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Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102-115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys111 in the loop, is readily oxidized and alkylated. We have found that modification of this Cys111 with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly108 to Cys111 in loop VI. One loop VI of the dimer forms a 310-helix (Gly108 to His110) within a unique β-bridge stabilized by a hydrogen bond between Ser105-NH and His110-CO, while the other forms a β-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.

Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2',4'-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2',4'-BNA/LNA in its sense strand and with 2'-methoxy (2'-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2',4'-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5'-UG-3' in the sense strand would ameliorate the negative immune response.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.32; published online 18 September 2012.

In its element: Zn(2+) at the M7 site of MFI-type zeolites activates H(2), via ZnH and OH species, and leads to Zn(0) species. The Zn(0) species returns to its original state, a Zn(2+) ion, upon evacuation of the zeolite at 873 K (see picture). The formation of the Zn(0) species is supported by DFT calculations., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Silver turns up the A-C: In the presence of Ag(I) ions, a DNA polymerase incorporated deoxyadenosine (from dATP) at the site opposite cytosine in the template strand to afford the full-length product (see scheme), meaning that DNA polymerases prefer a C-Ag(I)-A base pair to the more thermodynamically stable C-Ag(I)-C base pair., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2',4'-BNA (also called as locked nucleic acid (LNA)) and 2',4'-BNA(NC) chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2',4'-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2',4'-BNA-AON, 2',4'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine-thymine (T-T) and cytosine-cytosine (C-C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine-pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil-5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine-thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).

We found from DFT calculations that Ag-Ag orbital interactions as well as Ag-O electrostatic interactions determine the structures of three silver cations inside a nanometer-sized cavity of ZSM-5 (Ag(3)-ZSM-5) in lower and higher spin states. Both interactions strongly depend on the number of Al atoms substituted for Si atoms on the ZSM-5 framework (ZSM-5(Al(n))), where n ranges from 1 to 3. In smaller n, stronger Ag-Ag orbital interactions and weaker Ag-O electrostatic interactions operate. Accordingly, there are significant dependencies of the structures of three silver cations on the number of Al atoms. In lower spin states of Ag(3)-ZSM-5(Al(1)) and Ag(3)-ZSM-5(Al(2)), D(3h)-like triangle clusters are contained inside ZSM-5 whereas their higher spin states have triangle clusters distorted significantly from the D(3h) structure. In lower spin states, the totally symmetric orbital consisting of 5s(Ag) orbitals is responsible for cluster formation, whereas in higher spin states occupation of a 5s(Ag)-based orbital with one node results in significant distortion of the triangle clusters. The distortion can be partially understood by analogies to Jahn-Teller distortion of the bare D(3h) Ag(3)(+) cluster in the triplet spin state. When n is 3, we found that three silver cations are isolated in a lower spin state and that a linear cluster consisting of two silver cations is formed in a higher spin state. Thus, we demonstrate from DFT calculations that the number of Al atoms can control the properties of three silver cations inside a ZSM-5 cavity. Since the structural and electronic features of the enclosed silver clusters can link to their catalytic properties, the DFT findings can help us to understand the catalytic activity of Ag-ZSM-5.

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metal-mediated base pairs formed by the interaction between metal ions and artificial bases in oligonucleotides have been developed for potential applications in nanotechnology. We recently found that a natural C:C mismatched base pair bound to an Ag(+) ion to generate a novel metal-mediated base pair in duplex DNA. Preparation of the novel C-Ag-C base pair involving natural bases is more convenient than that of metal-mediated base pairs involving artificial bases because time-consuming base synthesis is not required. Here, we examined the thermodynamic properties of the binding between the Ag(+) ion and each of single and double C:C mismatched base pair in duplex DNA by isothermal titration calorimetry. The Ag(+) ion specifically bound to the C:C mismatched base pair at a 1:1 molar ratio with 10(6) M(-1) binding constant, which was significantly larger than those for nonspecific metal ion-DNA interactions. The specific binding between the Ag(+) ion and the single C:C mismatched base pair was mainly driven by the positive dehydration entropy change and the negative binding enthalpy change. In the interaction between the Ag(+) ion and each of the consecutive and interrupted double C:C mismatched base pairs, stoichiometric binding at a 1:1 molar ratio was achieved in each step of the first and second Ag(+) binding. The binding affinity for the second Ag(+) binding was similar to that for the first Ag(+) binding. Stoichiometric binding without interference and negative cooperativity may be favorable for aligning multiple Ag(+) ions in duplex DNA for applications of the metal-mediated base pairs in nanotechnology.

