Biomedical subjects

T Washio

Publications and source records attributed to T Washio.

At least 19 recordsLinked to original sources

PubMed2003

Comparative study of translation termination sites and release factors (RF1 and RF2) in procaryotes.

Translation termination is catalyzed by release factors that recognize stop codons. However, previous works have shown that in some bacteria, the termination process also involves bases around stop codons. Recently, Ito et al. analyzed release factors and identified the amino acids therein that recognize stop codons. However, the amino acids that recognize bases around stop codons remain unclear. To identify the candidate amino acids that recognize the bases around stop codons, we aligned the protein sequences of the release factors of various bacteria and searched for amino acids that were conserved specifically in the sequence of bacteria that seemed to regulate translation termination by bases around stop codons. As a result, species having several highly conserved residues in RF1 and RF2 showed positive correlations between their codon usage bias and conservation of the bases around the stop codons. In addition, some of the residues were located very close to the SPF motif, which deciphers stop codons. These results suggest that these conserved amino acids enable the release factors to recognize the bases around the stop codons.

Amino Acid Sequence↗

PubMed2002

On biased distribution of introns in various eukaryotes.

We conducted comprehensive analyses on intron positions in the Mus musculus genome by comparing genomic sequences in the GenBank database and cDNA sequences in the mouse cDNA library recently developed by Riken Genomic Sciences Center. Our results confirm that introns have a tendency to be located toward the 5' end of the gene. The same type of analysis was conducted in the coding region of seven eukaryotes (Saccharomyces cerevisiae, Plasmodium falciparum, Caenorhabditis elegans, Drosophila melanogaster, M. musculus, Homo sapiens, Arabidopsis thaliana). Introns in genes with a single intron have a locational bias toward the 5' end in all species except A. thaliana. We also measured the distance from the start codon to the position of the intron, and found that single introns prefer the location immediately after the start codon in S. cerevisiae and P. falciparum. We discuss three possible explanations for these findings: (1) they are the consequence of intron loss by reverse-transcriptase; (2) they are necessary to accommodate the function; and (3) they are concerned with the mechanism of pre-mRNA splicing.

Animals↗

PubMed2002

Comprehensive sequence analysis of translation termination sites in various eukaryotes.

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.

3' Untranslated Regions↗

PubMed2001

Preferential usage of some minor codons in bacteria.

In many bacterial species, such as Deinococcus radiodurans, Haemophilus influenzae, and Methanobacterium thermoautotrophicum, some minor codons are preferentially used near the initiation codon. Among these codons, there are some minor codons that have strong preference for the initiation site in the high codon adaptation index (CAI) group (comprising of highly expressed genes) rather than in the low CAI genes group (comprising of low expressing genes). In the present study, codon usage in the initiation site and in the rest of the gene was systematically compared in the 27 complete bacterial genomes and Saccharomyces cerevisiae genome. Furthermore, we classified genes into two groups according to the CAI values and conducted the same analysis for each of the two groups. Our results suggest a role for some minor codons in the initiation site of the regulating translation system in many bacteria. We have summarized codons that are preferentially used in the initiation site and probably play a role in regulating genes expression in these organisms.

Amino Acids↗

PubMed2001

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes.

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.

5' Untranslated Regions↗

PubMed2001

An experimental study on the interface strength between titanium mesh cage and vertebra in reference to vertebral bone mineral density.

