Biomedical subjects

K Taira

Publications and source records attributed to K Taira.

At least 127 records · Page 7Linked to original sources

PubMed1999

Functional analysis of the transcriptional coactivators p300 and CBP using ribozyme.

p300 and CERB binding protein (CBP) are transcriptional coactivator which participate many signal transduction pathways via interaction with DNA binding proteins. In this study, we investigated functions of p300 and CBP during the retinoic acid (RA) induced F9 cell differentiation using hammerhead ribozymes. In wild type F9 cells, induction of c-jun gene was observed at 18 h after treatment with RA. Interestingly, RA-dependent expression of c-jun gene was not induced in cells expressing the p300-directed ribozyme. By contrast, the CBP-directed ribozyme did not affect expression of the c-jun gene. These results suggest that p300 is critical for the induction of c-jun gene during RA-induced differentiation.

Animals↗

PubMed1999

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.

Base Sequence↗

PubMed1999

Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes.

Recently, hammerhead ribozyme-mediated cleavage was analyzed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role could be monitored of metal ions that are directly involved in the cleavage reaction. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies demonstrated that binding of a metal ion to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and N7 of nucleotide G10.1 is critical for efficient catalysis. In order to clarify the effect of this metal ion, we chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1.

Catalysis↗

PubMed1999

Selection of intracellularly active ribozymes in mammalian cells.

Ribozymes are expected to be useful as antiviral agents and powerful tools of functional analysis of unknown gene products in vivo. For use of ribozymes in vivo, they must be fully functional in the intracellular environment. Not all ribozymes selected in vitro would be expected to work in vivo, whereas ribozymes selected in the intracellular environment should retain their function in vivo. With the eventual aim of using ribozymes as antiviral agents or biological tools in mammalian cells, we then devised a novel selection system in mammalian cells of active ribozymes by targeting at a gene for the cyclin dependent kinase inhibitor (CDKI), p16INK4a. In this system, we found that p16INK4a-knockdown cells became malignant and they formed foci. In the mammalian system, we confirmed that the selected cells harbored the active ribozyme, indicating that our positive selection systems in vivo were operational.

3T3 Cells↗

PubMed1999

Factors governing the activity in vivo of ribozymes transcribed by RNA polymerase III.

In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAVal ribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.

Animals↗

PubMed1999

Detection of abnormal high-frequency components in the QRS complex by the wavelet transform in patients with idiopathic dilated cardiomyopathy.

In order to investigate whether increased fine, fractionated signals within the QRS complex can detect arrhythmogenic substrates and how these fine signals link with ventricular mechanical dysfunction, wavelet analysis was performed on averaged QRS complexes obtained from the left precordial lead in 26 patients with idiopatic dilated cardiomyopathy (IDCM) and in 12 normal subjects. The number of local maxima and the duration of the wavelet transform were significantly greater in patients with IDCM than in normal subjects; the number at 100 Hz was 8.8+/-3.1 vs 6.0+/-1.1 (p<0.01), and the duration at 100Hz was 93+/-15 vs 75+/-7ms (p<0.01). Both of these indices were greater in the patients with than in those without late potentials, repetitive ventricular premature beats or cardiac death. In addition, significant inverse curvilinear relationships were observed between the left ventricular ejection fraction and both the number of local maxima and the duration of the wavelet transform. In conclusion, fine fragmented signals in the QRS complex detected by wavelet analysis would be an important marker for potentially arrhythmogenic substrates and seemed to progress in parallel with left ventricular mechanical dysfunction in IDCM.

Adolescent↗

PubMed1999

[Clinical retrospective study on outpatients at clinic for oral implant].

A retrospective study was made on 1,000 outpatients visiting the Clinic for Oral Implant, University Hospital, Faculty of Dentistry, Tokyo Medical and Dental University between April 1995 and June 1998. The following findings were obtained according to sex, age group, reason for visit, classification of edentulous area, indication and contraindication for implant therapy, and pre-implant surgical treatment. Forty percent of the outpatients were male and 60% were female. The largest number was in the 50-59-year-old group. The reason for visit of 822 patients was request for implant therapy. 123 patients with uncomfortable implants done at other clinics, 12 patients undergoing maintenance of implants at other clinics and 43 other patients. There were 505 upper jaw cases and 529 lower jaw cases. Most anterior edentulous cases were in the upper jaw group. Most of the posterior edentulous cases were unilateral cases and many of them were free-end saddle cases. Indication patients were 447 of the 822 outpatients. The main contraindication was insufficient volume of bone. Two hundred twenty-one of the 477 indication patients did not need the pretreatment which consisted of extraction, bone graft, free gingival graft, provisional prosthetics, sinus lift, and so on.

