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T Wakasugi

Publications and source records attributed to T Wakasugi.

At least 19 recordsLinked to original sources

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

Amino Acid Sequence

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron.

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequence is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5' and 3' splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3' splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3' splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.

Amino Acid Sequence

Comparison of defect size between thallium-201 and technetium-99m tetrofosmin myocardial single-photon emission computed tomography in patients with single-vessel coronary artery disease.

Defect size on exercise-rest technetium (Tc)-99m tetrofosmin myocardial perfusion imaging was compared with that on exercise-reinjection thallium-201 imaging with 20 patients with 1-vessel coronary artery disease. In each patient, exercise-reinjection thallium-201 single-photon emission computed tomography (SPECT) and exercise-rest Tc-99m tetrofosmin SPECT imaging were performed. For visual analysis of the obtained SPECT images, the left ventricular myocardium was divided into 20 segments based on 3 short-axis slices from the apical, middle, and basal ventricular levels. For quantitative analysis, a square region of interest was placed on the center of each segment which was used for visual analysis, and relative regional activity to the normal reference region was calculated for each segment. By visual interpretation of the images, exercise Tc-99m tetrofosmin imaging showed a smaller defect size than exercise thallium-201 imaging (6.9 +/- 3.9 vs 8.8 +/- 3.0 segments, p <0.01). In contrast, rest Tc-99m tetrofosmin imaging showed a defect size similar to that on reinjection thallium-201 imaging (5.9 +/- 3.6 vs 5.6 +/- 3.9 segments, p = NS). Similarly, the mean defect sizes during exercise determined by quantitative analysis were smaller on Tc-99m tetrofosmin SPECT than those on thallium-201 SPECT at all tested threshold cutoff points ranging from 50% to 70%, whereas there were no significant differences in defect sizes between rest Tc-99m tetrofosmin and reinjection thallium-201 imaging. These data indicate that exercise Tc-99m tetrofosmin SPECT defect size determined either by visual analysis or by quantitative analysis may be smaller than on exercise thallium-201 SPECT.

Aged

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC_ to CUU_), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing as another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

Amino Acid Sequence

Impaired fatty acid uptake in ischemic but viable myocardium identified by thallium-201 reinjection.

Iodine 123-labeled 15-iodophenyl-3-methyl-pentadecanoic acid (BMIPP) has been proposed as a potential myocardial fatty acid probe. We studied BMIPP uptake in ischemic myocardium identified by thallium reinjection. Fifty-five patients with coronary artery disease who had persistent defects on standard exercise-redistribution thallium imaging were investigated. Patients underwent exercise-redistribution-reinjection thallium and resting BMIPP imaging. BMIPP uptake less than that seen with thallium on reinjection imaging was observed in 105 (82%) of 128 myocardial segments with new fill-in after thallium reinjection and 87 (37%) of 238 segments with reversible thallium defects. In contrast, only 32 (19%) of 166 segments with no fill-in showed discordantly decreased BMIPP uptake. Quantitative analysis showed reduction in BMIPP activity compared with differential uptake of thallium, an index of resting myocardial perfusion, especially in the area of fill-in (53.5% +/- 15.0% vs 76% +/- 12.1% of peak; p<0.01). These observations are consistent with impaired fatty acid uptake in ischemic myocardium, particularly in the area of fill-in after thallium reinjection.

Adult

Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy.

We previously reported that the anti-tumour effect of OK-432 is considerably enhanced by its intratumoral injection together with fibrinogen. In the present study, we generated killer T cells by culturing tumour-infiltrating lymphocytes from thyroid cancer patients who had received this local immunotherapy. Phenotypic analysis revealed that the T cells were positive for CD3+, CD4+, Leu8-, CD45RO+ and T-cell receptor (TCR)alpha beta+, as well as showing strong surface expression of HLA-DR, CD25, LFA-1 and ICAM-1. The generated CD4+ T cells secreted interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, TNF-beta, and interleukin (IL)-6 (but not IL-4), and exhibited a high level of cytolytic activity against several tumour cell lines. The cytolytic activity of these T cells for Daudi cells was inhibited by preincubation with an anti-intercellular adhesion molecule (ICAM)-1 antibody, but not by preincubation with anti-TCR alpha beta, anti-CD2, or anti-LFA-1 antibodies. Pretreatment with anti-ICAM-1 antibody inhibited T-cell cytolytic activity, but not conjugation with target cells. In addition, incubation with immobilised anti-ICAM-1 enhanced the secretion of IFN-gamma by T cells. We conclude that ICAM-1 expressed on the effector cytotoxic CD4+ T lymphocytes delivers regulatory signals that enhance IFN-gamma secretion.

