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Biomedical subjects

K Taira

Publications and source records attributed to K Taira.

At least 163 records · Page 9Linked to original sources
PubMed1996

Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.

Antisense Elements (Genetics)↗
PubMed1996

Characterization of several kinds of dimer minizyme: simultaneous cleavage at two sites in HIV-1 tat mRNA by dimer minizymes.

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 activity forms a dimeric structure with a common stem II. Because of their dimeric structure, minizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In order to examine the properties of different kinds of minizyme, we constructed a number of minizymes with short oligonucleotide linkers (2-5 bases) instead of stem-loop II and examined their cleavage activities against HIV-1 tat mRNA. Analyses of melting curves, as well as Arrhenius plots, revealed that, in general, the longer the oligonucleotide linkers, the more stable and more active were the dimer minizymes. All minizymes examined cleaved the target substrate at two sites simultaneously. The activity of the dimer minizyme with a 5 nt linker was higher than that of the parental hammerhead ribozyme because the latter full-sized ribozyme was able to cleave at one site only.

Base Sequence↗
PubMed1996

Dependence on Mg2+ ions of the activities of dimeric hammerhead minizymes.

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 (1996) J. Am. Chem. Soc. 118, 1624-1628]. We now demonstrate that the stability of the dimeric structure is influenced by Mg2+ ions. We found that the dependence on Mg2+ ions of the activity of homodimeric minizyme (a dimer with two identical binding sites) has composite biphasic characteristics. When the concentration of Mg2+ ions reached a specific critical level, the dependence on the concentration of Mg2+ ions lost its tendency to reach a plateau. In the case of the heterodimeric minizyme (a dimer with two different binding sites), we investigated the kinetic behavior of two different forms of the dimer, namely, free dimer and the complex of the dimer with an uncleavable substrate. The kinetic behavior of the free heterodimer was very similar to that of the homodimeric minizyme. In contrast, in the presence of the uncleavable substrate at a concentration as high as that of the minizyme, the curve for the dependence on Mg2+ ions showed normal saturation kinetics. While, at low concentrations of Mg2+ ions, the activity of the heterodimers was much higher when the dimeric structure was stabilized by the presence of the uncleavable substrate, at high concentrations of Mg2+ ions, this difference in activity became less and less significant. Thus, high concentrations of Mg2+ ions were able to stabilize the dimeric minizymes in the absence of the uncleavable substrate.

Base Sequence↗
PubMed1996

Magnesium-mediated conversion of an inactive form of a hammerhead ribozyme to an active complex 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 3-base pair duplex and a 4-base pair duplex that included a wobble G:U 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. Elevated concentrations (1.0 M) of monovalent Na+ ions could not induce the formation of the R32-RdC11 complex. 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 Composition↗
PubMed1996

Detection of undegraded oligonucleotides in vivo by fluorescence resonance energy transfer. Nuclease activities in living sea urchin eggs.

A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer between two different fluorochromes attached to a single oligonucleotide. Ten-mer oligodeoxyribonucleotides labeled with fluorescein at one end and with rhodamine X at the other end were used. The oligomer had a specific absorption spectrum with peaks at 497 and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, the oligomer had a specific fluorescence spectrum with peaks at 523 and 610 nm. The fluorescence intensity at 610 nm was 6-8 times higher than that at 523 nm. After digestion of the oligomer with an endonuclease, the fluorescence at 523 nm increased more than 12-15-fold but its fluorescence peak at 610 nm almost completely disappeared. To examine effects in vivo, sea urchin eggs were injected with a solution of the oligomer and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. The ratio of the intensities of red to green fluorescence decreased in dependence on time after injection of the oligomer. These changes were not observed in eggs that had been injected with a solution of similarly double-labeled, phosphorothioate oligomer. These results indicated that unfertilized sea urchin eggs had nucleolytic activity. Analysis in vitro on supernatant of the egg homogenate indeed demonstrated the existence of nucleases. All together, our results indicate that the integrity of oligonucleotides can be estimated in living cells by monitoring the fluorescence resonance energy transfer of the double-labeled oligonucleotide.

Animals↗
PubMed1996

Receiving grooming as a reinforcer for the monkey.

