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Biomedical subjects

Atsushi Maruyama

Publications and source records attributed to Atsushi Maruyama.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed2004

Targeted gene delivery to sinusoidal endothelial cells: DNA nanoassociate bearing hyaluronan-glycocalyx.

Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). To develop a system for targeting foreign DNA to SECs, comb-type polycations having HA side chains were prepared by coupling HA to poly(L-lysine) (PLL). The HA-grafted-PLL copolymer (PLL-g-HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains. To determine whether the PLL-g-HA/DNA complexes were recognized by SEC HA receptors in vivo, we injected Wistar rats i.v. via the tail vein with PLL-g-HA complexed to a beta-galactosidase expression plasmid (pSV beta-Gal) labeled with 32P. One hour postinjection, >90% of the injected radioactivity remained in the liver. Administration of the PLL-g-HA complexed to an FITC-labeled DNA revealed that the carrier-DNA complex was distributed exclusively in SECs. A large number of SECs expressing beta-galactosidase was detected along the sinusoidal lining after transfection with PLL-g-HA/pSV beta-Gal. Moreover, PLL-g-HA effectively stabilized DNA triplex formation. In conclusion, the new PLL-g-HA/DNA carrier system permits targeted transfer of exogenous genes selectively to the SECs.

Animals↗
PubMed2004

Brain-derived neurotrophic factor acutely depresses excitatory synaptic transmission to GABAergic neurons in visual cortical slices.

Brain-derived neurotrophic factor (BDNF) acutely modulates synaptic transmission to excitatory neurons in hippocampus and neocortex. The question of whether BDNF acts similarly on excitatory synaptic transmission to GABAergic neurons was eluded in previous studies using cortical slices. To address this question, we used transgenic mice in which expression of green fluorescence protein (GFP) is regulated by glutamic acid decarboxylase 67 (GAD67) promoter. In cortical slices prepared from these GAD67-GFP knock-in mice, we could detect GABAergic neurons under a fluorescent microscope. An application of BDNF rapidly depressed excitatory postsynaptic currents (EPSCs) evoked by layer IV stimulation in most GFP-positive neurons in layer II/III of the cortex. This effect was seen at synapses activated during the BDNF application and blocked by anti-TrkB IgG, indicating that the acute inhibitory action of BDNF is activity-dependent and mediated through TrkB. Paired-pulse ratios of the amplitude of EPSCs to paired stimulation at intervals of 10-100 ms were not significantly changed after BDNF application, suggesting that the site of depression may be postsynaptic. Responses to directly applied glutamate were also depressed by BDNF in most of neurons, being consistent with the interpretation of postsynaptic action of BDNF. The depressive action of BDNF was blocked by an intracellular injection of a Ca(2+) chelator, suggesting that a rise in Ca(2+) is involved in the acute depression of EPSCs. This action of BDNF was seen in 67% of parvalbumin (PV)-positive neurons, but in only 19% of PV-negative neurons, indicating that the depressive action is biased to PV-positive GABAergic neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2004

Preparation of cationic comb-type copolymer having guanidino moieties and its interaction with DNAs.

In order to evaluatet effects of different cationic moieties, such as primary amino and guanidino groups, on interactions between DNAs and cationic comb-type copolymers, comb-type copolymers having guanidino groups were prepared. The copolymers (GPLL-g-Dex) were obtained by guanidination of poly(L-lysine)-graft-dextran copolymers (PLL-g-Dex) using 1-guanyl-3,5-dimethylpyrazole nitrate under weak basic conditions. The resulting copolymers were characterized using NMR spectroscopy and size-exclusion chromatography-multiangle light scattering (SEC-MALS). The primary amino groups of the PLL backbones were thoroughly replaced with guanidino ones without any detectable side reactions, including fragmentation of PLL or Dex chains. The interactions of GPLL-g-Dex and PLL-g-Dex with DNAs were assessed by UV-melting curve measurements. These copolymers diversly affected the melting behavior of double-stranded DNA (dsDNA). GPLL-g-Dex has a lower ability to increase the Tm of dsDNA than PLL-g-Dex and also exhibits a higher affinity for dsDNA. The results suggest that the stability of dsDNA may be affected not only by ionic interaction between the copolymers and DNAs, but also by other modes of interactions, such as hydrogen-bondinginteractions.