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.

Metal-mediated base pair formation, resulting from the interaction between metal ions and artificial bases in oligonucleotides, has been developed for its potential application in nanotechnology. We have recently found that the T:T mismatched base pair binds with Hg(II) ions to generate a novel metal-mediated base pair in duplex DNA. The thermal stability of the duplex with the T-Hg-T base pair was comparable to that of the corresponding T:A or A:T. The novel T-Hg-T base pair involving the natural base thymine is more convenient than the metal-mediated base pairs involving artificial bases due to the lack of time-consuming synthesis. Here, we examine the specificity and thermodynamic properties of the binding between Hg(II) ions and the T:T mismatched base pair. Only the melting temperature of the duplex with T:T and not of the perfectly matched or other mismatched base pairs was found to specifically increase in the presence of Hg(II) ions. Hg(II) specifically bound with the T:T mismatched base pair at a molar ratio of 1:1 with a binding constant of 10(6) M(-1), which is significantly higher than that for nonspecific metal ion-DNA interactions. Furthermore, the higher-order structure of the duplex was not significantly distorted by the Hg(II) ion binding. Our results support the idea that the T-Hg-T base pair could eventually lead to progress in potential applications of metal-mediated base pairs in nanotechnology.

Large-scale density functional theory (DFT) calculations found significant preferences of two-coordinated copper cations as Xe binding site in ZSM-5. Such site-preferences cannot be seen in usual adsorbents such as the CO or NO molecule inside Cu-ZSM-5 as well as the Xe atom inside alkali-metal exchanged ZSM-5s. A key factor in the specificity of the inner Xe atom is that interactions of the Xe atom with the extraframework copper cation are substantial relative to the extraframework alkali-metal cases, but weak relative to the CO and NO cases. Since the Xe atom can distinguish two-coordinated copper cations from others, it can be utilized to track sensitively the location of active sites of Cu-ZSM-5.

Stabilization of nucleic acid assemblies, such as duplex and triplex, is quite important for their wide variety of potential applications. Various stabilization methods, including molecular designs of chemically modified nucleotides and hybrid stabilizers, and combinations of different stabilization methods have been developed to increase stability of nucleic acid assemblies. However, combinations of two stabilizing methods have not always yielded desired synergistic effects. In the present study, to propose a strategy for selection of a rational combination of stabilizing methods, we demonstrate synergistic stabilization of triplex by 2'-O,4'-C-methylene-bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide and addition of poly(l-lysine)-graft-dextran copolymer [poly(l-lysine) grafted with hydrophilic dextran side chains]. Each of these methods increased the binding constant for triplex formation by nearly 2 orders of magnitude. However, their kinetic contributions were quite distinct. The copolymer increased the association rate constant, whereas the 2',4'-BNA modification decreased the dissociation rate constant for triplex stabilization. The combination of both stabilizing methods increased the binding constant by nearly 4 orders of magnitude. Kinetic analyses revealed that the successful synergistic stabilization resulted from kinetic complementarity between increased association rate constants by the copolymer and decreased dissociation rate constants by the 2',4'-BNA modification. The stabilizing effect of one stabilization method did not alter that of the other stabilization method. We propose that kinetic analyses of each stabilizing effect permit selection of a rational combination of stabilizing methods for successful synergy in stabilizing nucleic acid assemblies.

We examined the effect of 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA(NC)) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 2',4'-BNA(NC) modified TFO was significantly higher than that observed with unmodified TFO. The 2',4'-BNA(NC) modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 2',4'-BNA(NC) backbone modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

It is difficult and time-consuming to distinguish Haemophilus influenzae from the genotypically similar Haemophilus parainfluenzae, which is a commensal of the human oral cavity. The novel nucleic acid amplification technique of loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was evaluated for H. influenzae detection. A H. influenzae-specific LAMP primer set was designed for the outer membrane protein P6 gene. Primer set specificity was validated using 4 Haemophilus spp. and 13 other species. Within 60 min, LAMP detected 100 or more copies of purified DNA with a sensitivity that was 10-fold higher than that of conventional PCR. This method can be used to differentiate H. influenzae from H. parainfluenzae strains. Thus, LAMP may represent a sensitive and reliable means of diagnosing H. influenzae infection.