The present study was intended to evaluate whether receiving grooming, given to a monkey by an experimenter, can be used as a positive reinforcer in operant conditioning. When the monkey touched the surface of the correct pattern in a visual discrimination task after a tone cue, the experimenter groomed the monkey's face, neck, and head with his hand. To test whether the discrimination behavior depended on the shape of the stimuli or on the position of the pattern, these experimental parameters were changed in the different tasks. When the square pattern was assigned as correct and presented on the animal's left side, the average score for correct discrimination was 90% in the last 10 sessions out of 30 sessions, and this was statistically significant at a confidence level of p < 0.005 (Grant's table). Correct discrimination was statistically significant when the position of the square was randomly changed to the right and left side of the monkey, and also when the correct pattern was reversed from the square to the cross and its position was again randomly changed. Therefore, it was concluded that the grooming that an experimenter gives to a monkey can be applied as a positive reinforcer in operant conditioning. This experimental paradigm is considered to be useful for neurophysiological analysis of brain mechanisms underlying reward derived from somatosensory input in nonhuman primates.

Animals↗
PubMed1996

Specific regulation of gene expression by antisense nucleic acids: a summary of methodologies and associated problems.

Gene therapy based on gene-specific nucleic acids has moved from theory to a practical possibility in a very short time. The new DNA and RNA therapeutic reagents are intended to stop the growth of cancerous cells or the production of viruses. At the practical level, the efficacy of antisense oligomers as therapeutic reagents has been carefully examined in various clinical contexts. For the efficient use of antisense nucleic acids as pharmaceutical agents, a complete analysis of their mechanisms of action is necessary. The use of antisense oligomers always involves the following problems: basepair specificity, stereoisomer specificity, stability and resistance to nucleases of sense-antisense duplexes, permeability of the cell membrane and targeting of the oligomer, safety, and the preparation of large amounts of oligomer. Herein, we review the basic concepts and problems associated with the exploitation of antisense technology. We have identified a new transcription factor triple-helix-binding zinc-finger protein-1 (THZif-1) induced by antisense c-myc RNA in the antisense-transformed HL60 cells. The encoded protein functions as the repressor of c-myc to achieve the reduction of the endogenous expression of c-myc gene. Therefore, the introduction of THZif-1 gene into HL60 cells in conjunction with antisense c-myc oligomers may result in the efficient repression of the expression of the c-myc gene. The molecular features of this factor are herein discussed.

Amino Acid Sequence↗
PubMed1996

[Prevalence of depressive symptoms and its relation to demographic variables in high school students].

In order to determine the prevalence of depressive symptoms and to examine its relationship to demographic variables in late adolescents, questionnaires were administered on 3,254 high school students in Okinawa. Depressive symptoms were assessed using the Zung Self-rating Depression Scale (SDS). The results were as follows: The SDS score distribution curve was near-normal. The mean SDS scores of males and females were 40.4 and 41.7, respectively. Overall, 53.4% of males and 61.4% of females had depressive symptomatology according to an SDS cut-off point of 40 or greater. There were significant differences in mean SDS scores and prevalence by gender. Females had higher depressive symptom scores. Both indicators of depressive symptomatology were higher than those in U.S. adolescents and Japanese adults. When controlling for the effect of school type, both male and female students living in urban ureas of the main island had more depressive symptoms than students living in rural areas of remote islands. When controlling for the effect of region, female students attending vocational high schools showed more depressive symptoms than other high school students. However, there was no difference by school type among male students. The high prevalence of depressive symptoms suggests that it is necessary to modify the assessment cut-off point for high school students and to promote further school mental health care.

Adolescent↗
PubMed1995

Extraordinary enhancement of the cleavage activity of a DNA-armed hammerhead ribozyme at elevated concentrations of Mg2+ ions.

As part of an ongoing effort to characterize structure-function relationships, activities of all-RNA and DNA-armed hammerhead ribozymes were examined. An analysis of the dependence on the concentration of Mg2+ ions of cleavage rates revealed that, whereas the kcat of the reaction catalyzed by the all-RNA ribozyme reached a maximum value of about 18 min-1 at a concentration of about 200 mM Mg2+ ions, that of the DNA-armed ribozyme increased linearly as the concentration of Mg2+ ions was increased above 300 mM, finally reaching a value of more than 100 min-1 at 700 mM Mg2+ ions. These results suggest that the potential activity of a hammerhead ribozyme might be greater than is usually recognized.

Base Sequence↗
PubMed1995

Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes.

