Biomedical subjects

M Ota

Publications and source records attributed to M Ota.

At least 19 recordsLinked to original sources

PubMed2000

DNA data bank of Japan (DDBJ) in collaboration with mass sequencing teams.

We at DDBJ (http://www.ddbj.nig.ac.jp) process and publicise the massive amounts of data submitted mainly by Japanese genome projects and sequencing teams. It is emphasised that the collaboration between data producing teams and the data bank is crucial in carrying out these processes smoothly. The amount of data submitted in 1999 is so large that it alone exceeds the total amount submitted in the preceding 10 years. To cope with this situation, we have developed tools not only for processing such massive amounts of data but also for efficiently retrieving data on demand.

Animals↗

PubMed1999

Strong hydrophobic nature of cysteine residues in proteins.

The differences between disulfide-bonding cystine (Cys_SS) and free cysteine (Cys_SH) residues were examined by analyzing the statistical distribution of both types of residue in proteins of known structure. Surprisingly, Cys_SH residues display stronger hydrophobicity than Cys_SS residues. A detailed survey of atoms which come into contact with the sulfhydryl group (sulfur atom) of Cys_SH revealed those atoms are essentially the same in number and variety as those of the methyl group of isoleucine, but are quite different to those of the hydroxyl group of serine. Moreover, the relationships among amino acids were also determined using the 3D-profile table of known protein structures. Cys_SH was located in the hydrophobic cluster, along with residues such as Met, Trp and Tyr, and was clearly separated from Ser and Thr in the polar cluster. These results imply that free cysteines behave as strongly hydrophobic, and not hydrophilic, residues in proteins.

Algorithms↗

PubMed1999

Design and synthesis of a globin fold.

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.

Amino Acid Sequence↗

PubMed1999

The Protein Mutant Database.

Currently the protein mutant database (PMD) contains over 81 000 mutants, including artificial as well as natural mutants of various proteins extracted from about 10 000 articles. We recently developed a powerful viewing and retrieving system (http://pmd.ddbj.nig.ac.jp), which is integrated with the sequence and tertiary structure databases. The system has the following features: (i) mutated sequences are displayed after being automatically generated from the information described in the entry together with the sequence data of wild-type proteins integrated. This is a convenient feature because it allows one to see the position of altered amino acids (shown in a different color) in the entire sequence of a wild-type protein; (ii) for those proteins whose 3D structures have been experimentally determined, a 3D structure is displayed to show mutation sites in a different color; (iii) a sequence homology search against PMD can be carried out with any query sequence; (iv) a summary of mutations of homologous sequences can be displayed, which shows all the mutations at a certain site of a protein, recorded throughout the PMD.

Amino Acid Sequence↗

PubMed1999

Cooperative approach for the protein fold recognition.

We, four independent predictors, organized a team and tackled blind protein structure predictions using fold recognition methods. We tried to assign the homologous or analogous folds in the protein structure database for a number of target sequences that showed no apparent sequence homology to the proteins of known folds. After primary analyses by conventional softwares, these sequences were threaded through the structural library using three different programs developed by ourselves, which employed different compatibility functions. Collecting the results of our individual analyses, and the available biological knowledge about the target, we held meetings and discussed all plausible structures for the target. For 25 target sequences, we submitted 56 models including NONE: This was the first time the fold was determined. At the time of the meeting (CASP3), 19 protein structures (21 domains) categorized as the threading targets were available. We succeeded in predicting eight out of 18 targets (20 domains) that we submitted; however, alignment accuracies were not satisfactory for some of the models. We often obtained correct answers even if some of us missed the right prediction; therefore it would appear that our threaders compensated each other. When all the information is managed effectively, the prediction gains more accuracy.

Algorithms↗

PubMed1999

Enzymatic ligation for synthesis of single-chain analogue of monellin by transglutaminase.

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C alpha-carboxyl group at the C-terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the gamma-amide group of the Gln residue and the epsilon-amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C-terminus (B-LLQG) was synthesized by solid-phase synthesis. The monellin A chain analogue in which KGK was elongated at the N-terminus (KGK-A) was synthesized by the same method as that of the B chain analogue. The KGK-A chain and the B-LLQG chain were coupled by MTGase to give single-chain analogue of monellin. The single-chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single-chain analogue of monellin was more heat stable than natural monellin.

Amino Acid Sequence↗

PubMed1999

Association analysis between the MIC-A and HLA-B alleles in Japanese patients with Behçet's disease.

