Biomedical subjects

Ken Kurokawa

Publications and source records attributed to Ken Kurokawa.

16 recordsLinked to original sources

PubMed2026

Cohesin prevents local mixing of condensed euchromatic domains in living human cells.

The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, how cohesin organizes these domains in living cells, especially in active euchromatin, remains elusive. Here, to address this question, we combined single-nucleosome imaging/tracking and super-resolution three-dimensional structured illumination microscopy with euchromatin-specific labeling of histone variant H3.3. Using this nanoscopic approach, we revealed that euchromatin forms condensed domains that are constrained by cohesin-mediated loops. This organization refines the classical view of euchromatin as largely open, in line with emerging evidence. Transcription machinery appears to be located near the condensed domain surfaces/borders. Cohesin loss increased nucleosome-level fluidity within these domains without altering their overall compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings suggest a physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.

Humans↗

PubMed2006

Escherichia coli histone-like protein H-NS preferentially binds to horizontally acquired DNA in association with RNA polymerase.

Heat-stable nucleoid-structuring protein (H-NS) is one of the main nucleoid proteins expressed in exponentially growing Escherichia coli cells. In addition to a role in nucleoid organization, H-NS functions as a pleiotropic regulator of gene expression. The genome-wide distribution of H-NS, compared with the distribution of RNA polymerase and transcriptionally active genes, was investigated using a high-density oligonucleotide chip. The new approach utilized in this study revealed that H-NS binds specifically to approximately 250 loci, covering >1000 genes, to maintain transcriptional inactivation. RNA polymerase was detected in >65% of H-NS binding sites with low or no transcriptional activity, indicating that the association of RNA polymerase to promoter regions is a general mode of transcription repression by H-NS. This study also revealed that most H-NS bound DNA have been horizontally acquired, which indicates that repression of inappropriate gene expression by H-NS plays an important role in the diversification of the E. coli genome. This study presents a comprehensive assessment of the distribution of H-NS within the E. coli genome, sheds light on the mechanism underlying the transcriptional regulation by H-NS, and provides new insight into bacterial genome evolution.

Bacterial Proteins↗

PubMed2006

Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH).

Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol-anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGA-mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3' untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow.

Biomarkers, Tumor↗

PubMed2006

PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria.

Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.

Amino Acid Sequence↗

PubMed2006

Development and implementation of an algorithm for detection of protein complexes in large interaction networks.

BACKGROUND: After complete sequencing of a number of genomes the focus has now turned to proteomics. Advanced proteomics technologies such as two-hybrid assay, mass spectrometry etc. are producing huge data sets of protein-protein interactions which can be portrayed as networks, and one of the burning issues is to find protein complexes in such networks. The enormous size of protein-protein interaction (PPI) networks warrants development of efficient computational methods for extraction of significant complexes. RESULTS: This paper presents an algorithm for detection of protein complexes in large interaction networks. In a PPI network, a node represents a protein and an edge represents an interaction. The input to the algorithm is the associated matrix of an interaction network and the outputs are protein complexes. The complexes are determined by way of finding clusters, i. e. the densely connected regions in the network. We also show and analyze some protein complexes generated by the proposed algorithm from typical PPI networks of Escherichia coli and Saccharomyces cerevisiae. A comparison between a PPI and a random network is also performed in the context of the proposed algorithm. CONCLUSION: The proposed algorithm makes it possible to detect clusters of proteins in PPI networks which mostly represent molecular biological functional units. Therefore, protein complexes determined solely based on interaction data can help us to predict the functions of proteins, and they are also useful to understand and explain certain biological processes.

Algorithms↗

PubMed2006

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.

Combinatorial Chemistry Techniques↗

PubMed2005

The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny.

Botulinum neurotoxins (BoNTXs) produced by Clostridium botulinum are among the most poisonous substances known. Of the seven types of BoNTXs, genes for type C1 and D toxins (BoNTX/C1 and D) are carried by bacteriophages. The gene for exoenzyme C3 also resides on these phages. Here, we present the complete genome sequence of c-st, a representative of BoNTX/C1-converting phages. The genome is a linear double-stranded DNA of 185,682 bp with 404-bp terminal direct repeats, the largest known temperate phage genome. We identified 198 potential protein-coding regions, including the genes for production of BoNTX/C1 and exoenzyme C3. Very exceptionally, as a viable bacteriophage, a number of insertion sequences were found on the c-st genome. By analyzing the molecular structure of the c-st genome in lysogens, we also found that it exists as a circular plasmid prophage. These features account for the unstable lysogeny of BoNTX phages, which has historically been called "pseudolysogeny." The PCR scanning analysis of other BoNTX/C1 and D phages based on the c-st sequence further revealed that BoNTX phages comprise a divergent phage family, probably generated by exchanging genomic segments among BoNTX phages and their relatives.

Bacteriophages↗

PubMed2005

Cell lysis directed by sigmaE in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli.

It has been shown that Escherichia coli cells with increased expression of the rpoE gene encoding sigma(E) exhibit enhanced cell lysis in early stationary phase. Further analysis of the lysis phenomenon was performed using a transient expression system of the rpoE gene and by DNA microarray. The former analysis revealed a sigma(E)-directed cell lysis, specific for early stationary phase but not for the exponential phase. The microarray analysis with RNAs from exponential and early stationary phase cells revealed that a large number of genes were up- or down-regulated when the rpoE gene was induced, and that several genes were induced in a phase-specific manner. The upregulated genes include many previously identified sigma(E) regulon genes, suggesting that a large number of genes are under the control of sigma(E) in this organism. These genes are involved in various cellular activities, including the cell envelope, cellular processes, regulatory functions, transport and translation. Genes that are presumably related to phase-specific cell lysis in E. coli are discussed.