STUDY DESIGN: Using human cadaver spines, the authors investigated mechanical properties of the interface between titanium mesh cage and vertebra in respect to vertebral bone mineral density. OBJECTIVES: The objective of this study is to examine the effects of the size of the mesh cage and an internal end ring system on interface mechanical properties in reference to vertebral bone mineral density. SUMMARY OF BACKGROUND DATA: A titanium mesh cage has recently been developed for anterior spinal reconstruction. The cage provides immediate postoperative stability and facilitates bony union with cancellous bone packed in the cage itself. Mechanical properties of the interface between the cage and vertebra, however, are yet to be clarified in osteoporotic spine. METHODS: Twenty-five lumbar vertebrae harvested from embalmed human cadavers (n = 20) were used. The vertebrae were divided into four experimental groups according to the applied cage conditions: Phi25 mm cage without internal end ring (L-), Phi19 mm cage without internal end ring (S-), Phi25 mm cage with internal end ring (L+), and Phi19 mm cage with internal end ring (S+). Bone mineral density of whole vertebral body was measured by dual energy radiograph absorptiometer (DXA). Peripheral quantitative computed tomography was used to determine local bone mineral density of subchondral cancellous bone of vertebral body. Each cage was compressed on vertebral endplate via a specially designed device connected to a material testing machine. Maximum load and stiffness of the interface between the cage and vertebra were measured from load-deformation data in quasi-static compression loading with a loading rate of 0.5 mm/min. Relationships between the mechanical properties and vertebral bone mineral densities were evaluated. In 11 specimens acoustic emission during compression loading was measured and simultaneously recorded in load-deformation data. After the mechanical test microradiograms of midsagittal sections of the vertebrae were taken to observe failure patterns of endplate or trabecular bone. RESULTS: Vertebrae compressed with large cages (group: L- or L+) showed greater maximum load than those compressed with small cages (group: S- or S+). The internal end ring contributed to higher maximumload. The size of the cage or the internal end ring, however, did not have any effect on stiffness. Maximum load and stiffness were positively correlated with whole vertebral bone mineral density measured by dual energy radiograph absorptiometer or local cancellous bone mineral density of subchondral bone measured by peripheral quantitative computed tomography. Correlation coefficient and P value were more significant in the association of the mechanical properties and subchondral bone mineral density measured by peripheral quantitative computed tomography than in the association of the parameters and whole vertebral bone mineral density measured by DXA. A load-deformation curve with an acoustic emission event count rate showed that significant acoustic emission signals were generated around maximum load. On microradiographic study most vertebrae compressed with the cage showed encroachment of the cage spikes into the endplate or trabecular structure, preserving structures of the most central portion of the vertebrae. CONCLUSION: A titanium mesh cage with larger diameter and/or augmentation of internal end ring produces a significant increase of the interface strength between the cage and the vertebra. A positive correlation between the interface strength and vertebral bone mineral density suggests that vertebral bone mineral density is an important parameter for successful spinal reconstruction, and also implies that in severe osteoporotic spine the stability of the cage is declined, and other instrumentation should be combined.

Aged↗

PubMed2001

Functional annotation of a full-length mouse cDNA collection.

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Animals↗

PubMed2001

Correlation between Shine--Dalgarno sequence conservation and codon usage of bacterial genes.

In this study, we analyzed the correlation between codon usage bias and Shine--Dalgarno (SD) sequence conservation, using complete genome sequences of nine prokaryotes. For codon usage bias, we adopted the codon adaptation index (CAI), which is based on the codon usage preference of genes encoding ribosomal proteins, elongation factors, heat shock proteins, outer membrane proteins, and RNA polymerase subunit proteins. To compute SD sequence conservation, we used SD motif sequences predicted by Tompa and systematically aligned them with 5'UTR sequences. We found that there exists a clear correlation between the CAI values and SD sequence conservation in the genomes of Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii, and no relationship is found in M. genitalium, M. pneumoniae, and Synechocystis. That is, genes with higher CAI values tend to have more conserved SD sequences than do genes with lower CAI values in these organisms. Some organisms, such as M. thermoautotrophicum, do not clearly show the correlation. The biological significance of these results is discussed in the context of the translation initiation process and translation efficiency.

Base Sequence↗

PubMed2001

Identification of human drug-metabolizing enzymes involved in the metabolism of SNI-2011.