Base Sequence↗
PubMed2003

Cationic comb-type copolymers for DNA analysis.

Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.

Aryl Hydrocarbon Hydroxylases↗
PubMed2003

A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes.

BACKGROUND: Staphylococcus aureus is a major human pathogen and causes a serious hospital infection due to the acquired multidrug resistance. Unlike the well-studied bacteria such as Escherichia coli and Bacillus subtilis, which have seven and 18 sigma factors, respectively, only two sigma factors have been known for S. aureus. We searched for possible sigma factor genes by examining the S. aureus genome with a special attention to the gene arrangement around the sigma factor genes of a close relative, B. subtilis. RESULTS: A new sigma factor gene was identified in Staphylococcus. The gene constituted a conserved gene cluster with other genes including translation- and transcription-related genes. Phylogenetic analysis and comparison of the gene sequences among species indicated that the staphylococcal sigma factor originated from a common ancestor of B. subtilis SigH. An over-expression of this sigma factor in S. aureus resulted in a drastic induction of the expression of the com operons that encode proteins required for the natural genetic competence. CONCLUSIONS: We demonstrated that the newly identified staphylococcal sigma factor participated in a regulatory network of transcription that controlled the genetic competence genes. In our phylogenetic tree, the factor was classified as a single group with a common function.

Bacillus subtilis↗
PubMed2003

Cationic comb-type copolymers as an artificial nucleic acid-chaperone: spectroscopic analyses for DNA/copolymer interaction.

We have reported previously that the cationic comb-type copolymer, PLL-g-Dex, accelerated DNA strand exchange reaction while stabilizing DNA duplexes. In this research, we showed the effects of cationic PLL-g-Dex copolymer on hybridization of self-complementary octamer oligonucleotides. NMR studies suggested that PLL-g-Dex copolymer preferentially interacted with a duplex DNA rather than a single-stranded DNA, and did not perturb local structures of DNA duplexes. CD studies also supported inert nature of the copolymer upon hybrid structure. From these results we concluded that the accelerating mechanisms of the copolymer did not involved partial perturbation of duplex structure.

Biopolymers↗
PubMed2003

Design of artificial nucleic acid chaperones for DNA engineering.

We have shown that the comb-type copolymer consisting of a polycation backbone and hydrophilic side chains stabilizes DNA hybrids. Furthermore, the copolymers showed the activity to accelerate DNA strand exchange reactions between double helical DNA and its complementary DNA. The copolymer was considered to stimulate breakage and reassociation of base pairing and act as an artificial nucleic acid chaperone. The polymer's chaperoning activity was elaborated for rapid and precise judging of a subtle difference in DNA sequences. One base alternation out of 20mer DNA was quickly detected by the strand exchange assay employing the copolymer. From these, we conclude that the copolymer would be a useful material in DNA engineering that employs rapid and precise DNA folding.

Biopolymers↗
PubMed2003

Rational combination of strategies to achieve synergistic stabilization of triplex.

The combination of two stabilizing strategies to increase stability of nucleic acid assembly is not always resulted in synergistic effect. In the present study, to explore the rational way to select the combination for synergistically stabilizing strategies, we examined the kinetic effects of different triplex-stabilizing strategies, poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). The former increased the association rate constant, whereas the latter decreased the dissociation rate constant in triplex equilibrium. Each strategy increased the binding constant of the triplex formation by nearly two orders of magnitude. The combination of both stabilizing strategies, which was the triplex formation with PN TFO in the presence of the copolymer, increased the binding constant by nearly four orders of magnitude, implying successful synergy of their activities. The kinetically orchestrated effects in which the copolymer and the PN modification contribute to distinct ingredients in triplex equilibrium achieved the observed synergistic stabilization. We conclude that kinetic analyses of stabilizing effects enable us to select a rational combination of stabilizing strategies.

