Biomedical subjects

S Kuhara

Publications and source records attributed to S Kuhara.

At least 19 recordsLinked to original sources

PubMed2001

Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Animals↗

PubMed2001

In synergy with noggin and follistatin, Xenopus nodal-related gene induces sonic hedgehog on notochord and floor plate.

In early development of vertebrates, sonic hedgehog functions in dorsal-ventral patterning of dorsal tissue (nervous system and somites). In Xenopus, sonic hedgehog (Xshh) is first expressed in the Spemann organizer/notochord and floor plate. We report here the mechanism governing Xshh mRNA induction in these regions. In animal cap assays, the antagonizing BMPs signal was not sufficient to induce Xshh mRNA expression; however, it could induce Xshh mRNA expression in the presence of Xnr-1. In whole embryos, when secondary axes were induced by coexpressing noggin and Xnr-1 or follistatin and Xnr-1, Xshh mRNA expression was observed in the notochord and floor plate within the induced axes. It seems apparent that spatially restricted Xshh mRNA expression is determined as intersection of the two signals.

Animals↗

PubMed2001

Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12.

Escherichia coli O157:H7 is a major food-borne infectious pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Here we report the complete chromosome sequence of an O157:H7 strain isolated from the Sakai outbreak, and the results of genomic comparison with a benign laboratory strain, K-12 MG1655. The chromosome is 5.5 Mb in size, 859 Kb larger than that of K-12. We identified a 4.1-Mb sequence highly conserved between the two strains, which may represent the fundamental backbone of the E. coli chromosome. The remaining 1.4-Mb sequence comprises of O157:H7-specific sequences, most of which are horizontally transferred foreign DNAs. The predominant roles of bacteriophages in the emergence of O157:H7 is evident by the presence of 24 prophages and prophage-like elements that occupy more than half of the O157:H7-specific sequences. The O157:H7 chromosome encodes 1632 proteins and 20 tRNAs that are not present in K-12. Among these, at least 131 proteins are assumed to have virulence-related functions. Genome-wide codon usage analysis suggested that the O157:H7-specific tRNAs are involved in the efficient expression of the strain-specific genes. A complete set of the genes specific to O157:H7 presented here sheds new insight into the pathogenicity and the physiology of O157:H7, and will open a way to fully understand the molecular mechanisms underlying the O157:H7 infection.

Bacterial Proteins↗

PubMed2000

Glycine-rich region of mitochondrial processing peptidase alpha-subunit is essential for binding and cleavage of the precursor proteins.

Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off amino-terminal extension peptides. The alpha-subunit has a characteristic glycine-rich segment in the middle portion. To elucidate the role of the region in processing functions of the enzyme, deletion or site-directed mutations were introduced, and effects on kinetic parameters and substrate binding of the enzyme were analyzed. Deletion of three residues of the region, Phe(289) to Ala(291), led to a dramatic reduction in processing activity to practically zero. Mutation of Phe(289), Lys(296), and Met(298) to alanine resulted in a decrease in the activity, but these mutations had no apparent effect on interactions between the two subunits, indicating that reduction in processing activity is not due to structural disruption at the interface interacting with the beta-subunit. Although the mutant enzymes, Phe289Ala, Lys296Ala, and Met298Ala, had an approximate 10-fold less affinity for substrate peptides than did that of the wild type, the deletion mutant, delta 289-291, showed an extremely low affinity. Thus, shortening of the glycine-rich stretch led to a dramatic reduction of interaction between the enzyme and substrate peptides and cleavage reaction, whereas mutation of each amino acid in this region seemed to affect primarily the cleavage reaction.

Amino Acid Sequence↗

PubMed2000

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis.

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18. 3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B. halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 sigma factors which belong to the extracytoplasmic function family, 10 are unique to B. halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.

ATP-Binding Cassette Transporters↗

PubMed2000

Gene responses in bean leaves induced by herbivory and by herbivore-induced volatiles.

Plant-plant interactions via herbivory-induced leaf volatiles could result in the induction of defense responses against aggressive biotic agents in plants. In this study, cDNA microarray technology showed comprehensive gene activation in lima bean leaves that were exposed to volatiles released from the neighboring leaves infested with spider mites. The infestation with spider mites and the herbivory-induced volatiles enhanced 97 and 227 gene spots on the microarray tip printed with 2032 lima bean cDNA, respectively. These genes are related to such broad functions as responses to pathogenesis, wounding, hormones, ethylene biosynthesis, flavonoid biosynthesis, (post) transcriptional modifications, translations, chaperones, secondary signaling messengers, membrane transports, protein/peptide degradations, and photosynthesis. We therefore conclude that herbivorous damage and herbivory-induced volatiles elicit comprehensive and drastic changes of metabolisms in leaves.

Animals↗

PubMed2000

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217.

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.

Amino Acid Motifs↗

PubMed2000

Comparison of whole genome sequences of Chlamydia pneumoniae J138 from Japan and CWL029 from USA.

