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Biomedical subjects

Atsushi Maruyama

Publications and source records attributed to Atsushi Maruyama.

At least 19 recordsLinked to original sources
PubMed2007

Spectroscopic investigation of cationic comb-type copolymers/DNA interaction: interpolyelectrolyte complex enhancement synchronized with DNA hybridization.

We have demonstrated that cationic comb-type copolymers consisting of a polycation backbone and abundant grafts of water-soluble polymers stabilize DNA hybrids. Furthermore, the copolymers were found to accelerate strand exchange reaction between a double-stranded DNA and its complementary single-stranded DNA. In this article, we investigated the effects of PLL-g-Dex on base pairs of a self-complementary DNA octamer, d(GGAATTCC). The soluble interpolyelectrolyte complex (IPEC) between the DNA and copolymer allowed us to characterize the complex by using spectroscopic methods under physiological ionic condition. Chemical shifts of nucleobase proton signals were not changed by PLL-g-Dex. Furthermore, the copolymer slightly changed the von't Hoff DeltaH accompanying the helix-coil transition of the octamer. These results indicated that the base pairs of the duplex DNA in the IPEC were not perturbed by the polycationic copolymer. It was obviously shown by temperature dependencies of proton and phosphorus NMR spectra that DNA/copolymer interaction was considerably enhanced in response to ds DNA formation. An increase in the density and total number of DNA negative charges upon hybrid formation likely caused the higher affinity of the copolymer with the ds form over that of the copolymer with the ss form. The IPEC formation of CCCs with DNA, however, seems highly sensitive to the coil-helix transition of the DNA.

Cations↗
PubMed2007

Cationic comb-type copolymers for boosting DNA-fueled nanomachines.

For the better applications and developments of DNA nanomachines, their responding kinetics, output, and sequence-selectivity need to be improved. Furthermore, the DNA nanomachines currently have several limitations in operating conditions. Here we show that a simple addition of a cationic comb-type copolymer, poly(l-lysine)-graft-dextran, produces the robust and quick responses of DNA nanomachines under moderate conditions including physiologically relevant conditions even at very low strand concentrations (nanomoles per liter range) through hybrid stabilization and DNA strand exchange acceleration.

Base Sequence↗
PubMed2006

BRG1 interacts with Nrf2 to selectively mediate HO-1 induction in response to oxidative stress.

NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.

Animals↗
PubMed2006

Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.

Cell Nucleus↗
PubMed2006

Structural effect of cationic copolymers on nucleic acid-chaperoning activity.

In order to evaluate the effect of cationic copolymer structures on their nucleic acid-chaperoning activity, we prepared various copolymers having different cationic residues or backbone molecular weight. It was revealed that nucleic acid-chaperoning activity increases with increasing molecular weight of the copolymer backbone and that the copolymer having the guanidino groups is effective for increasing nucleic acid-chaperoning activity. Compared with PLL-g-Dex, GPLL-g-Dex has weak activity to stabilize ds DNA. This weak stabilization effect of GPLL-g-Dex may contribute to the higher accelerating effect.

DNA↗
PubMed2006

Electrochemical detection of DNA single base mismatch by the use of strand exchange reaction.

The electrochemical method based on the strand exchange reaction (SER) for the detection of DNA single-base mismatch has been developed. Different electrochemical responses due to the slower SER rates for mismatch containing DNA than fully matched DNA were observed by using the redox-modified partial duplex DNA immobilized on gold electrode as an analytical probe.

Base Pair Mismatch↗
PubMed2006

Single-base mismatch recognition using partially double-stranded probes having various lengths.

We have focused on the nucleation-synchronized DNA strand exchange reaction (ns-SER) as a format of a single-base mismatch detection. We employed a partially double-stranded (PDS) DNA probe having a single-stranded (ss) portion. We could rapidly resolve a single-base mismatch by the ns-SER rates of the PDS probe with target DNAs. Here, we reported the influence of probe length and operating conditions, such as temperature and buffer conditions, on a single-base mismatch recognition using the PDS probes. We could reliably recognize a single-base mismatch even with a 61mer long probe. However, the ns-SER rates were decreased with increasing probe length. We considered that a rate-limiting step of the ns-SER might be sifted from nucleation to branch migration processes. By an addition of cationic comb-type copolymers (PLL-g-Dex) in the buffer, the reaction rate was considerably increased 1-2 orders without disordering the resolution power.

Base Pair Mismatch↗
PubMed2006

Bacterial nucleoid dynamics: oxidative stress response in Staphylococcus aureus.

A single-molecule-imaging technique, atomic force microscopy (AFM) was applied to the analyses of the genome architecture of Staphylococcus aureus. The staphylococcal cells on a cover glass were subjected to a mild lysis procedure that had maintained the fundamental structural units in Escherichia coli. The nucleoids were found to consist of fibrous structures with diameters of 80 and 40 nm. This feature was shared with the E. coli nucleoid. However, whereas the E. coli nucleoid dynamically changed its structure to a highly compacted one towards the stationary phase, the S. aureus nucleoid never underwent such a tight compaction under a normal growth condition. Bioinformatic analysis suggested that this was attributable to the lack of IHF that regulate the expression of a nucleoid protein, Dps, required for nucleoid compaction in E. coli. On the other hand, under oxidative conditions, MrgA (a staphylococcal Dps homolog) was over-expressed and a drastic compaction of the nucleoid was detected. A knock-out mutant of the gene encoding the transcription factor (perR) constitutively expressed mrgA, and its nucleoid was compacted without the oxidative stresses. The regulatory mechanisms of Dps/MrgA expression and their biological significance were postulated in relation to the nucleoid compaction.