To understand the telomere regulation mechanism in relation to cell aging and cancer, we examined the single-stranded telomeric DNA binding domain (ssDBD) of fission yeast telomere-binding protein Pot1 by constructing a series of deletion mutants. We found that Pot1(1-182) (amino acids 1-182) stably expressed in Escherichia coli without any degradation retained a stable folded structure and functional telomeric DNA binding activity, indicating that Pot1(1-182) corresponds to ssDBD. We investigated the amino acids of Pot1(1-182) involved in single-stranded telomeric DNA recognition by constructing a series of site-directed mutants. Although the previously reported X-ray crystallographic structure suggests that 12 amino acids contact the telomeric DNA, an electrophoretic mobility shift assay and isothermal titration calorimetry analyses of the binding ability of the site-directed mutants indicated that only five amino acids significantly contributed to telomeric DNA recognition. We conclude that the contribution to recognition is quite different in magnitude among the amino acids judged to contact the target by X-ray crystallographic structure.

Addition of mercury (II) cation to fluorescent-labeled duplex involving a T:T mismatch base pair and silver (I) cation to fluorescent-labeled duplex involving a C:C mismatch base pair significantly changed the fluorescence intensity, but no significant change in the fluorescence intensity was observed for duplexes involving the other base pairs. The fluorescence spectral change upon addition of the metal cation can discriminate T:T and C:C mismatch base pairs from the other base pairs. Our results certainly support the idea that the fluorescence spectral change upon addition of the metal cation could be a convenient strategy for the mismatch base pair detection by the heteroduplex analysis, and may eventually lead to progress in single nucleotide polymorphism genotyping.

To understand the regulation mechanism of fission yeast telomeric DNA, we analyzed the structural properties of 4Gn: d(G(n)TTAC)(4) (n = 3, 4) and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n=2-6) and 4Gn: d(GnTTAC)4 (n=3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.

A triplex formation-based artificial transcription factor to recognize any upstream sequence of target genes was developed to regulate the target gene expression. The artificial transcription factor contains a single-stranded RNA to bind with duplex DNA of the upstream sequence of the target gene to form triplex, and an effecter domain, such as activation or repression domain, of transcription factor. Reporter beta-galactosidase activity in yeast was increased 1.5-2 times by introduction of the artificial transcription factor. The novel artificial transcription factor may be a useful tool to regulate the target gene expression and reveal unknown function of the target genes.

An 80-year-old man, who had been taking the antiarrhythmic drug amiodarone for chronic atrial fibrillation since October 2004, developed dyspnea and fever in June 2005, and was admitted to our hospital. Chest X-ray and CT scan showed ground-glass opacities in all lung fields. Arterial blood gas analysis revealed hypoxemia. These findings led to a diagnosis of acute respiratory distress syndrome. Oral amiodarone was discontinued, and steroid pulse therapy was started; however, the disease progressed rapidly, and the patient died on the fourth hospital day. Autopsy showed diffuse alveolar damage, accumulation of edema fluid in the alveolar spaces, and the presence of foamy macrophages, leading to a diagnosis of amiodarone-induced pulmonary damage.