Adult↗

PubMed1998

Explanation by a putative triester-like mechanism for the thio effects and Mn2+ rescues in reactions catalyzed by a hammerhead ribozyme.

Divalent metal ion-dependent hammerhead ribozymes can cleave any RNA with a NUX triplet, wherein the N can be any residue and X can be C, U or A. In recent literature on the mechanism of action of hammerhead ribozymes, one important role of divalent metal ions is generally suggested to be an electrophilic catalyst by directly coordinating with the pro-Rp oxygen of the scissile phosphate to stabilize the transition state. This proposal was made on the basis of thio effects and the proposed electrophilic catalyst is very attractive as an explanation for the catalytic activity of metalloenzymes. Reexamination of thio effects with substrates having a GUA triplet at the cleavage site shows that, in agreement with the previous finding, the cleavage rate, in the presence of Mg2+ ions, is significantly reduced in the case of the phosphorothioate substrate (RpS), wherein the pro-Rp oxygen at the scissile phosphate is replaced by sulfur, while the cleavage rate is reduced to a much lesser extent for the other isomer (SpS), wherein the pro-Sp oxygen at the scissile phosphate is replaced by sulfur. However, more careful examination of the rescue ability of Mn2+ ions with these isomers demonstrates that more thiophilic Mn2+ ions rescue the reaction not only with the RpS isomer but also with the SpS isomer and, importantly, to a greater extent for the SpS isomer. These results argue against the previous conclusion that a metal ion is directly coordinating with the pro-Rp oxygen of the scissile phosphate to stabilize the transition state. In this paper we try to elucidate the possible origin of the thio effects and propose a 'triester-like' mechanism in reactions catalyzed by hammerhead ribozymes.

Catalysis↗

PubMed1998

Effects of helical structures formed by the binding arms of DNAzymes and their substrates on catalytic activity.

As a part of our efforts to clarify structure-function relationships in reactions catalyzed by deoxyribozymes (DNAzymes), which were recently selected in vitro , we synthesized various chimeras and analyzed the kinetics of the corresponding cleavage reactions. We focused on the binding arms and generated helices composed of binding arms and substrates that consisted of RNA and RNA, of RNA and DNA or of DNA and DNA. As expected for the rate limiting chemical cleavage step in reactions catalyzed by DNAzymes, a linear relationship between log( k cat) and pH was observed. In all cases examined, introduction of DNA into the binding helix enhanced the rate of chemical cleavage. Comparison of CD spectra of DNAzyme. substrate complexes suggested that higher levels of B-form-like helix were associated with higher rates of cleavage of the substrate within the complex. To our surprise, the enhancement of catalytic activity that followed introduction of DNA into the binding helix (enhancement by the presence of more B-form-like helix) was very similar to that observed in the case of the hammerhead ribozymes that we had investigated previously. These data, together with other observations, strongly suggest that the reaction mechanism of metal-ion-dependent DNAzymes is almost identical to that of hammerhead ribozymes.

Base Sequence↗

PubMed1998

Distinct roles of the co-activators p300 and CBP in retinoic-acid-induced F9-cell differentiation.

The related proteins p300 and CBP (cAMP-response-element-binding protein (CREB)-binding protein)) are transcriptional co-activators that act with other factors to regulate gene expression and play roles in many cell-differentiation and signal transduction pathways. Both proteins have intrinsic histone-acetyltransferase activity and may act directly on chromatin, of which histone is a component, to facilitate transcription. They are also involved in growth control pathways, as shown by their interaction with the tumour suppressor p53 and the viral oncogenes E1A and SV40 T antigen. Here we report functional differences of p300 and CBP in vivo. We examined their roles during retinoic-acid-induced differentiation, cell-cycle exit and programmed cell death (apoptosis) of embryonal carcinoma F9 cells, using hammerhead ribozymes capable of cleaving either p300 or CBP messenger RNAs. F9 cells expressing a p300-specific ribozyme became resistant to retinoic-acid-induced differentiation, whereas cells expressing a CBP-specific ribozyme were unaffected. Similarly, retinoic-acid-induced transcriptional upregulation of the cell-cycle inhibitor p21Cip1 required normal levels of p300, but not CBP, whereas the reverse was true for p27Kip1. In contrast, both ribozymes blocked retinoic-acid-induced apoptosis, indicating that both co-activators are required for this process. Thus, despite their similarities, p300 and CBP have distinct functions during retinoic-acid-induced differentiation of F9 cells.

Apoptosis↗

PubMed1998

A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of a fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase.

