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Hiroshi Mizuno

Publications and source records attributed to Hiroshi Mizuno.

At least 19 recordsLinked to original sources
PubMed2006

An RNA aptamer that discriminates bovine factor IX from human factor IX.

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.

Animals↗
PubMed2006

Structure of an RNA duplex r(GGCGBrUGCGCU)2 with terminal and internal tandem G.U base pairs.

The crystal structure of a self-complementary RNA duplex r(GGCG(Br)UGCGCU)(2) with terminal G.U and internal tandem G.U base pairs has been determined at 2.1 Angstroms resolution. The crystals belong to the tetragonal space group P4(3), with unit-cell parameters a = b = 37.69, c = 96.28 Angstroms and two duplexes in the asymmetric unit. The two strands of each duplex are related by a pseudodyad axis. The structure was refined to final R(work) and R(free) values of 20.9 and 25.3%, respectively. The duplexes stack in an end-to-end manner, forming infinite columns along the c axis. This is the first structural study of an RNA duplex containing G.U pairs at the termini. The stacking overlaps of the terminal G.U base pairs with their adjacent Watson-Crick base pairs are larger than those of Watson-Crick base pairs of the 5'-YR-3'/3'-RY-5' type. The terminal G.U base pairs of neighbouring duplexes are also stacked with each other. An alternating underwound-overwound pattern of the twist angles is seen at each step along the duplex. This observation is typical for internal tandem G.U pairs, while the terminal G.U base pairs exhibit high twist angles with the adjacent Watson-Crick pairs. The 3'-side of U of the internal G.U base pair, which is unstacked, appears to be stabilized by pi-cation interaction with an Mg(2+) ion.

Base Pairing↗
PubMed2006

A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells.

A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781-bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin.

Actins↗
PubMed2006

Structural basis of HutP-mediated transcription anti-termination.

Bacteria often use anti-terminator proteins to sense a specific metabolite signal and direct RNA polymerase to either terminate transcription or transcribe the downstream genes of an operon. Although many proteins that regulate various operons using this mechanism have been identified, insights into their activation processes before cognate mRNA binding have remained obscure. HutP from Bacillus subtilis regulates the hut operon by an anti-termination mechanism. Recently, several crystal structures of HutP [apo-HutP, HutP-L-histidine (and histidine analog), HutP-L-histidine-Mg(2+) and HutP-L-histidine-Mg(2+)-RNA] have been reported. These structural and functional studies of HutP have revealed how the protein undergoes conformational changes in response to two key components: L-histidine and Mg(2+) ions.

Bacillus subtilis↗
PubMed2006

Molecular dynamics calculations of wild type vs. mutant protein C: relationship between binding affinity to endothelial cell protein C receptor and hereditary disease.

Molecular dynamics simulations of the protein C gamma-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 --> Lys) in the protein C Gla domain. Our results suggest that the Gla 25 --> Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca2+ ion.

1-Carboxyglutamic Acid↗
PubMed2006

Squamous cell carcinoma of the auricle arising from keloid after radium needle therapy.

The squamous cell carcinoma (SCC) of the auricle was one of the common tumors of not only skin but also ear. SCC of the auricle was described to be the carcinoma of old age with some predisposing factors and keloid was known to be one of the predisposing factors for malignant tumors. Keloid and radium needle therapy were risk factors, latter the major, independently from each other and were reported to conclude in SCC depending on the duration for the keloid and the dose for the radium needle therapy. The increased age with the long duration of these predisposing factors should have to be kept in mind in every situation when these treatment modalities had been planned to. Surgical interventions should always be the first choice in the treatment of not only the carcinomas but also in keloids in selected cases.

Aged, 80 and over↗
PubMed2006

Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S.

Telomeres, which are important for chromosome maintenance, are composed of long, repetitive DNA sequences associated with a variety of telomere-binding proteins. We characterized the organization and structure of rice telomeres and adjacent subtelomere regions on the basis of cytogenetic and sequence analyses. The length of the rice telomeres ranged from 5.1 to 10.8 kb, as revealed by both fibre-fluorescent in situ hybridization and terminal restriction-fragment assay. Physical maps of the chromosomal ends were constructed from a fosmid library. This facilitated sequencing of the telomere regions of chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The resulting sequences contained conserved TTTAGGG telomere repeats, which indicates that the physical maps partly covered the telomere regions of the respective chromosome arms. These repeats were organized in the order of 5'-TTTAGGG-3' from the chromosome-specific region, except in chromosome 7S, in which seven inverted copies also existed in tandem array. Analysis of the telomere-flanking regions revealed the occurrence of deletions, insertions, or chromosome-specific substitutions of single nucleotides within the repeat sequences at the junction between the telomere and subtelomere. The sequences of the 500-kb regions of the seven chromosome ends were analysed in detail. A total of 598 genes were predicted in the telomeric regions. In addition, repetitive sequences derived from various kinds of retrotransposon were identified. No significant evidence for segmental duplication could be detected within or among the subtelomere regions. These results indicate that the rice chromosome ends are heterogeneous in both sequence and characterization.