Chlamydia pneumoniae is a widespread pathogen of humans causing pneumonia and bronchitis. There are many reports of an association between C.PNEUMONIAE: infection and atherosclerosis. We determined the whole genome sequence of C.PNEUMONIAE: strain J138 isolated in Japan in 1994 and compared it with the sequence of strain CWL029 isolated in the USA before 1987. The J138 circular chromosome consists of 1 226 565 nt (40.7% G+C) with 1072 likely protein-coding genes that is 3665 nt shorter than the CWL029 genome. Plasmids, phage- or transposon-like sequences were not identified. The overall genomic organization, gene order and predicted proteomes of the two strains are very similar, suggesting a high level of structural and functional conservation between the two unrelated isolates. The most conspicuous differences in the J138 genome relative to the CWL029 genome are the absence of five DNA segments, ranging in size from 89 to 1649 nt, and the presence of three DNA segments, ranging from 27 to 84 nt. The complex organization of these 'different zones' may be attributable to a unique system of recombination.

Base Composition↗

PubMed2000

Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins.

Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATalpha strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of approximately 4 x 10(6) different combinations, constituting approximately 10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

DNA, Fungal↗

PubMed2000

Comparative analysis of the whole set of rRNA operons between an enterohemorrhagic Escherichia coli O157:H7 Sakai strain and an Escherichia coli K-12 strain MG1655.

Two primer sets for direct sequence determination of all seven rRNA operons (rrn) of Escherichia coli have been developed; one is for specific-amplification of each rrn operon and the other is for direct sequencing of the amplified operons. Using these primer sets, we determined the nucleotide sequences of seven rrn operons, including promoter and terminator regions, of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai strain. To elucidate the intercistronic or intraspecific variation of rrn operons, their sequences were compared with those for the K-12 rrn operons. The rrn genes and the internal transcribed spacer regions showed a higher similarity to each other in each strain than between the corresponding operons of the two strains. However, the degree of intercistronic homogeneity was much higher in the EHEC strain than in K-12. In contrast, promoter and terminator regions in each operons were conserved between the corresponding operons of the two strains, which exceeded intercistronic similarity.

Base Sequence↗

PubMed2000

Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States.

Chlamydia pneumoniae is a widespread pathogen of the respiratory tract that is also associated with atherosclerosis. The whole genome sequence was determined for a Japanese isolate, C. pneumoniae strain J138. The sequence predicted a variety of genes encoding outer membrane proteins (OMPs) including ompA and porB, another 10 predicted omp genes, and 27 pmp genes. All were detected in the whole genome sequence of strain CWL029, a strain isolated and sequenced in the United States. A comparative study of the OMPs of the two strains revealed a nucleotide sequence identity of 89.6%-100% (deduced amino acid sequence identity, 71.1%-100%). The overall genomic organization and location of genes are identical in both strains. Thus, a few unique sequences of the OMPs may be essential for specific attributes that define the differential biology of two C. pneumoniae strains.

Bacterial Outer Membrane Proteins↗

PubMed2000

Algorithms for identifying Boolean networks and related biological networks based on matrix multiplication and fingerprint function.

Due to the recent progress of the DNA microarray technology, a large number of gene expression profile data are being produced. How to analyze gene expression data is an important topic in computational molecular biology. Several studies have been done using the Boolean network as a model of a genetic network. This paper proposes efficient algorithms for identifying Boolean networks of bounded indegree and related biological networks, where identification of a Boolean network can be formalized as a problem of identifying many Boolean functions simultaneously. For the identification of a Boolean network, an O(mnD+1) time naive algorithm and a simple O (mnD) time algorithm are known, where n denotes the number of nodes, m denotes the number of examples, and D denotes the maximum in degree. This paper presents an improved O(momega-2nD + mnD+omega-3) time Monte-Carlo type randomized algorithm, where omega is the exponent of matrix multiplication (currently, omega < 2.376). The algorithm is obtained by combining fast matrix multiplication with the randomized fingerprint function for string matching. Although the algorithm and its analysis are simple, the result is nontrivial and the technique can be applied to several related problems.

Algorithms↗

PubMed2000

Inferring qualitative relations in genetic networks and metabolic pathways.

MOTIVATION: Inferring genetic network architecture from time series data of gene expression patterns is an important topic in bioinformatics. Although inference algorithms based on the Boolean network were proposed, the Boolean network was not sufficient as a model of a genetic network. RESULTS: First, a Boolean network model with noise is proposed, together with an inference algorithm for it. Next, a qualitative network model is proposed, in which regulation rules are represented as qualitative rules and embedded in the network structure. Algorithms are also presented for inferring qualitative relations from time series data. Then, an algorithm for inferring S-systems (synergistic and saturable systems) from time series data is presented, where S-systems are based on a particular kind of nonlinear differential equation and have been applied to the analysis of various biological systems. Theoretical results are shown for Boolean networks with noises and simple qualitative networks. Computational results are shown for Boolean networks with noises and S-systems, where real data are not used because the proposed models are still conceptual and the quantity and quality of currently available data are not enough for the application of the proposed methods.