In vitro studies were conducted to identify human drug-metabolizing enzymes involved in the metabolism of SNI-2011 ((+/-)-cis-2-methylspiro [1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline hydrochloride hydrate). When 14C-SNI-2011 was incubated with human liver microsomes, SNI-2011 trans-sulfoxide and cis-sulfoxide were detected as major metabolites. These oxidations required NADPH, and were markedly inhibited by SKF-525A, indicating that cytochrome P450 (CYP) was involved. In a chemical inhibition study, metabolism of SNI-2011 in liver microsomes was inhibited (35-65%) by CYP3A4 inhibitors (ketoconazole and troleandomycin) and CYP2D6 inhibitors (quinidine and chlorpromazine). Furthermore, using microsomes containing cDNA-expressed CYPs, it was found that high rates of sulfoxidation activities were observed with CYP2D6 and CYP3A4. On the other hand, when 14C-SNI-2011 was incubated with human kidney microsomes, SNI-2011 N-oxide was identified as a major metabolite. This N-oxidation required NADPH, and was completely inhibited by thiourea, indicating that flavin-containing monooxygenase (FMO) was involved. In addition, microsomes containing cDNA-expressed FMO1, a major isoform in human kidney, mainly catalyzed N-oxidation of SNI-2011, but microsomes containing FMO3, a major isoform in adult human liver, did not. These results suggest that SNI-2011 is mainly catalyzed to sulfoxides and N-oxide by CYP2D6/3A4 in liver and FMOI in kidney, respectively.

Animals↗

PubMed2000

Causal analysis of CpG suppression in the Mycoplasma genome.

Some bacterial genomes are known to have low CpG dinucleotide frequencies. While their causes are not clearly understood, the frequency of CpG is suppressed significantly in the genome of Mycoplasma genitalium, but not in that of Mycoplasma pneumoniae. We compared orthologous gene pairs of the two closely related species to analyze CpG substitution patterns between these two genomes. We also divided genome sequences into three regions: protein-coding, noncoding, and RNA-coding, and obtained the CpG frequencies for each region for each organism. It was found that the observed/expected ratio of CpG dinucleotides is low in both the protein-coding and noncoding regions; while that ratio is in the normal range in the RNA-coding region. Our results indicate that CpG suppression of the Mycoplasma genome is not caused by (1) biased usage amino acid; (2) biased usage of synonymous codon; or (3) methylation effects by the CpG methyltransferase in the genomes of their hosts. Instead, we consider it likely that a certain global pressure, such as genome-wide pressure for the advantages of DNA stability or replication, has the effect of decreasing CpG over the entire genome, which, in turn, resulted in the biased codon usage.

Base Composition↗

PubMed2000

An experimental study of porcine lumbar segmental stiffness by the distraction-compression principle using a threaded interbody cage.

The objectives of this study were to quantify changes in stiffness and disk height of porcine functional spinal units (FSUs) by installation of a threaded interbody cage and those by gradual resection of the annulus fibrosus. Flexion, extension, bending, and torsion to the FSUs were performed in four sequential stages: stage I, intact FSU; stage II, the FSUs are fitted with a threaded fusion cage; stage III, the FSUs are fitted with a threaded fusion cage with the anterior one third of the annulus fibrosus excised, including excision of the anterior longitudinal ligament; and stage IV, in addition to stage III, the bilateral annulus fibrosus is excised. Segmental stiffness in each loading in the four stages and a change of disk height induced by the instrumentation were measured. After instrumentation, stiffnesses in all loading modes (p < 0.005) and disk height (p = 0.002) increased significantly. The stiffnesses of FSUs fixed by the cage decreased with gradual excision of the annulus fibrosus in flexion, extension, and bending. These results suggest that distraction of the annulus fibrosus and posterior ligamentous structures by installation of the cage increases the soft-tissue tension, resulting in compression to the cage and a stiffer motion segment.

Animals↗

PubMed1999

Two-stage method for protein-ligand docking.

A two-stage method for the computational prediction of the structure of protein-ligand complexes is proposed. Given an experimentally determined structure of the protein, in the first stage a large number of plausible ligand conformations is generated using the fast docking algorithm FlexX. In the second stage these conformations are minimized and reranked using a method based on a classical force field. The two-stage method is tested for 10 different protein-ligand complexes. For 9 of them experimentally determined structures are known. It turns out that the two-stage method strongly improves the predictive power as compared to that of the fast docking stage alone. The tenth case is a bona fide prediction of a complex of thrombin with a new inhibitor for which no experimentally determined structure is available so far.

