Biomedical subjects

Takashi Horiuchi

Publications and source records attributed to Takashi Horiuchi.

At least 19 recordsLinked to original sources

PubMed2007

Escherichia coli with a linear genome.

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.

Cell Proliferation↗

PubMed2006

Intercellular localization of occludins and ZO-1 as a solute transport barrier of the mesothelial monolayer.

A hyperpermeable state has been observed in patients on long-term peritoneal dialysis. To understand the causes of the structural or functional changes and the progression of the fibrotic process, it is important to determine which region of the peritoneum exhibits these changes. The objectives of this study were to determine the solute permeability associated with cell-cell adhesion of human peritoneal mesothelial cells (HPMCs), to study the relationship between solute permeability and localizations of tight junction-associated proteins (TJPs: occludins and ZO-1), and to assess the effect of exogenous H2O2 supplementation. HPMCs were cultured on a Transwell until the transmesothelial electrical resistance (TER) reached a plateau. Solute permeation tests were conducted using fluorescein isothiocyanate - labeled dextran (molecular weight: 4, 10, 70, and 150 kDa) to calculate the solute permeability coefficient (SPC). Localization of TJPs was observed by a confocal laser scanning microscope after immunofluorescent staining. TER levels increased steadily, beginning at 97.5 +/- 0.7 ohms.cm2 and leveling off at 128 +/- 3.6 ohms.cm2 (n = 4). This was accompanied by the confluence of cells and the appearance of localized TJPs. SPC levels of the HPMC monolayer on the Transwell were reduced compared to those of the Transwell itself, indicating that the HPMC monolayer provided resistance against solute permeation. Exogenous H2O2 supplementation revealed an increased permeability accompanied with delocalization of TJPs, particularly occludins. The delocalization of occludins and ZO-1 at the intercellular space led to a decrease in intercellular binding capacity and thus triggered an increase in the solute permeability.

Cells, Cultured↗

PubMed2006

Development of a new method for endovascular aortic repair: combination therapy of cell transplantation and stent grafts with a drug delivery system.

BACKGROUND: Endovascular aortic repair by stent grafts (S/Gs) has been developed as a less invasive treatment for aortic aneurysms. However, some aneurysmal cavities can remain without organization, causing re-expansion. We demonstrated previously that transplantation of a cell combination (myoblasts and fibroblasts) promoted thrombus organization in a rat model. We also developed basic fibroblast growth factor (bFGF) slow-delivery S/Gs coated with elastin and impregnated with bFGF. Here, we evaluated the effects of cell transplantation combined with bFGF slow release on canine thoracic aortic aneurysmal sacs after S/Gs repair. METHODS AND RESULTS: Thoracic aortic aneurysms were surgically created with jugular vein patches in 15 beagles. Myoblasts and fibroblasts of autologous skeletal muscle were isolated and cultured for cell transplantation. The S/Gs had 6 holes and produced endoleaks in the excluded cavities. Collagen gel (gel group, n=5) or a mixture of skeletal myoblasts and fibroblasts with collagen gel (cell group, n=5) were injected into the aneurysmal sacs excluded by the S/Gs. We also studied the effects of combined therapy of bFGF slow-release S/Gs and cell transplantation (hybrid group, n=5). After 14 days, histological analyses revealed that the excluded aneurysmal cavities of the gel group were filled with fresh thrombus, whereas the excluded cavities in the cell-transplanted groups were occupied by organized tissue. The percentages of the organized areas relative to the excluded cavities, evaluated by Masson's trichrome staining, were 18.1+/-4.0%, 52.6+/-4.0%, and 77.1+/-6.9% in the gel, cell, and hybrid groups, respectively. Collagen fibers had already appeared, and increased numbers of alpha-smooth muscle actin-positive cells were observed in the hybrid group. CONCLUSIONS: Cell transplantation accelerated thrombus organization. Moreover, slow release of bFGF enhanced the effects of cell transplantation. Cell transplantation into unorganized spaces may improve the outcomes of endovascular treatments of aortic aneurysms.

Animals↗

PubMed2006

Arabidopsis TEBICHI, with helicase and DNA polymerase domains, is required for regulated cell division and differentiation in meristems.