Escherichia coli↗

PubMed2004

Distinct genomic sequence of the CNR/Pcdhalpha genes in chicken.

CNR/Pcdhalpha family proteins have been first identified as a receptor family that corporate with Fyn, a family of the Src family of tyrosine kinase, and known as synaptic cadherins. Here we report the complete genomic sequence and organization of the chicken (Gallus gallus) CNR/Pcdhalpha The total length of chicken CNR/Pcdhalpha is 177kb. The chicken CNR/Pcdhalpha cluster encodes 12 variable and 3 constant exons. The genomic organizations of the chicken, rat, mouse, and human CNR/Pcdhalpha are basically orthologous. The constant-region exons (CP1, CP2, and CP3) are highly conserved between chicken and mammals, with percent identities of 90.9%, 90.7%, and 91.8% at the amino-acid level for chicken versus rat, mouse, and human, respectively. In contrast, the percent identities of the variable-region exons between chicken and mammals were lower: 51.8%, 51.3%, and 52.7%, on average, for chicken versus rat, mouse, and human, respectively, at the amino-acid level. Moreover, the chicken variable-region exons (from v1 to v12) are highly conserved paralogously (91.4%: nucleic acid, 92.4%: amino acid) in comparison with those of mammals. The CG content of each variable exon in the chicken (v1 to v12) is 74% on average and the CpG dinucleotide frequency in each variable-region exon is twice that of mammals. Due to the high CG content, chicken variable exons (from v1 to v12) encode 3 to 4 frame-shifted open reading frames, which span 1.5-3.0kb, in both the sense and anti-sense orientations.

Animals↗

PubMed2004

Application of revised version of neural independent component analysis to classification problems of confiscated methamphetamine.

Recently, profiling the chemical substances in illegally distributed drugs has been needed in order to reveal the drug channels. However, this kind of profiling is often difficult because such drugs contain various kinds of impurities and the quantity of these impurities changes. Due to these circumstances, several methods, including a slightly revised ICA (Independent Component Analysis) by a Hebbian learning artificial neural network, were applied for profiling illegally distributed methamphetamine. Eventually, better classification results with the ICA than with other methods were obtained. These results show that ICA could make it easier to profile illegally distributed methamphetamine.

Algorithms↗

PubMed2003

Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae.

BACKGROUND: Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. METHODS: The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. FINDINGS: The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. INTERPRETATION: The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.

Cholera↗

PubMed2003

Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution.

Group Astreptococcus (GAS) is a gram-positive bacterial pathogen that causes various suppurative infections and nonsuppurative sequelae. Since the late 1980s, streptococcal toxic-shock like syndrome (STSS) and severe invasive GAS infections have been reported globally. Here we sequenced the genome of serotype M3 strain SSI-1, isolated from an STSS patient in Japan, and compared it with those of other GAS strains. The SSI-1 genome is composed of 1,884,275 bp, and 1.7 Mb of the sequence is highly conserved relative to strain SF370 (serotype M1) and MGAS8232 (serotype M18), and almost completely conserved relative to strain MGAS315 (serotype M3). However, a large genomic rearrangement has been shown to occur across the replication axis between the homologous rrn-comX1 regions and between two prophage-coding regions across the replication axis. Atotal of 1 Mb of chromosomal DNA is inverted across the replication axis. Interestingly, the recombinations between the prophage regions are within the phage genes, and the genes encoding superantigens and mitogenic factors are interchanged between two prophages. This genomic rearrangement occurs in 65% of clinical isolates (64/94) collected after 1990, whereas it is found in only 25% of clinical isolates (7/28) collected before 1985. These observations indicate that streptococcal phages represent important plasticity regions in the GAS chromosome where recombination between homologous phage genes can occur and result not only in new phage derivatives, but also in large chromosomal rearrangements.

Attachment Sites, Microbiological↗

PubMed2002

Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning.

Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse XbaI-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage.

Chromosomes, Bacterial↗

PubMed2002

Plasmodium falciparum phospholipase C hydrolyzing sphingomyelin and lysocholinephospholipids is a possible target for malaria chemotherapy.

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is approximately 25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Amides↗

PubMed2002

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells.

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.

Amino Acid Sequence↗

PubMed2002

Study of transcripts from AC010088, a 199,485 bp fragment of the human Y chromosome located in the azoospermia factor region c.

Deletions on the long arm of the human Y chromosome are associated with male infertility. In this work, we studied transcripts of a 199,485 bp long fragment of the Yq11 region (GenBank accession number, AC010088) located in the AZFc (azoospermia factor region c), and characterized their gene structures. After masking repetitive elements, we searched human mRNA Refseqs (reference sequences), a dbEST (database of expressed sequence tags) and a non-redundant nucleic acid database for the mRNAs and ESTs corresponding to the AC010088 using the BLAST programs at the NCBI (National Center for Biotechnology Information) site. Our findings are summarized as follows: i) BPY2 (testis basic protein on Y, 2), DAZ1 (deleted in azoospermia 1), TTY4 (testis transcript Y 4) mRNAs and 23 ESTs were found; ii) Eighteen of 23 ESTs were transcripts of the DAZ gene(s), one EST was a transcript of TTY4 gene, and the remaining 4 probably corresponded to 4 different pseudogenes; iii) DAZ gene(s) were expressed not only in testis, but also in lung carcinoma cells, stomach and Ewing's sarcoma cells; iv) beta-satellite clusters were present around and within the BPY2 and TTY4 gene region; v) In this study, TTY4, BPY2 and DAZ1 genes were mapped precisely to the AC010088 region.

Chromosome Mapping↗