OBJECTIVE: Behçet's disease is known to be strongly associated with HLA-B51 in many different ethnic groups. Recently, by association analysis using refined microsatellite mapping, the critical region for Behçet's disease was identified as a 46-kb segment centromeric to the HLA-B gene. No expressed gene has been detected in this segment to date except the MIC-A (major histocompatibility complex class I chain-related gene A) and HLA-B genes. The present study was undertaken to analyze allelic distribution of the MIC-A gene among Japanese patients with Behçet's disease. METHODS: Ninety-five Japanese patients with Behçet's disease and 116 ethnically matched healthy controls were enrolled in this study. MIC-A genotyping was performed by direct sequencing of polymerase chain reaction products from exons 2, 3, and 4 of the MIC-A gene, using an automated DNA sequencer. RESULTS: The MIC-A009 allele was significantly more frequent in the patient group (69.5%) compared with the healthy controls (31.0%) (relative risk 5.06, corrected P = 0.00000024). In stratification analysis on the confounding effect of MIC-A009 on HLA-B*51 association and vice versa, Behçet's disease was distinctively associated only with HLA-B*51. Further, MIC-A009 was found to be strongly associated not only with HLA-B51, but also with HLA-B52, which was not increased in the patient group to any degree. CONCLUSION: These results imply that the real disease susceptibility gene involved in the development of Behçet's disease is the HLA-B*51 allele itself and that the significant increase of the MIC-A009 allele in the patient group results secondarily from a strong linkage disequilibrium with HLA-B*51.

Adult↗

PubMed1999

MIC-A allele profiles and HLA class I associations in Behçet's disease.

Recently a new family of non-classical MHC molecules, the MHC class I chain-related protein (MIC), encoded by genes located in the major histocompatability complex have been identified. On the basis of the location of MIC genes and the structure and expression of MIC molecules it has been postulated that MIC may be a susceptibility factor in Behçet's disease (BD). We investigated the association of the 16 described external domain alleles and the transmembrane triplet repeats of MIC-A with BD in a Middle Eastern population. DNA from ninety-five patients and 102 age- and sex-matched controls were analyzed by polymerase chain reaction using allele specific primers. Our results show an increase of MIC-A*009 in the BD patient group 44/95 (46%) compared with controls 24/102 (24%) (chi2=11.3, OR=2.8, P=0.00078). MIC-A*009 was also found to be strongly associated with HLA-B51 in the patients 39/44 (88%) when compared with controls 10/24 (42%) (chi2=4, P=0.04). MIC-A*009 was also found in linkage disequilibrium with HLA-B52, but only in controls. The A6 form of a MIC-A transmembrane triplet repeat was found to be significantly raised in the patients (80/95; 84%;) compared with controls (58/102, 57%) (chi2=17.5, OR=4, P=0.000028). Although the MIC-A associations described are highly significant, the association with HLA-B51 independently remains the most significant factor (chi2=56.8, P<10(-6)). The data suggests that as both MIC-A*009 and A6 are in strong linkage disequilibrium with HLA-B51, they are unlikely to be the susceptibility gene for BD but may be markers for additional risk factors.

Adolescent↗

PubMed1999

Triplet repeat polymorphism in the MICA gene in HLA-B27 positive and negative caucasian patients with ankylosing spondylitis.

Previously, we reported a triplet repeat polymorphism in the transmembrane region within the MICA gene closely linked to HLA-B in a limited number of B27-positive Caucasian patients with ankylosing spondylitis (AS) (N = 48). In this study, we enrolled much more patients including some negative for B27, 162 AS subjects consisting of 140 B27-positive, and 22 B27-negative patients. The microsatellite allele consisting of 4 repetitions of (GCT/AGC) (A4 allele) was present at a significantly higher phenotype frequency in the patient group than in the ethnically matched control group (Pc < 0.000001). However, the frequency of the A4 allele was not significantly higher in the B27-positive and B27-negative patient groups, as compared to the B27-positive and B27-negative control groups, respectively. The higher phenotype frequency of the A4 allele in the patient group was supposed to be due to a strong linkage disequilibrium between the MICA and HLA-B genes. Thus, the possibility that the MICA gene is involved in the pathogenesis of AS can be excluded, supporting the hypothesis of a primary association of AS with HLA-B27.

Case-Control Studies↗

PubMed1999

T cell repertoire in the liver of patients with autoimmune hepatitis.

Despite a large number of T cells infiltrating into the liver of patients with autoimmune hepatitis (AIH), little is known about their roles or target antigens. To investigate the roles of these T cells in the pathogenesis of AIH, we have studied the clonality of alphabeta T cell populations in liver tissue by size spectratyping the complementarity-determining region (CDR)3 size lengths of T cell receptor (TCR) Vbeta-chain transcripts. Analysis of nine AIH patients who had the HLA DR4 haplotype showed clonal expansion in all samples. More than two T cell clones expanded in most patients. Although the expression of the TCR Vbeta genes was different among the nine patients, clonal expansion of T cells expressing either TCR Vbeta2, 3, 4, 16, or 22 was observed in two patients or more. TCR Vbeta4 clones expanded in 5 cases. Cloning and sequencing of TCR Vbeta CDR3 from PCR products revealed no whole CDR3-shared clones among different patients. In conclusion, several T cell clonotypes first recognize target antigens, then expand and accumulate in the liver of AIH patients. These suggest heterogeneity of autoantigens and the complexity of AIH immunopathogenesis in individual patients.