In plant meristems, each cell divides and differentiates in a spatially and temporally regulated manner, and continuous organogenesis occurs using cells derived from the meristem. We report the identification of the Arabidopsis thaliana TEBICHI (TEB) gene, which is required for regulated cell division and differentiation in meristems. The teb mutants show morphological defects, such as short roots, serrated leaves, and fasciation, as well as defective patterns of cell division and differentiation in the meristem. The TEB gene encodes a homolog of Drosophila MUS308 and mammalian DNA polymerase theta, which prevent spontaneous or DNA damage-induced production of DNA double strand breaks. As expected from the function of animal homologs, teb mutants show constitutively activated DNA damage responses. Unlike other fasciation mutants with activated DNA damage responses, however, teb mutants do not activate transcriptionally silenced genes. teb shows an accumulation of cells expressing cyclinB1;1:GUS in meristems, suggesting that constitutively activated DNA damage responses in teb lead to a defect in G2/M cell cycle progression. Furthermore, other fasciation mutants, such as fasciata2 and tonsoku/mgoun3/brushy1, also show an accumulation of cells expressing cyclinB1;1:GUS in meristems. These results suggest that cell cycle progression at G2/M is important for the regulation of the pattern of cell division and of differentiation during plant development.

Arabidopsis↗

PubMed2006

Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110.

With the goal of solving the whole-cell problem with Escherichia coli K-12 as a model cell, highly accurate genomes were determined for two closely related K-12 strains, MG1655 and W3110. Completion of the W3110 genome and comparison with the MG1655 genome revealed differences at 267 sites, including 251 sites with short, mostly single-nucleotide, insertions or deletions (indels) or base substitutions (totaling 358 nucleotides), in addition to 13 sites with an insertion sequence element or defective prophage in only one strain and two sites for the W3110 inversion. Direct DNA sequencing of PCR products for the 251 regions with short indel and base disparities revealed that only eight sites are true differences. The other 243 discrepancies were due to errors in the original MG1655 sequence, including 79 frameshifts, one amino-acid residue deletion, five amino-acid residue insertions, 73 missense, and 17 silent changes within coding regions. Errors in the original MG1655 sequence (<1 per 13,000 bases) were mostly within portions sequenced with out-dated technology based on radioactive chemistry.

Base Sequence↗

PubMed2006

Escherichia coli K-12: a cooperatively developed annotation snapshot--2005.

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

Congresses as Topic↗

PubMed2006

Condensin loaded onto the replication fork barrier site in the rRNA gene repeats during S phase in a FOB1-dependent fashion to prevent contraction of a long repetitive array in Saccharomyces cerevisiae.

An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.

Adenosine Triphosphatases↗

PubMed2006

[Residue analysis of metoclopramide in bovine and swine tissues by HPLC and administration experiment].

A reversed-phase ion-pair HPLC method with ultraviolet detection has been developed for determination of metoclopramide (MCP) in bovine and swine muscle, liver, kidney, fat and intestine. MCP was extracted from samples with acetonitrile, and the extracts were cleaned up on an Oasis HLB cartridge (60 mg) after liquid-liquid extraction. The limit of detection of MCP was 0.002 microg/g and the limit of quantitation was 0.007 microg/g. Recoveries of MCP spiked at 0.03 ppm ranged from 74.1 to 93.3% for bovine tissues and from 86.1 to 92.7% for swine tissues. The present method was used for the analysis of bovine and swine tissues 1 day after withdrawal following drug administration. The MCP concentrations in all tissues were lower than the Japanese provisional MRLs.

Adipose Tissue↗

PubMed2006

[Determination of glycarbylamide residues in chicken tissues by HPLC with post-column reaction].

A newly developed HPLC method for determination of glycarbylamide (GB) in chicken liver was applied to other tissues (muscle, fat and kidney). The recoveries of GB in muscle, fat and kidney were 87.2% (CV 0.5), 91.3% (CV 4.7), 79.7% (CV 1.0), respectively. The detection limit of GB was 0.01 ppm. GB concentrations were determined by this method in tissues (muscle, fat, liver and kidney) from chickens sacrificed 5 days after oral administration of GB mixed in feed at 60 mg/kg of feed for 7 days. GB was not detected in these chicken tissues.

Adipose Tissue↗

PubMed2005

Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting.