Bacterial Proteins↗
PubMed2005

Whole genome sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection.

Staphylococcus saprophyticus is a uropathogenic Staphylococcus frequently isolated from young female outpatients presenting with uncomplicated urinary tract infections. We sequenced the whole genome of S. saprophyticus type strain ATCC 15305, which harbors a circular chromosome of 2,516,575 bp with 2,446 ORFs and two plasmids. Comparative genomic analyses with the strains of two other species, Staphylococcus aureus and Staphylococcus epidermidis, as well as experimental data, revealed the following characteristics of the S. saprophyticus genome. S. saprophyticus does not possess any virulence factors found in S. aureus, such as coagulase, enterotoxins, exoenzymes, and extracellular matrix-binding proteins, although it does have a remarkable paralog expansion of transport systems related to highly variable ion contents in the urinary environment. A further unique feature is that only a single ORF is predictable as a cell wall-anchored protein, and it shows positive hemagglutination and adherence to human bladder cell associated with initial colonization in the urinary tract. It also shows significantly high urease activity in S. saprophyticus. The uropathogenicity of S. saprophyticus can be attributed to its genome that is needed for its survival in the human urinary tract by means of novel cell wall-anchored adhesin and redundant uro-adaptive transport systems, together with urease.

Animals↗
PubMed2005

Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells.

The Nrf2-Keap1 system coordinately regulates cytoprotective gene expression via the antioxidant responsive element (ARE). The expression of several ARE-regulated genes was found to be up-regulated in endothelial cells by laminar shear stress, suggesting that Nrf2 contributes to the anti-atherosclerosis response via the ARE. To gain further insight into the roles that Nrf2 plays in the development of atherosclerosis, we examined how Nrf2 regulates gene expression in response to anti-atherogenic laminar flow (L-flow) or pro-atherogenic oscillatory flow (O-flow). Exposure of human aortic endothelial cells (HAECs) to L-flow, but not to O-flow, induced the expression of cytoprotective genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1) by 5-fold and heme oxygenase-1 by 8-fold. The critical contribution of Nrf2 to the expression induced by L-flow was ascertained in siRNA-mediated knock-down experiments. Two cyclooxygenase-2 (COX-2) specific inhibitors attenuated Nrf2 nuclear accumulation in the acute phase of L-flow exposure. A downstream product of COX-2, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), activated the Nrf2 regulatory pathway in HAECs through binding to the cysteines of Keap1. These results demonstrate that 15d-PGJ2 is essential for L-flow to activate Nrf2 and induce anti-atherosclerotic gene expression. Whereas both L-flow and O-flow induced the nuclear accumulation of Nrf2 to comparable levels, chromatin immunoprecipitation analysis revealed that Nrf2 binding to the NQO1 ARE was significantly diminished in the case of O-flow compared with that of L-flow. These results suggest that O-flow inhibits Nrf2 activity at the DNA binding step, thereby suppressing athero-protective gene expression and hence predisposing the blood vessels to the formation of atherosclerosis.

Aorta↗
PubMed2005

Extracellular signal-dependent nuclear import of STAT3 is mediated by various importin alphas.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. STAT3 is activated by cytokines and growth factors via the phosphorylation of a tyrosine residue, dimerization, and subsequent nuclear translocation. However, the mechanism of its nuclear translocation is unclear. A study of the cytokine-stimulated import of STAT3 into the nucleus is reported herein. An oncostatin M (OSM)-dependent nuclear import assay system was first established in living cells. Using this system, we demonstrated that the microinjection of the importin alpha5/NPI-1 mutant, an anti-importin beta antibody, and the RanQ69L mutant inhibited the nuclear import of STAT3. Second, we showed that tyrosine-phosphorylated STAT3 associates, not only with importin alpha5/NPI-1 but also with other importin alphas, as a result of OSM stimulation, as evidenced by a solution binding assay. These findings suggest that the extracellular signal-dependent nuclear transport of STAT3 is mediated by various importin alphas, importin beta, and Ran.

Active Transport, Cell Nucleus↗
PubMed2005

The effect of backbone structure on polycation comb-type copolymer/DNA interactions and the molecular assembly of DNA.