Algorithms↗

PubMed1999

Comparative study of overlapping genes in the genomes of Mycoplasma genitalium and Mycoplasma pneumoniae.

Overlapping genes are defined, in this paper, as a pair of adjacent genes whose coding regions are partly overlapping. We systematically analyzed all overlapping genes in the genomes of two closely related species: Mycoplasma genitalium and Mycoplasma pneumoniae. Careful comparisons were made for homologous genes that are overlapped in one species but not in the other. This comparative analysis allows us to propose a model of how overlapping genes emerged in the course of evolution. It was found that overlapping genes were generated primarily due to the loss of a stop codon in either gene, in many cases, the absence of which resulted in elongation of the 3' end of the gene's coding region. More specifically, the loss of the stop codon took place as a result of the following events: deletion of the stop codon (64.4%), point mutation at the stop codon (4.4%), and frame shift at the end of the coding region (6.7%). Overlapping genes, in a sense, can be thought of as the results of evolutionary pressure to minimize genome size. However, our analysis indicates that many overlapping genes, at least in the genomes of M.genitalium and M.pneumoniae, are due to incidental elongation of the coding regions.

Genes, Bacterial↗

PubMed1998

Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination.

Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 shows a sharp drop, as expected, at 30 bp downstream of the stop codon, presumably due to hairpin-forming sequences. Similar drops are observed for Haemophilus influenzae Rd, Bacillus subtilis and Chlamydia trachomatis, suggesting that these organisms also form hairpins at their transcription termination sites. On the other hand, 12 other prokaryotes- Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis PCC6803, Helicobacter pylori, Borrelia burgdorferi, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Aquifex aeolicus, Pyrococcus horikoshii, Mycobacterium tuberculosis and Treponema pallidum -show no apparent decrease in free energy value at the corresponding regions. This result suggests that these prokaryotes, or at least some of them, may never form hairpins at their transcription termination sites.

Base Composition↗

PubMed1998

Fracture risk during pedicle screw insertion in osteoporotic spine.

Although using a larger screw can enhance the stability of screw-bone interface in pedicular screw instrumentation, it may cause pedicle fracture during screw insertion in osteoporotic spine. We investigated structural changes of the pedicle with the advance of osteoporosis and its implication for the risk of pedicle fracture. Bone mineral density of trabecular, subcortical, and cortical bone of pedicle in an osteoporotic group, assessed by peripheral quantitative computed tomography, was significantly lower than that of those in the normal group. Cortical thickness also declined. There were no pedicle fractures in normal vertebrae, but seven (41.2%) fractures were observed among 17 pedicles in osteoporotic vertebrae in which bone mineral density measured with dual-energy x-ray absorptiometry was <0.7 g/cm2 and screw diameter >70% of the outer diameter of the pedicle. Our results suggest that screw diameter should not exceed 70% of the outer diameter of the pedicle in the case of osteoporosis in which vertebral bone mineral density is <0.7 g/cm2.

Aged↗

PubMed1997

Structural characteristics of the pedicle and its role in screw stability.

STUDY DESIGN: Cross-sectional regional bone mineral density of the pedicle was measured by peripheral quantitative computed tomography. Biomechanical tests were performed to clarify the role of the pedicle in screw stability. OBJECTIVES: To identify the structural characteristics of the pedicle that supports pedicle screw stability and the differences in these characteristics between normal and osteoporotic vertebrae. SUMMARY OF BACKGROUND DATA: The pedicle screw is an essential component of many systems used to align the spine. The contribution of the pedicle to screw stability, however, has not been fully investigated. METHODS: Trabecular, subcortical, and cortical bone mineral density and the area of the pedicle were measured by peripheral quantitative computed tomography. Bone mineral density also was recalculated in four circumferential layers. These parameters were compared between normal and osteoporotic individuals. The relative contribution of the pedicle to screw stability was evaluated by caudocephalad and pull-out loading in a vertebra with or without its body. RESULTS: Inner trabecular, middle subcortical, and outer cortical bone mineral density and cortical bone area in the pedicle were significantly lower in osteoporotic vertebrae than those in normal vertebrae. In the pedicle, bone mineral density increased close to the outer layer. Bone mineral density not as thick even in the outer layer in osteoporotic subjects. Approximately 80% of the caudocephalad stiffness and 60% of the pullout strength of the pedicle screw depended on the pedicle rather than on the vertebral body. CONCLUSION: Screw stability depends on the structural characteristics of the pedicle. The pedicle was denser in the subcortical bone, in which the threads of the screw engage, than in trabecular bone. In osteoporosis, bone mineral density was not as dense even in the outer layer, and the cortex was thinner than normal. A larger screw would not enhance screw stability and may break the thin cortex in osteoporotic vertebrae.