In order to study in detail the generality of the NUX (N = A, U, G, or C; X = A, U, or C) rule for the GUC triplet adjacent to the cleavage site in hammerhead ribozymes, two kinetic parameters, namely, kcat and Km, were determined for substrates with mutations in this triplet, which included double mutants with mutations of both N and X. All substrates with mutated cleavage sites were cleaved with reduced efficiency compared to the wild type. However, some mutations mainly affected kcat and others mainly affected Km, a phenomenon that could not have been predicted from previous results. A as the first or third base increased Km by 35- or 30-fold, respectively, while the effect on kcat was small. U as the first or third base decreased kcat by 8- or 15-fold, respectively, while the effect on Km was small. The effect of C as the first base on kinetic parameters was relatively small. The kinetic parameters of double mutants generally were determined by the effects of both individual point mutations. The AUA triplet gave a very much higher kcat than the other double mutants tested. In general, all of the mutants except for the mutant substrate with the CUC triplet had very low cleavage efficiencies, which ranged from 0.6% to 8% of the wild-type value, as a result of the deleterious effects of the mutations on kcat, Km, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Temperature-dependent change in the rate-determining step in a reaction catalyzed by a hammerhead ribozyme.

To characterize the reaction catalyzed by a hammerhead ribozyme, the dependence on temperature of the reaction was examined. An Arrhenius plot revealed a transition that indicated a temperature-dependent change in the activation energy at around 25 degrees C. Thermodynamic parameters of the reaction were estimated at 10 and 35 degrees C. The analyses led to the following conclusions. At 25-50 degrees C, the chemical cleavage step (kcleav) was the rate-determining step, and the cleaved fragments dissociated from the ribozyme at a higher rate than the rate of the chemical reaction. When the temperature was below 25 degrees C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate-determining step. Above 50 degrees C, the rate of the reaction decreased because, at such high temperatures, the formation of the Michaelis-Menten complex (duplex formation) was hampered by thermal melting. A conformational change in the ribozyme-substrate complex was not the rate-determining step at any of the temperatures examined.

Base Sequence↗
PubMed1995

Ribozymes: from mechanistic studies to applications in vivo.

The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs. However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the catalytic domain captures magnesium ions and magnesium ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes. In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state. When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme. It is likely, therefore, that hammerhead ribozymes exploit the general double-metal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties. Since the hammerhead ribozyme is one of the smallest RNA enzymes known and has potential as an antiviral agent, thus ribozyme has been extensively investigated for applications in vivo. Ribozymes are described that have possible utility as agents against HIV-1.

Animals↗
PubMed1995

A case of trichorrhexis nodosa developed in winter.

A young Japanese man developed localized trichorrhexis nodosa (LTN) of the scalp hair in the winter season. To investigate the roles of shampoo, severe sunlight exposure and/or mechanical injuries, we performed the following studies. Hair was collected from the patient and from a control. The study was performed in two steps. In the first step, hair was put into shampoo, rinsed with saline water, and then exposed to ultraviolet B (UVB) radiation once a day for one week. In the second step, the hair was similarly treated, but each shaft was bent gently with forceps before UVB exposure. Scanning electron microscopic studies revealed cuticular changes when the hair was treated only with shampoo and UVB. When it was treated with shampoo, UVB, and mechanical bending, the patient's hair developed longitudinal and transverse fractures of the hair shafts, while the control hair showed only partial damage to the hair shaft. On the basis on the above findings, we conclude that mechanical bending may damage the hair shaft.

Adolescent↗
PubMed1995

Detection of undegraded oligonucleotides in living sea urchin eggs by fluorescence resonance energy transfer.

A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. A 10-meric oligodeoxynucleotide labeled with fluorescein at its 5'-end and with rhodamine X at its 3'-end (F-ODN-R) was used. The oligomer had a specific absorption spectrum with peaks at 497 nm and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, F-ODN-R had a specific fluorescence spectrum with peaks at 523 nm and 610 nm. The digestion of F-ODN-R with an endonuclease caused the increase in light intensity at 523 nm and the decrease at 610 nm. To examine effects in vivo, living sea urchin eggs were injected with a solution of F-ODN-R and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. Eggs injected with the digested F-ODN-R emitted only green fluorescence. These results indicated that the integrity of oligonucleotides can be estimated in living cells by monitoring FRET after double-labeling of the oligonucleotides with fluorescein and rhodamine X.

Animals↗
PubMed1995

In vitro selection analysis of trans-acting HDV ribozyme.

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.

Base Sequence↗