A series of comb-type copolymers comprised of various polycation backbones and dextran (Dex) side chains were prepared to study the DNA/copolymer interaction. While the cationic copolymers with a lower degree of dextran grafts maintained an ability to condense DNA molecules into a globule form those with a higher degree of dextran grafting interacted with DNA without inducing DNA condensation. The structural differences in cationic backbones diversely influenced DNA hybridization as evaluated by circular dichroism (CD) spectrometry and UV-melting analyses. The copolymer having a polyallylamine (PAA) backbone induced B-->A-type transformation of DNA duplex, whereas the copolymers having either alpha-poly(l-lysine) (alpha PLL) or epsilon-poly(l-lysine) (epsilon PLL) backbone induced B-->C-type transformation. The PAA copolymer is the first example of the artificial polymer that induces B-->A-type transformation under physiologically relevant condition. UV-melting analyses of DNA strands indicated that the alpha PLL copolymers showed the highest stabilization efficacy toward poly(dA).poly(dT) duplex and poly(dA).2poly(dT) triplex without affecting reversibility of inter DNA association. Melting temperatures (T(m)) of the triplex increased from 38 degrees Celsius to 99 degrees Celsius by the addition of the alpha PLL copolymer with an appropriate grafting degree. While the PAA copolymers had higher density of cationic groups along the backbone than alpha PLL copolymers, these copolymers moderately increased T(m) of the DNA triplex. The PAA copolymer caused considerable hysteresis in thermal melting/reassociation processes. Note that the PLL copolymers increased T(m) of the DNA triplex and not the duplex, suggesting their potential as a triplex selective stabilizer. Chemical structures of the cationic backbones of the copolymers were characteristically affected on the copolymer/DNA interaction even if their backbones were surrounded by abundant side chains (> wt%) of dextran. The study suggested that tailor-made design of "functional polycounterion" is a strategy to engineer molecular assembling of DNA.

Binding Sites↗
PubMed2005

Discrimination of single nucleotide polymorphisms by strand exchange assay using partially double-stranded probes.

Previously we designed the partially double-stranded (PDS) probes that have protruding single-stranded portion for a single-base mismatch analysis. The single-stranded portion is engineered to sense existences of mismatches in the counterparts and to transduce it in strand exchange rates. Here we report the influence of probe length and operating conditions, such as temperature and buffer conditions, on the mismatch resolution using the PDS probes. Reliable detection of single-base mismatches was achieved even with a 45mer-long probe. By lowering operating temperature, the higher and faster discrimination of the mismatches was demonstrated. Addition of cationic comb-type copolymers (CCCs) in the buffer increased the reaction rate 3-4 orders without disordering the resolving power.

Base Pair Mismatch↗
PubMed2005

The molecular structure effect of cationic comb-type copolymers on nucleic acid chaperone activity.

We have shown that the cationic comb-type copolymers (CCCs) accelerate DNA hybridization and increase stability of DNA duplexes and triplexes. The CCCs were considered to act as "nucleic acid chaperones," promoting the formation of the most stable hybrids. In this study, CCCs with primary amino or guanidino groups were prepared to evaluate the effects of different cationic moieties upon the nucleic acid chaperone activity. CCCs having guanidino groups have higher accelerating effect on DNA strand exchange reactions than that having primary amino groups. It was suggested that some modes of interactions, such as hydrogen bonding, other than ionic interactions between the copolymers and DNAs may be involved upon the strand exchange activation.

DNA↗
PubMed2005

Modulation of highly ordered structures of human telomeric sequence by cationic copolymers.

Human telomeric DNA, d(GGGTTA)n, tends to adopt complex intramolecular G-quadruplex structure in solution. The sequence folds into G-quadruplex with the antiparallel orientation in the presence of sodium ions, while antiparallel and parallel conformations were coexisted in solution containing relatively high concentration of potassium ions. Here, we describe that cationic comb-type copolymer, poly(l-lysine)-graft-dextran (PLL-g-Dex), triggers selective folding of d[GGG(TTAGGG)3] into G-quadruplex with the parallel orientation. The parallel G-quadruplex formed in the presence of the copolymer has the melting temperature > 90 degrees C, including strong stabilization effect of the copolymer.

Base Sequence↗
PubMed2004

Nucleotide substitutions in Staphylococcus aureus strains, Mu50, Mu3, and N315.

A specific phenotype of Staphylococcus aureus strains Mu50 and Mu3 is characterized by thickened cell wall and moderate resistance to vancomycin. The N315 strain is a prototype of methicillin-resistant S. aureus (MRSA), but it is methicillin susceptible, despite carrying the mecA resistance gene. Here, we revised differences in the sequences of Mu50 and N315, referencing that of Mu3 which were assumed to be of one lineage. The 362 ORFs diverse between Mu50 and N315 were picked up, and the corresponding ones in three strains were re-sequenced. This defined 213 ORFs diverse between Mu50 and N315, and 9 between Mu50 and Mu3. The fixed diversities of 174 ORFs (except for 39 silent ORFs from 213), including nucleotide substitution (NSs), frame shift, and truncation were grouped into three major functional categories, which were transport (14.9% in the 174 diverse ORFs), metabolism of carbohydrates (5.7%), and RNA synthesis (9.6%). The other gene categories had small diversities. These gene categories seemed to be functionally decisive for the Mu50-specific characters, the thickened cell wall and moderate vancomycin resistance. All of the diverse genes and the high quality sequence of Mu50 can be viewed at the web site (http://133.5.48.239/VRSA/).

Bacterial Proteins↗