Adjuvants, Immunologic

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A).

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (Rs alpha I, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.

Base Sequence

The rps3-rpl16-nad3-rps12 gene cluster in rice mitochondrial DNA is transcribed from alternative promoters.

The two gene clusters rps3-rpl16 and nad3-rps12 are separated from each other in the mitochondrial genome and are expressed as the individual transcription units in many plants. In rice mitochondrial DNA (mtDNA), the four genes rps3, rpl16, nad3 and rps12 are located within a region of 6 kbp. Northern-blot analysis revealed that a large transcript (6.6 kb) hybridized to both the rps3-rpl16 and the nad3-rps12 gene clusters. Using RT-PCR, we amplified a fragment of anticipated size (790 bp) from two primers that corresponded to sequences in the coding regions of rpl16 and nad3, demonstrating that at least two of the four genes, namely rpl16 and nad3, were co-transcribed. These results together indicated that all four genes, namely, rps3, rpl16, nad3 and rps12, were co-transcribed in rice mitochondria. Transcription initiation sites were determined by an in vitro capping/ribonuclease protection assay and primer extension analysis. Two initiation sites were identified in the rps3-rpl16-nad3-rps12 gene cluster: one was located upstream of rps3 and the other was located between rpl16 and nad3. This evidence indicates that the rps3-rpl16-nad3-rps12 gene cluster is transcribed from two alternative promoters.

Base Sequence

Myocardial viability assessment with technetium-99m-tetrofosmin and thallium-201 reinjection in coronary artery disease.

UNLABELLED: Exercise-rest 99mTc-tetrofosmin myocardial perfusion images with a 2-day protocol was compared to exercise-redistribution-reinjection 201Tl images to assess the ability of 99mTc-tetrofosmin to detect viable myocardium. METHODS: We studied 25 patients with coronary artery disease and regional or global left ventricular dysfunction. Myocardial SPECT images with 99mTc-tetrofosmin were obtained 10 min after injection during exercise and 1 and 3 hr after rest injection. Within 1 wk of the 99mTc-tetrofosmin study, exercise-redistribution-reinjection 201Tl SPECT imaging was performed. RESULTS: Visual analysis demonstrated concordance between 201Tl and 99mTc-tetrofosmin imaging for defect reversibility in 126 of 209 segments (60%), with initial defects on both exercise 201Tl and 99mTc-tetrofosmin images. In the remaining discordant 83 segments (40%), 73 (88%) appeared nonreversible on 99mTc-tetrofosmin imaging but were reversible on 201Tl imaging. CONCLUSION: On the basis of defect reversibility by visual analysis, exercise-rest 99mTc-tetrofosmin imaging underestimates myocardial viability compared to 201Tl reinjection imaging. The identification of viable myocardium with both 99mTc-tetrofosmin and 201Tl can be greatly enhanced to a similar degree if the severity of reduction in activity within nonreversible defects is considered. These two agents may provide comparable information about myocardial viability by quantitative analysis of defect severity.

Coronary Disease

Improved myocardial fatty acid utilization after percutaneous transluminal coronary angioplasty.

Exercise-reinjection 201Tl imaging and resting 20-min and 3-hr BMIPP imaging were performed before and 4 mo after percutaneous transluminal coronary angioplasty (PTCA) in a patient with effort angina. Before PTCA, exercise 201Tl imaging showed decreased 201Tl activity in the septal wall, with significant fill-in on the reinjection 201Tl image. The resting 20-min BMIPP image showed decreased activity in the septal wall, with a slight redistribution on the 3-hr BMIPP image. The 201Tl and BMIPP images 4 mo after PTCA showed significant improvement in the 201Tl pattern and BMIPP uptake in the septal wall with no abnormally decreased activities.

Aged

Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii.

The complete nucleotide sequence (119,707 bp) of the black pine (Pinus thunbergii) chloroplast genome has been determined. It contains 4 rRNA genes and 32 tRNA genes. To our knowledge, the tRNAPro (GGG) gene has not been found in any other chloroplast genome analyzed. Sixty-one genes encoding proteins and 11 conserved open reading frames are also found. Extensive rearrangements are apparent in the chloroplast genome relative to those of other land plants. The most striking feature is the loss of all 11 functional genes (ndh genes) for subunits of a putative NADH dehydrogenase that are found in the chloroplast genomes of angiosperms and a bryophyte. Four ndh genes were completely lost and the other 7 genes remain as obvious pseudogenes. This unexpected finding raises the possibility that all ndh genes have been transferred to the nucleus or that an NADH dehydrogenase is not essential in black pine chloroplasts.