Adult↗

PubMed1999

Sequencing based typing for genetic polymorphisms in exons, 2, 3 and 4 of the MICA gene.

We have established a sequencing based typing (SBT) method for detection of genetic polymorphism in the exon 2 to 4 domains of the major histocompatibility complex (MHC) class I chain-related gene A (MICA) and applied it to allele typing of 130 healthy Japanese individuals. A 2.2-kb segment including exons 2, 3 and 4 of the MICA gene was amplified by a pair of generic primers followed by cycle sequencing using exon-specific nested primers. In total, 8 alleles were observed in a Japanese population and the most frequent allele was MICA008 with the gene frequency of 30.8%. MICA009 was the second most frequent (16.5%), while the rarest one was MICA007 (1.2%). MICA alleles displayed strong linkage equilibria with HLA-B antigens (i.e. MICA008 with B7, B48, B60 and B61; MICA009 with B51 and B52; MICA002 with B35, B39, B58 and B67; MICA004 with B44, MICA007 with B13 and B27; MICA010 with B46, B62 and B48, MICA012 with B54, B55, B56 and B59; MICA019 and B70, B71 and B62). Recently, the B48 haplotype has been reported to lack the entire MICA gene by a large-scale deletion in a Japanese population. Among 8 serologically B48 homozygous individuals, 4 were found to represent this MICA null allele as assessed by no polymerase chain reaction (PCR) amplification using MICA-specific primers, while the remaining four possessed the intact MICA gene with MICA008 or MICA010.

Asian People↗

PubMed1999

New polymorphic microsatellite markers in the human MHC class I region.

The human major histocompatibility complex (MHC) class I region is believed to contain a large number of genes encoding susceptible factors for diseases such as Behcet's disease, Graves disease and psoriasis vulgaris. To identify the causative genes of those diseases, we have conducted large-scale genomic sequencing and determined the 1.8 Mb entire HLA class I region from the MICB gene to the HLA-F gene. During the course of genomic sequencing, a total of 731 microsatellite sequences with dinucleotide to pentanucleotide repeats were found in this region. Previously, we reported that 26 microsatellites between MICB and S on the most centromeric side of the class I region, and between HSR1 and HLA-92/L in the midst of the class I region were highly polymorphic, and served as excellent genetic markers. In this paper, in order to fill the gaps with no known polymorphic microsatellites available in the HLA class I region, 12 new polymorphic microsatellite markers were recruited from the 1.8 Mb region including the remaining class I segments, namely between S and HSR1, and between HLA-92/L and HLA-F The average number of alleles at these new microsatellite loci was 8.2 with a polymorphism content value (PIC) of 0.63. These 38 markers in total almost uniformly interspersed in the HLA class I region will enable us to search precisely for the location of disease susceptible loci within the HLA class I region by association and for linkage analyses.

DNA↗

PubMed1999

HLA class I and II typing of the patients with Behçet's disease in Saudi Arabia.

Thirteen Saudi Arabian patients with Behçet's disease (BD) were typed for HLA-A and -B alleles by the conventional serologic typing and for HLA-DRB1, -DQB1 and -DPB1 alleles by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. As a result, the phenotype frequency of the B51 antigen was significantly increased in the patient group as compared to the ethnically matched control group (76.9% in patients vs. 22.20% in controls), but no significant difference was observed in HLA-A, -DRB1, -DQB1 or -DPB1 alleles between the patients and controls, as previously observed in Japanese BD. Further, by HLA-B51 allelic genotyping performed by the polymerase chain reaction-sequence specific primers (PCR-SSP) method, all of the B51-positive patients and controls were found to carry one particular allele, B*5101, except one patient with B*5108.

Behcet Syndrome↗

PubMed1999

Two cases of pyoderma gangrenosum complicated with nasal septal perforation.

We report two patients with pyoderma gangrenosum complicated with nasal septal perforation. An 18-year-old woman and a 65-year-old man had typical lesions of pyoderma gangrenosum on the legs that responded well to oral prednisolone. Both patients complained of mild nasal discharge, and nasal fibroscopy revealed nasal septal perforation. Biopsy of the nasal lesions showed an active inflammatory infiltrate, mainly of neutrophils. Systemic investigations failed to show any pulmonary or renal lesions of Wegener's granulomatosis. Cytoplasmic immunofluorescent pattern antineutrophil cytoplasmic antibody was negative. In both cases, intense neutrophilic infiltration was observed not only in skin lesions but also in nasal lesions, which may indicate that the nasal lesions had a pathogenesis in common with the skin lesions.

