 5'-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.
Animals↗
Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA.
Hepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategy in vitro to isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6-16 and G6-19 RNA, were found to inhibit NS3 in vitro. Kinetic studies of the inhibition revealed that the aptamer G6-16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 microM. We also analyzed aptamers G6-16 and G6-19 for their action with a longer protein substrate (amino acid region 2203-2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6-16 and G6-19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads.
Base Sequence↗
Inhibition of transcription by the TAR RNA of HIV-1 in a nuclear extract of HeLa cells.
Regulation of transcription of human immunodeficiency virus type-1 (HIV-1) requires specific interaction of Tat protein with the trans-activation response region (TAR). Inhibition of replication of HIV-1 has previously been achieved with a TAR decoy, namely a short RNA oligonucleotide that corresponded to the sequence of the authentic TAR RNA. Since TAR RNA has the potential to interact with cellular factors, we examined the effect of TAR RNA on efficiency of transcription in nuclear of HeLa cell extracts. We performed an in vitro transcription assay in the presence of authentic TAR RNA using a template that was driven by the CMV (cytomegalovirus) early promoter in a HeLa nuclear extract and found, for the first time, that TAR RNA inhibited transcription by approximately 60-70% independently of the Tat-TAR interaction. Furthermore, we evaluated inhibition of transcription by variants of TAR RNA and found that the TAR RNA loop, bases surrounding the loop, the triple base bulge and the 'lower' stem region of TAR RNA were responsible for the inhibition of transcription. Taken together, earlier reports on proteins that bind to TAR RNA and the present results suggest that integrity of TAR RNA is important for efficient binding to cellular transcription factors. As judged from the significant inhibition observed in this study, the TAR decoy might sequester transcription factors and thus it might potentially be able to inhibit transcription of housekeeping genes that are unrelated to Tat function.
Base Sequence↗
Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.
With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo , especially for the treatment of CML, if we can develop exogenous delivery strategies.
Base Sequence↗
Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli.
For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.
Base Sequence↗
Effect of taurine on angiotensin II-induced hypertrophy of neonatal rat cardiac cells.
The effect of taurine on angiotensin II-induced hypertrophy of cultured neonatal rat heart cells (myocytes and nonmyocytes) was examined. Angiotensin II (1-100 nM) alone caused an increase in the rate of protein synthesis of myocytes without changing the rate of DNA synthesis and cell number. It mediated increases in DNA synthesis and cell number in nonmyocytes. Furthermore, at the lower concentration of 1 nM, it induced c-fos and c-jun expression in both cultured myocytes and nonmyocytes. Exposure of the cells to taurine (20 mM) in the absence of angiotensin II had no effect on either hyperplastic growth or immediate early response gene expression by the two types of cultured cardiac cells. However, myocytes pretreated for 24 h with 20 mM taurine exhibited reduced responsiveness to angiotensin II (1 nM), resulting in lower levels of angiotensin II-mediated stimulation in protein synthesis, and immediate early response gene expression was attenuated. Similarly, taurine treatment of nonmyocytes reduced the degree of hyperplastic growth (DNA synthesis and cell number) and immediate early response gene expression stimulated by angiotensin II. Finally, taurine partially prevented the increase in intracellular free calcium [Ca2+]i mediated by angiotensin II in cardiac cells. Our results indicate that taurine is an effective inhibitor of angiotensin II action.
Angiotensin II↗
Analysis of giant cell tumor of bone with pulmonary metastases.
The authors reviewed 6 cases of giant cell tumor of bone with pulmonary metastases, analyzed the growth rate of the metastases, and performed flow cytometry on paraffin blocks from primary and metastatic lesions. Surgery on the pulmonary metastases was done in 3 cases. Chemotherapy was administered in all 6 cases. If the doubling time of the pulmonary metastases was more than 80 days, the case was considered to be slow growing or in regression. However, if the doubling time was approximately 30 days, the prognosis was poor. The cases were divided into 3 types according to the prognosis. After chemotherapy (cyclophosphamide), the pulmonary metastases decreased in size in 2 cases. In 2 other cases, the metastases were slow growing, and the remaining 2 patients died early. Flow cytometric analysis was performed on primary and metastatic lesions, which showed a diploid pattern in most cases, but a tetraploid pattern in 1 recurrent case. Pulmonary metastatic lesions displayed a diploid pattern in 4 cases from which materials were taken either at surgery or autopsy.
Adolescent↗
A population study on risk factors for insomnia among adult Japanese women: a possible effect of road traffic volume.
In an effort to identify risk factors for insomnia and determine the contribution of nightime road traffic volume to insomnia in the general population, a questionnaire survey was carried out among 3,600 adult Japanese women living in eight urban residential areas. The crude prevalence rate of insomnia was 11.2%. Multivariate analysis revealed that aging, living with a child/children aged six or younger, undergoing medical treatment, experiencing major life events, having an irregular bedtime, having a sleep apnealike symptom, and living near a road with a heavy volume of traffic are risk factors for insomnia. Taking into account other risk factors, there was a level-response relationship between the nighttime traffic volume of main roads and the risk of insomnia in the subjects living in the zones 0-20 m from these roads. These results suggest that road traffic noise raises the sound level in bedrooms in such zones, and consequently the prevalence rate of insomnia among the residents, and that noise-induced insomnia is an important public health problem, at least in highly urbanized areas. To confirm this, a further study on noise exposure is needed.
Adult↗
Structural analysis of nucleic acids by using fluorescence resonance energy transfer (FRET).
We examined changes in the extent of fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In our system, FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. While, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. It should help us to estimate the suitability of designed functional molecules, such as antisense DNA and RNA and ribozymes, that target to specific sites.
DNA↗
Comparison of activities between hammerhead ribozymes and DNA enzymes targeted to L6 BCR-ABL chimeric (b2a2) mRNA.
For the treatment of chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using DNA enzymes, that was newly selected by Santoro & Joyce and that can cleave RNA molecules with high activity, to cleave of L6 BCR-ABL fusion (b2a2) mRNA. By contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction, without any cleavage of the normal ABL or BCR mRNA.
Base Sequence↗
Activities of tRNA-embedded dimeric minizymes.
Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.
Base Sequence↗
Constructing an efficient trans-acting genomic HDV ribozyme.
We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).
Base Sequence↗
Catalytic activities of hammerhead ribozymes with a triterpenoid linker instead of stem/loop II.
A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem/loop II. In a previous study we demonstrated that a minizyme with high-level activity forms a dimeric structure with a common stem II (Amontov and Taira, J. Am. Chem. Soc., 118 (1996) 1624-1628). As a continuation of this study, we recently examined whether a short oligonucleotide linker at stem/loop II could be replaced by a triterpenoid linker and whether the resulting minizymes with bulky hydrophobic groups would form dimeric structures. In contrast to the minizyme with high-level activity that we characterized in the previous study, minizymes that contained a triterpenoid group had low cleavage activities. The nature of the dependence of the activity on the concentration of ribozyme revealed that these minizymes with a triterpenoid group do not form dimeric structures. Thus, the low activities of these structures can be attributed to their failure to form dimers.
Base Sequence↗