Nucleic acid-based drugs, including antisense RNA and DNA, ribozymes and decoys appear to have potential for the suppression of the expression of specific genes. To allow the examination of the potential of such agents in vivo as anti-HIV drugs in standard laboratories, where facilities for handling live virions are not available, we constructed a simple assay system (HIV-1 model) that allows measurement of the extent of inhibition of Tat-mediated transcription of HIV-1 by nucleic acid-based drugs and other agents. In cells that harbor a stable chimeric long terminal repeat (LTR)-Luc construct (a fusion gene consisting of the LTR of HIV-1 and the gene for luciferase), total luciferase activity in an aliquot of cell lysate is dose- and promoter-dependent on transfection with a Tat expression plasmid, reflecting the character of the LTR promoter of HIV. When HeLa cells were co-transfected with the Tat expression plasmid and another plasmid that encoded the U6 promoter or the promoter of the gene for tRNA(Val) linked to the trans-activating response (TAR) sequence, total luciferase activity was inhibited by 60 or 40%, respectively. The inhibition was also dependent on the dose of the TAR expression plasmid. These results demonstrate the usefulness of this simple assay system for detection of the efficacy of a decoy RNA or a ribozyme in vivo, without a requirement for HIV-infected cells, by measurement of luciferase activity in vitro.

Artificial Gene Fusion↗

PubMed1998

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, 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. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. 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. This conclusion based on changes in FRET was confirmed by gel-shift assays.

DNA↗

PubMed1998

p300 and ATF-2 are components of the DRF complex, which regulates retinoic acid- and E1A-mediated transcription of the c-jun gene in F9 cells.

Transcriptional activation of the c-jun gene is a critical event in the differentiation of F9 cells. In our previous studies we characterized an element [differentiation response element (DRE)] in the c-jun promoter that is both necessary and sufficient to confer the capacity for differentiation-dependent up-regulation. This element binds the differentiation regulatory factor (DRF) complex, of which one component is the adenovirus E1A-associated protein p300. We have now identified activation transcription factor-2 (ATF-2) as a DNA-binding subunit of the DRF complex. p300 and ATF-2 interact with each other in vivo and in vitro. The bromodomain and the C/H2 domain of p300 mediate the binding to ATF-2, which in turn requires a proline-rich region between amino acids 112 and 350 for its interaction with p300. The phosphorylation of the serine residue at position 121 of ATF-2 appears to be induced by protein kinase C alpha (PKC alpha) after treatment of cells with retinoic acid (RA) or induction with E1A. In cotransfection assays, wild-type ATF-2 enhanced the transcription of an E2/tk-luciferase construct, in conjunction with p300-E2. However, a mutant form of ATF-2 with a mutation at position 121 (pCMVATF-2(Ser121-Ala)) did not. These results suggest that ATF-2 and p300 cooperate in the control of transcription by forming a protein complex that is responsive to differentiation-inducing signals, such as RA or E1A, and moreover, that the phosphorylation of ATF-2 by PKC alpha is probably a signaling event in the pathway that leads to the transactivation of the c-jun gene in F9 cells.

Activating Transcription Factor 2↗

PubMed1998

Successful gene transfer to the porcine liver in vivo with an adenoviral vector.

In vivo gene transfer to the porcine liver was tested with adenoviral vector to achieve molecular biological graft modulation. In adult female pigs immunosuppressed with cyclophosphamide, cyclosporine, and prednisolone, the liver was surgically isolated and flushed out with cold lactate Ringer solution (4 degrees C), by means of a pump-controlled bypass of the portal vein and the inferior vena cava in Groups A and D. In Group A (n = 4), 2 x 1011 pfu of the adenoviral vectors (pAdexCALacZ) were injected through the left hepatic artery during cold ischemia. In Group (n = 4), 2 x 1011 pfu of adenovirus vectors were injected through the auricular vein in a one-shot manner without a laparotomy. In Group C (n = 4), 2 x 1011 pfu/ml of adenoviral vectors were injected through the hepatic artery in a one-shot manner, without a surgical isolation of the liver. Group D (n = 4) animals received the same protocol as Group A except for the fact that they did not receive the immunosuppressive regimen. In a polymerase chain reaction, a transfected LacZ sequence was detected until POD 28 in Group A, but not in the other groups. In 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, only the Group A animals revealed apparent staining predominantly in the portal area at POD 2, which then continued to be recognized until POD 28. The in situ perfusion of the liver combined with immunosuppression is thought to provide an ideal environment for the liver-directed adenovirus-mediated gene transfer to the porcine liver, by enabling a long contact with a high titer of the adenoviral vector.

Adenoviridae↗

PubMed1998