Absorptiometry, Photon↗

PubMed1997

An experimental study of a combination method using a pedicle screw and laminar hook for the osteoporotic spine.

STUDY DESIGN: Using human cadaver spines, the authors compared the effect of using a combination of pedicle screw and laminar hook on the same vertebra with that of using a pedicle screw alone in reference to bone mineral density of the vertebra under nondestructive cyclic loading. OBJECTIVES: To quantify stiffness obtained by pedicle screw alone and by the combination method and to clarify a relationship between stiffness obtained by each instrumentation method and bone mineral density of the vertebra. SUMMARY OF BACKGROUND DATA: The use of pedicle screws apparently improves the union rate of spinal fusion. Instrumentation failures sometimes occur, however, such as loosening or loss of correction of the spine, especially in patients with osteoporosis. Some augmentation method in instrumentation is necessary to overcome bone fragility in the osteoporotic spine. METHODS: Thirteen cadaver lumbar vertebrae were used for this study. Bone mineral density was measured by dual-energy X-ray absorptiometer. After separating each vertebrae, the pedicle screw was screwed into a vertebra. Five cycles of cephalocaudal loading were performed to the shank of the screw with a cross-head speed of 3 mm/min under a maximum load control of 29.4 N using an Instron type testing machine, and the stiffness obtained with the pedicle screw (Kj) was calculated from the load-deformation curve. Then, a laminar hook was set and connected to the screw via a rod. Mechanical testing was performed in the same way, and the stiffness obtained with the combination method (Kf) was determined. Kj and Kf were compared using the paired t test. The relationship between Kj, Kf, or the stiffness improvement ratio ([Kf-Kj]/Kj) by the combination method and bone mineral density was analyzed by linear regression analysis. RESULTS: Stiffness obtained by the combination method was significantly greater than that obtained by pedicle screw alone (89.8 +/- 35.0 N/mm by the combination method, 60.2 +/- 19.6 N/mm by pedicle screwing alone; P < 0.0001). Stiffness, whether obtained by pedicle screw alone or by the combination method, was positively correlated with bone mineral density (with pedicle screw alone, R2 = 0.614, P < 0.0001; with the combination method, R2 = 0.645, P < 0.0001). However, there was no significant correlation between stiffness improvement ratio and bone mineral density. CONCLUSION: Instrumentation stiffness obtained by the combination method was significantly greater than that obtained by the use of pedicle screw alone. There was no significant correlation between the improvement ratio by the combination method and bone mineral density. These results suggest that the combination method is valuable irrespective of the presence of spinal osteoporosis.

Aged↗

PubMed1995

Cytotoxic effects of several hopanoids on mouse leukemia L1210 and P388 cells.

The cytotoxic effects of hopanoids, including bacteriohopane-32, 33, 34, 35-tetrol (Tetrol), bacteriohopane-32-ol (Monol), diploptene, diplopterol and acetylated Monol (AcO-Monol) isolated from Acetobacter aceti, were tested against two leukemia cell lines. Tetrol and Monol have been shown to be toxic to mouse L1210 and P388 compared to the other hopanoids. By measuring the ESR spectra of the spin labeled membranes of these cells, it was shown that the incorporation of Monol resulted in a decrease in the fluidity of the membranes. The decrease in membrane fluidity may correlate, in part, with the cytotoxicity of hopanoids against the two cell lines.

Animals↗