Base Sequence↗
PubMed2003

Polycation-accelerated strand exchange (PASE) for SNPs typing.

In the previous study (Chem. Eur. J., 7, 176 (2001), J. Am. Chem. Soc., 124, 12676 (2002)), we demonstrated that the cationic comb-type copolymer (PLL-g-Dex, CCC) composed of a cationic poly(L-lysine) backbone and water-soluble side chains of dextran accelerated the DNA strand exchange reaction between double helical DNA and its homologous single strand. In this study, Polycation-Accelerated Strand Exchange (PASE) assay was applied to discriminate single-base mismatch in DNA sequence such as single nucleotide polymorphisms (SNPs). We could discriminate the 20mer DNA with a single-base mismatch from the full matched DNA with high efficacy by PASE.

Base Pair Mismatch↗
PubMed2003

Oxidative-stress-inducible qorA encodes an NADPH-dependent quinone oxidoreductase catalysing a one-electron reduction in Staphylococcus aureus.

This work characterized the putative quinone oxidoreductase gene (qorA) from Staphylococcus aureus. The deduced amino acid sequence indicated that the 333 aa protein contains an NAD(P)H-binding motif. A Northern blot analysis revealed that 2.6 kb and 1.4 kb signals were detected by using a qorA probe. Both the signals were enhanced under the presence of a redox-cycling agent, 9,10-phenanthrenequinone (PQ). It was also revealed that the expression of three genes, SA1988, SA1989 (qorA) and SA1990, was enhanced at the transcriptional level by PQ exposure. The results suggested that the 2.6 kb signal detected by the qorA probe was in two co-transcripts, i.e. SA1990-qorA and qorA-SA1988 were transcribed. Besides, primer extension analyses confirmed the enhancement of qorA and SA1990 transcripts. The GST (glutathione S-transferase)-tagged QorA protein was expressed in Escherichia coli and purified using a glutathione affinity column. In purification steps, a 36 kDa band co-purified with the GST-QorA, and it was detected even in the thrombin-cleaved fraction. N-terminal amino acid sequences for the 36 kDa protein revealed that it was an intact QorA. They showed that QorA formed a multimer under physiological conditions. The purified recombinant GST-QorA catalysed NADPH consumption in the presence of PQ as a substrate, but not NADH. To characterize the catalytic activity of QorA, superoxide anion that was generated through one-electron reduction of PQ and hydroquinone that was produced by two-electron reduction of PQ were measured. During reduction of PQ by GST-QorA, superoxide anion was generated, whereas a small amount of 9,10-dihydroxyphenanthrene (hydroquinone of PQ) was produced. These results suggest that the activity of QorA is similar to zeta-Crystallin, catalysing an NADPH-dependent one-electron reduction of quinone.

Amino Acid Sequence↗
PubMed2002

DNA strand exchange stimulated by spontaneous complex formation with cationic comb-type copolymer.

Cationic comb-type copolymers (CCCs) composed of a polycation backbone and water-soluble side chains accelerate by 4-5 orders the DNA strand exchange reaction (SER) between double helical DNA and its homologous single-strand DNA. The accelerating effect is considered due to alleviation of counterion association during transitional intermediate formation in sequential displacement pathway. CCCs stabilize not only matured hybrids but also the nucleation complex to accelerate hybridization.

Cations↗
PubMed2002

A double-strand decoy DNA oligomer for NF-kappaB inhibits TNFalpha-induced ICAM-1 expression in sinusoidal endothelial cells.

Altered gene expression of liver sinusoidal endothelial cells (SECs) is associated with impaired immune response. Here we report that the decoy technique effectively suppresses TNFalpha-induced ICAM-1 expression in SEC. An NF-kappaB decoy (NF-kappaB31: 5(')-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3), which contains a consensus sequence for NF-kappaB, was complexed to PLL-g-HA [hyaluronate-grafted poly(L-lysine) copolymer] that permits transfer of exogenous DNA selectively to the SEC. The PLL-g-HA/NF-kappaB31 complex was added to the culture media of LSE cells, a human SEC-derived cell line. Then, cells were stimulated with TNFalpha (5ng/mL). PLL-g-HA/NF-kappaB31, but not control oligodeoxynucleotides having a reverse or scrambled sequence, inhibited the intranuclear localization of NF-kappaB induced by TNFalpha, with almost complete inhibition at 2.5microg/mL as DNA. NF-kappaB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells. The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions.