Base Sequence

RNA editing of tobacco petB mRNAs occurs both in chloroplasts and non-photosynthetic proplastids.

We found an RNA editing site in the protein coding region of tobacco (Nicotiana tabacum) petB transcripts. This editing (CCA to CUA) leads to an amino acid conversion from proline to leucine. It is observed not only in chloroplasts isolated from tobacco leaves but also in non-photosynthetic proplastids isolated from a tobacco cell culture. Also unspliced pre-mRNA shows complete editing. The editing site is the same as that recently observed in the maize petB transcripts which restores the codon for a highly conserved leucine residue, suggesting that RNA editing at this site is critical for the protein (cytochrome b6) function.

Base Sequence

An atpE-specific promoter within the coding region of the atpB gene in tobacco chloroplast DNA.

The atpB and atpE genes encode beta and epsilon subunits, respectively, of chloroplast ATP synthase and are co-transcribed in the plant species so far studied. In tobacco, an atpB gene-specific probe hybridizes to 2.7- and 2.3-kb transcripts. In addition to these, a probe from the atpE coding region hybridizes also to a 1.0-kb transcript. The 5' end of the atpE-specific transcript has been mapped 430/431 nt upstream of the atpE translation initiation site, within the coding region of the atpB gene. In-vitro capping revealed that this transcript results from a primary transcriptional event and is also characterized by -10 and -35 canonical sequences in the 5' region. It has been found to share a common 3' end with the bi-cistronic transcripts that has been mapped within the coding region of the divergently transcribed trnM gene, approximately 236 nt downstream from the atpE termination codon. Interestingly, this transcript accumulates only in leaves and not in proplastid-containing cultured (BY-2) cells, indicating that, unless it is preferentially degraded in BY-2 cells, its expression might be transcriptionally controlled.

Base Sequence

A new gene encoding tRNA(Pro) (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts.

The chloroplast genome of black pine (Pinus thunbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA(Pro) (GGG) and tRNA(Arg) (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA(Gly) (UCC) gene and full copies of tRNA(His) (GUG), tRNA(Thr) (GGU) and tRNA(Ser) (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolution. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the "two-out-of-three" and the "U:N wobble" mechanisms.

Base Sequence

Nucleotide sequence of a 28-kbp portion of rice mitochondrial DNA: the existence of many sequences that correspond to parts of mitochondrial genes in intergenic regions.

The nucleotide sequence of a 27,588-bp region of rice mitochondrial DNA was determined. This sequence contains putative genes that encode initiator methionine tRNA (trnfM), subunits III (nad3) and IV (nad4) of the NADH dehydrogenase complex, and ribosomal proteins S3 (rps3), S12 (rps12) and L16 (rpl16). An open reading frame that contains sequences homologous to parts of rps2 and atpA is also present. In addition to these regions, there are many short sequences with homology to fragments of mitochondrial DNAs from rice or other plants. These sequences may be remnants of multiple rearrangements of the genome and their presence seems to explain, in part, the large sizes of the mitochondrial genomes of higher plants.

Base Sequence

Relationship between various parameters derived from 123I-labelled beta-methyl-branched fatty acid whole-body scintigraphy and left ventricular ejection fraction in patients with ischaemic heart disease.

Heart uptake and clearance of 123I-15-(p-iodophenyl)-3(R,S)-methyl- pentadecanoic acid (BMIPP) by whole-body scintigraphy in relation to left ventricular ejection fraction (LVEF) was evaluated in 34 patients with ischaemic heart disease. At 45 and 210 min after injection, anterior whole-body images were obtained. In each patient, multigated blood pool scintigraphy was also performed, and LVEF was calculated. The percentage heart uptake of BMIPP did not have a significant correlation with LVEF (R = -0.299, NS). In contrast, there was a positive correlation between LVEF measured by multigated blood pool scintigraphy and BMIPP count density of the heart (R = 0.467, P = 0.005). Myocardial BMIPP washout rate also positively correlated with LVEF (R = 0.472, P = 0.005). In addition to these observations, there was a positive correlation between the BMIPP count density of the heart and myocardial BMIPP washout rate (R = 0.0543, P = 0.001). Thus, the BMIPP count density of the heart and myocardial BMIPP washout rate derived from BMIPP whole-body scintigraphy directly correlated with LVEF. However, the correlations between these parameters and LVEF were not strong enough to allow left ventricular dysfunction to be assessed precisely.

Adult

[DNA sequencer].

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Automation