Adolescent↗

PubMed1999

Selective incorporation and specific cytocidal effect as the cellular basis for the antimelanoma action of sulphur containing tyrosine analogs.

Tyrosine analogs are good candidates for developing melanoma chemotherapy because melanogenesis is inherently toxic and uniquely expressed in melanocytic cells. Sulphur containing substrate (tyrosine) analogs, N-acetyl-4-S-cysteaminylphenol (NAcCAP) and N-propionyl-4-S-cysteaminylphenol (NPrCAP), have been shown to have potent antimelanoma activity in mice bearing melanoma. Both NAcCAP and NPrCAP show selective cytotoxicity towards melanoma cell lines. But the mechanism leading to selectivity is not clear as these drugs are also toxic to other cell lines to a lesser extent. Here we show that these drugs have both cytostatic and cytocidal effects, which could account for this. Cytostatic effect is suggested by DNA flow cytometry. The drug causes cell cycle changes in four human cell lines (normal skin fibroblasts, HeLa cells, and melanoma cell lines, C32 and SK-MEL-23) in a dose-dependent manner blocking cells in S phase with concomitant decrease in the number of cells in G1 phase. There is also a gradual decrease in cells in G2 + M phases. The dose-concentration curves give IC50 values in the range of 50-400 microM and the melanotic melanoma cell line SK-MEL-23 has the lowest IC50 value consistent with our hypothesis that these drugs are selective towards melanoma cells. The concentration-dependent accumulation of cells in S phase suggest a cytostatic effect as a consequence of inhibition of DNA synthesis in agreement with [3H] thymidine incorporation assay. There is a highly specific uptake of [14C]NAcCAP and irreversible damage to DNA synthesis machinery in SK-MEL-23 cells, indicating a melanotic-specific cytocidal effect as well. Trypan blue exclusion study and competitive inhibition assay indicated that visible cytocidal effect occurs slowly and oxidative stress resulting from tyrosinase mediated oxidation of the drug appears to be the underlying mechanism. The primary antimelanoma effect of cysteaminylphenols derives from a selective cytostatic effect, but is followed by a specific cytocidal action rendering the drugs useful for targeted melanoma chemotherapy.

Animals↗

PubMed1999

The critical region for Behçet disease in the human major histocompatibility complex is reduced to a 46-kb segment centromeric of HLA-B, by association analysis using refined microsatellite mapping.

The HLA-B51 allele is known to be associated with Behçet disease. Recently, we found a higher risk for Behçet disease in the MICA gene, 46 kb centromeric of HLA-B, by investigation of GCT repetitive polymorphism within exon 5 of MICA. The pathogenic gene causing Behçet disease, however, has remained uncertain. Here, eight polymorphic microsatellite markers, distributed over a 900-kb region surrounding the HLA-B locus, were subjected to association analysis for Behçet disease. Statistical studies of associated alleles detected on each microsatellite locus showed that the pathogenic gene for Behçet disease is most likely found within a 46-kb segment between the MICA and HLA-B genes. The results of this mapping study, and the results of an earlier study of ours, suggest that MICA is a strong candidate gene for the development of Behçet disease.

Behcet Syndrome↗

PubMed1999

Association analysis using refined microsatellite markers localizes a susceptibility locus for psoriasis vulgaris within a 111 kb segment telomeric to the HLA-C gene.

The HLA-Cw6 antigen has been associated with psoriasis vulgaris despite racial and ethnic differences. However, it remains unclear whether it is the HLA-Cw6 antigen itself or a closely linked, hitherto unidentified, locus that predisposes to the disease. Here, in order to map the susceptibility locus for psoriasis vulgaris precisely within the HLA class I region, 11 polymorphic microsatellite markers distributed throughout a 1060 kb segment surrounding the HLA-C locus were subjected to association analysis in Japanese psoriasis vulgaris patients. Statistical analyses of the distribution and deviation from Hardy-Weinberg equilibrium of the allelic frequency at each micro-satellite locus revealed that the pathogenic gene for psoriasis vulgaris is located within a reduced interval of 111 kb spanning 89-200 kb telomeric of the HLA-C gene. In addition to three known genes, POU5F1, TCF19 and S, this 111 kb fragment contains four new, expressed genes identified in the course of our genomic sequencing of the entire HLA class I region. Therefore, these seven genes are the potential candidates for susceptibility to psoriasis vulgaris.

Adult↗

PubMed1999

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.

Amino Acid Substitution↗