Active Transport, Cell Nucleus↗
PubMed2002

Myocardial viability assessment with gated SPECT Tc-99m tetrofosmin % wall thickening: comparison with F-18 FDG-PET.

OBJECT: This study was designed to assess the value of gated SPECT Tc-99m-tetrofosmin (TF) wall thickening (WT) in addition to TF exercise (Ex)/rest myocardial SPECT, in comparison with F-18 fluorodeoxyglucose (FDG)-PET. METHODS: The study population consisted of 33 patients with old myocardial infarction (27 men and 6 women; mean age, 62 +/- 8 years old). All patients underwent Ex/rest TF SPECT and glucose loading FDG-PET. Polar map images of Ex/rest TF were generated and divided into 24 segments for further analysis. We classified LV segments according to the exercise-rest perfusion scintigraphy. LV segments with less than 70% of the maximum TF activity on the exercise image were defined as stress-induced defects. Among these, the segments whose TF activity increased by 10% from exercise to rest images or exceeded 70% of the maximum uptake were defined as reversible (viable) defects. The remaining defects on the rest image were irreversible (non-viable) defect segments, and were considered for viability study on the basis of %WT. %WT was calculated according to the standard method: [(counts ES - counts ED)/counts ED] x 100. A viable segment on gated SPECT was defined as a segment whose %WT exceeded the lower limit of the normal value (mean - SD). PET viability was defined as FDG uptake exceeding 50% of the maximum count. RESULTS: Among the 792 segments evaluated in the 33 patients studied, there were 689 PET viable segments. Of the 689 segments analyzed, 198 (29%) were identified as having defects on Ex images. Among these defects, 55 (8%) were reversible or partially reversible, as evidenced by rest images, and 143 (21%) were irreversible. Of the irreversible segments on Ex/rest images, 106 (15%) demonstrated no apparent WT by gated TF SPECT, whereas 37 (6%) segments with irreversible defects did have apparent WT. Overall, the sensitivity of Ex/rest TF perfusion imaging was 79%. Sensitivity was improved from 79% to 85% by combining %WT and perfusion data, but specificity was reduced from 70% to 56%. CONCLUSION: %WT evaluated from gated TF imaging enhanced myocardial viability assessment in comparison with FDG-PET.

Aged↗
PubMed2002

Acceleration of DNA strand exchange reaction by cationic polymers.

In previous reports, we have demonstrated that the cationic comb-type copolymers (PLL-g-Dex) having a poly-L-lysine main chain and dextran side chains stabilized DNA duplex and triplex. Furthermore, the copolymer was found to accelerate more than 20,000 folds strand exchange reaction between 20 bp duplex and its homologous single strand (1). This study was designed to inspect structural factor of polycationic moieties in acceleration effects. To this end, polypeptides containing lysine or arginine moieties as cationic groups were examined in their potency to accelerating strand exchange reaction. It was shown that arginine-rich peptide showed higher accelerating effect than lysine peptide when total positive charges of peptides were kept constant.

Amino Acid Sequence↗
PubMed2002

Investigation of DNA strand exchange mechanisms mediated by cationic polymers.

It was observed that conjugation of dextran for PLL provided PLL-g-Dex with new properties of enhancing and accelerating inter-nucleotide recognition (1, 2, 3). In this paper, we report the effects of cationic PLL-g-Dex copolymers on base-pair formation of self-complementary octamer oligonucleotide by using NMR and CD analysis. NMR studies revealed that PLL-g-Dex copolymer did not affect hydrogen-bondings between bases in DNA duplex. CD studies also supported inert nature of the copolymer upon hybrid structure.

Base Sequence↗