Sequences involved in the regulation of genetic and genomic processes are primarily located in noncoding regions. Identifying such cis-acting sequences from sequence data is difficult because their patterns are not readily apparent, and, to date, identification has concentrated on regions associated with gene-coding functions. Here, we used phylogenetic footprinting to look for gene-independent noncoding elements in the ribosomal RNA gene repeats from several Saccharomyces species. Similarity plots of ribosomal intergenic spacer alignments from six closely related Saccharomyces species allowed the identification of previously characterized functional elements, such as the origin-of-replication and replication-fork barrier sites, demonstrating that this method is a powerful predictor of noncoding functional elements. Seventeen previously uncharacterized elements, showing high levels of conservation, were also discovered. The conservation of these elements suggests that they are functional, and we demonstrate the functionality of two classes of these elements, a putative bidirectional noncoding promoter and a series of conserved peaks with matches to the origin-of-replication core consensus. Our results paint a comprehensive picture of the functionality of the Saccharomyces ribosomal intergenic region and demonstrate that functional elements not involved in gene-coding function can be identified by using comparative genomics based on sequence conservation.

Base Sequence↗

PubMed2005

Effect of dielectric properties of solvents on the quality factor for a beyond 900 MHz cryogenic probe model.

A previous report by Kelly et al. [J. Am. Chem. Soc. 124 (2002) 12013] indicated that the ionic conductivity of aqueous solution produces a significant contribution to the sensitivity loss in high-resolution NMR equipped with a cryogenically cooled probe. The loss in a sample solution contains two contributions: one from the ionic conductivity and the other from the dielectric loss; the latter is especially important at high frequencies such as above 900 MHz. Here, we investigated the effect of the dielectric conductivity on the quality factor of a 930 MHz cryogenic probe model; in particular, it deals with the ionic aqueous solutions and organic solvents commonly used for NMR in biological research and the chemistry of natural compounds. The sample quality factor, Qs, at first increases with the real part of the relative dielectric permittivity epsilon' and then saturates. In the case of polar organic solvents, the transverse electric field on the sample decreases with epsilon', resulting in an increase of Qs. In the case of non-polar organic solvents, the dielectric conductivity is so small that the gradient of the increase is steep, resulting in much larger Qs though the epsilon' is small. The effect of the transverse electric field is negligible if the epsilon' becomes large, thus the loss for ionic aqueous solution is mainly governed by a loop current induced in the sample solution. As the induced electromotive force is independent of the epsilon', the Qs is saturated at high values of epsilon'. Based on the Qs obtained with the cryogenic probe model, the sensitivity for the cryogenic probe is expected to be as follows: the loss in sensitivity by loading water is more than 66%, i.e., the effect of the dielectric conductivity of water is remarkable at high frequencies; polar organic solvent suffers much larger losses, which is due to the enhancement of the effective sample resistance by the effect of epsilon'; a non-polar organic solvent is nearly free of the sensitivity loss as the dielectric conductivity is negligible; the reverse micelle behaves similarly.

Computer Simulation↗

PubMed2005

Enhancement of perineurial repair and inhibition of nerve adhesion by viscous injectable pure alginate sol.

BACKGROUND: Clinical treatment of recurrent symptoms following peripheral nerve decompression is problematic. Removal of scar tissue can produce an inappropriately pronounced healing response and thereby lead to more serious tethering or compression of the nerve. The goal of the present study was to test the ability of viscous injectable pure alginate sol to prevent perineurial adhesion using a rat neurolysis model. METHODS: The antiadhesive effect of viscous injectable pure alginate sol after neurolysis was evaluated histologically and biomechanically. A total of 40 rats were used in this study. The viscous injectable pure alginate sol was applied topically on the right side after external (six rats) or extensive internal (six rats) neurolysis, whereas the left side received no treatment. The nerves were harvested over 6 weeks, and histologic analysis was performed with hematoxylin and eosin staining and Masson trichrome staining. Functional analysis of the blood and perineurial barriers was also evaluated with Evans blue albumin (four rats). Sixteen rats were used for biomechanical analysis, and eight rats were used as a control group. The results were analyzed statistically using a post hoc test. RESULTS: Histologic examination showed that the use of viscous injectable pure alginate sol was associated with excellent biocompatibility and strong inhibition of perineurial granulation. Viscous injectable pure alginate sol was absorbed completely within 6 weeks without inducing inflammation. In addition, viscous injectable pure alginate sol enhanced repair of the perineurium associated with regeneration of epithelial-like cell layers, a key structure necessary for barrier function of the inner layer of the perineurium. Functional analysis with Evans blue albumin clearly demonstrated that, although blood nerve barrier function recovered by 6 weeks postoperatively in all nerves, the perineurial barrier function recovered only in the sol-treated nerves. Biomechanical study showed a significant antiadhesive effect of viscous injectable pure alginate sol. CONCLUSIONS: Viscous injectable pure alginate sol inhibited adhesion formation around nerves and enhanced regeneration of the perineurium with barrier function. Because excessive perineurial fibrosis and tethering at the neurolysis or neurorrhaphy site is a common postoperative problem in peripheral nerve surgery, viscous injectable pure alginate sol appears to have potentially broad clinical applications.