Algorithms↗

PubMed2000

Magnitude of structural changes of the T-cell receptor binding regions determine the strength of T-cell antagonism: molecular dynamics simulations of HLA-DR4 (DRB1*0405) complexed with analogue peptide.

In our model system, we generated T cell clones specific for the HLA-DR4 (DRB1*0405)-index peptide (YWALEAAAD) complex. Based on response patterns of the T cell clones, analogue peptides containing single amino acid substitutions of the index peptide were classified into three types, agonists, antagonists or null peptides (non-agonistic and non-antagonistic peptides). Subtle structural changes induced by the antagonists in the T-cell receptor (TCR) binding regions have already been explained using the root mean square (r.m.s.) deviations from the DR4-index peptide complex in the molecular dynamics (MD) trajectory. In this work, we performed additional MD simulations at 300 K with explicit solvent molecules to reveal the structural character of the HLA-DR4 complexed with the analogue peptides. We examined the r.m.s. deviations of the TCR-binding sites and the exposed areas of the bound peptides. Remarkable differences of the r.m.s. deviations among the DR4-antagonist complexes, together with our previous data, suggest that the magnitude of structural changes of TCR-binding regions would determine the strength of TCR antagonism. The simulations also indicate that TCR could discriminate null peptides from other ligands mainly through the changes of exposed side chains of the bound peptide, rather than the conformational changes of TCR-binding surfaces on HLA molecule.

Binding Sites↗

PubMed2000

Algorithms for inferring qualitative models of biological networks.

Modeling genetic networks and metabolic networks is an important topic in bioinformatics. We propose a qualitative network model which is a combination of the Boolean network and qualitative reasoning, where qualitative reasoning is a kind of reasoning method well-studied in Artificial Intelligence. We also present algorithms for inferring qualitative networks from time series data and an algorithm for inferring S-systems (synergistic and saturable systems) from time series data, where S-systems are based on a particular kind of nonlinear differential equation and have been applied to the analysis of various biological systems.

Algorithms↗

PubMed2000

An automated system for genome analysis to support microbial whole-genome shotgun sequencing.

We developed a semi-automated genome analysis system called GAMBLER in order to support the current whole-genome sequencing project focusing on alkaliphilic Bacillus halodurans C-125. GAMBLER was designed to reduce the human intervention required and to reduce the complications in annotating thousands of ORFs in the microbial genome. GAMBLER automates three major routines: analyzing assembly results provided by genome assembler software, assigning ORFs, and homology searching. GAMBLER is equipped with an interface for convenience of annotation. All processes and options are manipulatable through a WWW browser that enables scientists to share their genome analysis results without choosing computer platforms.

Automation↗

PubMed1999

Chromosome of the enterohemorrhagic Escherichia coli O157:H7; comparative analysis with K-12 MG1655 revealed the acquisition of a large amount of foreign DNAs.

A complete Xba I and Bln I cleavage map was constructed for the chromosome of an enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain isolated from an outbreak in Sakai City, Japan, in 1996. A comparative chromosome analysis with E. coli K-12 strain MG1655 was made. The EHEC chromosome was approximately 5600 kb in length, 1 Mb larger than that of MG1655. Despite the marked difference in chromosome length, the location and direction of seven rRNA operons of the EHEC strain were similar to those for MG1655. Overall organization of genes common in both strains is also highly conserved. Chromosome expansion was observed throughout the EHEC chromosome, albeit in an uneven manner. A large portion of the chromosome enlargement was observed in the region surrounding the replication terminus, particularly in a segment containing the terA locus. Sample sequencing of 3627 random shotgun clones suggested the presence of approximately 1550 kb strain-specific DNAs on the EHEC chromosome, most of which are likely to be of foreign origin.

Bacterial Toxins↗

PubMed1999

The hydrophobic cores of proteins predicted by wavelet analysis.

MOTIVATION: In the process of protein construction, buried hydrophobic residues tend to assemble in a core of a protein. Methods used to predict these cores involve use or no use of sequential alignment. In the case of a close homology, prediction was more accurate if sequential alignment was used. If the homology was weak, predictions would be unreliable. A hydrophobicity plot involving the hydropathy index is useful for purposes of prediction, and smoothing is essential. However, the proposed methods are insufficient. We attempted to predict hydrophobic cores with a low frequency extracted from the hydrophobicity plot, using wavelet analysis. RESULTS: The cores were predicted at a rate of 68.7%, by cross-validation. Using wavelet analysis, the cores of non-homologous proteins can be predicted with close to 70% accuracy, without sequential alignment. AVAILABILITY: The program used in this study is available from Intergalactic Reality (http://www.intergalact.com). CONTACT: hirakawa@grt.kyushu-u.ac.jp, kuhara@grt.kyushu-u.ac.jp

Computer Simulation↗