Base Sequence↗
PubMed2006

A potent anti-coagulant RNA aptamer inhibits blood coagulation by specifically blocking the extrinsic clotting pathway.

A potent antidote-controlled aptamer, as an anticoagulant, has reportedly overcome the complications of acute bleeding by the administration of available anticoagulants. In the present study, we provide evidence that the aptamer binds specifically to factors IX and IXa and inhibits their functions. Furthermore, we demonstrated that the aptamer inhibits blood coagulation by interfering with the extrinsic pathway, blocking the complex of factor VIIa and tissue factor interactions with factor IX. The results from the previous and present studies suggest that the aptamer probably binds in the vicinity of the EGF1 and EGF2 domains of factor IX.

Anticoagulants↗
PubMed2005

pH-Dependent structural changes at Ca(2+)-binding sites of coagulation factor IX-binding protein.

Coagulation factor IX-binding protein, isolated from Trimeresurus flavoviridis (IX-bp), is a C-type lectin-like protein. It is an anticoagulant consisting of homologous subunits, A and B. Each subunit has a Ca(2+)-binding site with a unique affinity (K(d) values of 14muM and 130muM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca(2+) binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca(2+) release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca(2+)-binding sites with each other and with those of the solved structures under alkaline conditions; pH 7.8 and pH 8.0 (complexed form). At pH 6.5, Glu43 in the Ca(2+)-binding site of subunit A displayed two conformations. One (minor) is that in the alkaline state, and the other (major) is that at pH 4.6. However, the corresponding Gln43 residue of subunit B is in only a single conformation, which is almost identical with that in the alkaline state. At pH 4.6, Glu43 of subunit A adopts a conformation similar to that of the major conformer observed at pH 6.5, while Gln43 of subunit B assumes a new conformation, and both Ca(2+) positions are occupied by water molecules. These results showed that Glu43 of subunit A is much more sensitive to protonation than Gln43 of subunit B, and the conformational change of Glu43 occurs around pH6.5, which may correspond to the step of Ca(2+) release.

Animals↗
PubMed2005

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis.

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP-RNA interactions. The best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+, while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP-RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein-RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.

Bacillus subtilis↗
PubMed2005

Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice.

Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.

Adipocytes↗
PubMed2005

Crystal structure of a CRISP family Ca2+ -channel blocker derived from snake venom.

The cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals, reptiles, amphibians and secernenteas, and are involved in a variety of biological reactions. Here we report the crystal structure of triflin, a snake venom derived blocker of high K(+)-induced artery contraction, at 2.4A resolution. Triflin consists of two domains. The first 163 residues form a large globular body with an alpha-beta-alpha sandwich core, which resembles pathogenesis-related proteins of group-1 (PR-1). Two glutamic acid-associated histidine residues are located in an elongated cleft. A Cd(2+) resides in this binding site, and forms a five-coordination sphere. The subsequent cysteine-rich domain adopts a rod-like shape, which is stabilized by five disulfide bridges. Hydrophobic residues, which may obstruct the target ion-channel, are exposed to the solvent. A concave surface, which is surrounded by these two domains, is also expected to play a significant role in the binding to the target receptor, leading to ion channel blockage. The C-terminal cysteine-rich region has a similar tertiary structure to voltage-gated potassium channel blocker toxins, such as BgK and ShK. These findings will contribute toward understanding the functions of the widely distributed CRISP family proteins.

Animals↗
PubMed2005

Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteine-rich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 A, and diffraction data were collected to 2.25 A resolution. Crystals of Pdc also belonged to space group P2(1)2(1)2(1), with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 A, and diffraction data were collected to 1.90 A resolution.

Animals↗
PubMed2005

Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements.

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)2 duplex. Moreover, the D positions in 27 D2O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 A resolution X-ray and 3.0 A resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.

Base Sequence↗