Alginates↗

PubMed2005

Effect of viscous injectable pure alginate sol on cultured fibroblasts.

BACKGROUND: Alginates have a wide variety of potential clinical applications, including use in cell encapsulation, drug delivery, and tissue engineering. Although the compounds are typically used in the form of a calcium hydrogel, alginates in this form possess several disadvantages, including low biodegradability, induction of foreign body reactions, and cytotoxicity secondary to Ca2+ efflux and contamination with bioincompatible substances. Thus, the goal of the present study was to develop a new method of obtaining sterilized, pure, highly viscous alginate sol from seaweed alginates and to determine its utility as an injectable antiadhesion drug. METHODS: Viscous injectable pure alginate sol was produced from a commercially available sodium alginate, and its molecular and physical characteristics were analyzed. The biological properties of the viscous injectable pure alginate sol were analyzed using cultured fibroblasts prepared from the dorsal skin of neonatal rats to determine its biocompatibility and its effects on cell proliferation, cell migration, and collagen lattice contraction. RESULTS: The mannuronic acid-to-glucuronic acid ratio of viscous injectable pure alginate sol, as determined by nuclear magnetic resonance studies, was 1.2, and its viscosity at 5 percent was 17,800 mPa. Purification used to produce viscous injectable pure alginate sol decreased contamination by insoluble particles by 20 percent and decreased polyphenol concentration by 17 percent. In vitro analyses with cultured fibroblasts demonstrated that viscous injectable pure alginate sol had excellent biodegradability and biocompatibility and that viscous injectable pure alginate sol inhibited fibroblast proliferation and migration. Furthermore, assessment of collagen contraction with floating fibroblast-loaded collagen lattices indicated that viscous injectable pure alginate sol enhanced wound healing in surrounding connective tissues. CONCLUSIONS: The authors conclude that viscous injectable pure alginate sol can inhibit scar formation by presenting a physical barrier to invading fibroblasts and by enhancing wound healing of surrounding tissues.

Alginates↗

PubMed2004

A novel gene amplification system in yeast based on double rolling-circle replication.

Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication. This system produced three types of amplification products. Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d. Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type-3 products are acentric multicopy minichromosomes carrying leu2d. Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.

Animals↗

PubMed2004

Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks.

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.

Adenosine Triphosphatases↗

PubMed2004

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI.

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).

Bacteriophages↗

PubMed2004

SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast.

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Cell Cycle Proteins↗

PubMed2004

Transcription-mediated hyper-recombination in HOT1.

Recombination hotspots are DNA sequences which enhance recombination around that region. HOT1 is one of the best-studied mitotic hotspots in yeast. HOT1 includes a RNA polymerase I (PolI) transcription promoter which is responsible for 35S ribosomal rRNA gene (rDNA) transcription. In a PolI defective mutant the HOT1 hotspot activity is abolished, therefore transcription of HOT1 is thought to be an important factor for the recombination stimulation. However, it is not clear whether the transcription itself or other pleiotropic phenotypes stimulates recombination. To investigate the role of transcription, we made a highly activated PolI transcription system in HOT1 by using a strain whose rDNA repeats are deleted (rdnDeltaDelta). In the rdnDeltaDelta strain, HOT1 transcription was increased about 14 times compared to wild-type. Recombination activity stimulated by HOT1 in this strain was also elevated, about 15 times, compared to wild-type. These results indicate that the level of PolI transcription in HOT1 determines efficiency of the recombination. Moreover, Fob1p, which is essential for both the recombination stimulation activity and transcription of HOT1, was dispensable in the rdnDeltaDelta strains. This suggests that Fob1p is functioning as a PolI transcriptional activator in the wild-type strain.

DNA, Ribosomal↗