The synthesis and detailed study of the new mononuclear spin crossover complex [Fe(II)H2L(2-Me)](ClO4)2 (where H2L(2-Me) = bis[((2-methylimidazol-4-yl)methylidene)-3-aminopropyl]ethylenediamine) are reported. Variable-temperature magnetic susceptibility measurements show the occurrence of a steep spin crossover centered at 171.5 K with a hysteresis loop of ca. 5 K width (T(/2)(increasing) = 174 K and T(1/2)(decreasing) = 169 K, for increasing and decreasing temperatures, respectively). The crystal structure has been resolved for the high-spin (HS) and low-spin (LS) states at 200 and 123 K, respectively, revealing a crystallographic phase transition that occurs concomitantly to the spin crossover: at 200 K, the complex crystallizes in the monoclinic system, space group P2(1)/n, while the space group is P2(1) at 123 K. The mean Fe-N distances are shortened by 0.2 A, but the thermal spin crossover is accompanied by significant structural changes: the rearrangement of the central atom C12 of a six-membered chelate ring of [Fe(II)H2L(2-Me)]2+ to two positions (C12A and C12B) and, consequently, the lack of an inversion center at 123 K (P2(1) space group). Both HS and LS supramolecular structures involve all possible hydrogen bonds between imidazole and amine NH functions, and perchlorate anions; however, the HS supramolecular structure is a one-dimensional (1D) network, and the LS phase may better be described as a two-dimensional (2D) extended structure of A and B molecules. The structural phase transition of [FeH2L(2-Me)](ClO4)2 seems to trigger the steep and hysteretic spin crossover. Discontinuities in the temperature dependence of the Mössbauer parameters (isomer shift and quadrupole splitting) at the spin crossover temperature confirmed the occurrence of a structural phase transition. The experimental enthalpy and entropy variations were determined by differential scanning calorimetry (DSC) as 7.5 +/- 0.4 kJ/mol and 45 +/- 3 J K(-1) mol(-1), respectively. The regular solution theory was applied to the experimental data, yielding an interaction parameter of Gamma = 3.36 kJ/mol, which is larger than 2RT(1/2), which fulfills the condition for observing hysteresis.

The patients were a 28-year-old man and a his 27-year-old wife. The husband smoked a cigarette immediately after using a waterproofing spray, and developed fever, cough, and dyspnea 15 min later. The wife had nausea 2 hours later. Nine hours later, the husband visited a local clinic, and was referred to our hospital because of hypoxemia. In addition, chest CT showed ill-defined areas of increased density, predominantly in the bilateral upper lung fields, with interlobular septal thickening, and he was hospitalized. Although the wife was asymptomatic at the time of examination, she had chest CT findings similar to those of her husband, and was also hospitalized. After admission, the husband received steroid pulse therapy and oxygen inhalation for his symptoms and hypoxemia, with return of arterial blood gas analysis results to normal on the third day. The wife had no symptoms or hypoxemia during her hospital stay. Their chest CT findings improved on the seventh day after admission, and they were discharged. Thus, it appears that the couple suffered from acute respiratory illness due to waterproofing spray exposure, and probably heat degradation products from cigarette smoking caused the husband to have severe symptoms.

In this randomized crossover study, 22 adult patients with moderate-to-severe persistent bronchial asthma were assigned to one of two groups. Patients in group 1 were administered fluticasone dry powder inhaler (DPI) for 8 weeks followed by a 2-week washout period, then hydrofluoroalkane-beclometasone dipropionate (HFA-BDP) for 8 weeks. After a further 2-week washout, they were again administered fluticasone DPI for 8 weeks. Patients in group 2 were assigned HFA-BDP followed by fluticasone PII and finally HFA-BDP over the same time periods. In both groups, no significant difference was observed in use of beta2-agonists and symptom score between the treatment periods; however, markers of pulmonary function were significantly higher when on HFA-BDP versus fluticasone DPI. Significant increases of morning peak expiratory flow (PEF) (p < 0.01), forced expiratory volume in 1 second (FEV1.0) (p < 0.01), V50 (p < 0.05), and V25 (p < 0.01) were observed at 18 weeks in group 1, whereas there were significant decreases of V50 (p < 0.05) at 18 weeks in group 2. No significant difference was noted in circulating eosinophil count and serum ECP between the 2 treatments; however, ECP in induced sputum and nitric oxide in expired gas were significantly lower (p < 0.05 and < 0.01, respectively) when on HFA-BDP versus fluticasone DPI. HFA-BDP might be delivered to small airways more effectively than fluticasone DPI.

A family of spin-crossover (SC) complexes, [Fe(II)H(3)L(Me)]Cl.X (X(-) = PF(6) (-), AsF(6) (-), SbF(6) (-), CF(3)SO(3) (-)), 1-4, has been synthesized, in which H(3)L(Me) denotes the hexadentate N(6) tripod-like ligand tris[2-{[(2-methylimidazol-4-yl)methylidene]amino}ethyl]amine, containing three imidazole groups, with a view to establishing the effect of the counter anion on the SC behavior. These complexes have been found to crystallize in the same monoclinic crystal system with similar cell dimensions. The general crystal structure consists of a two-dimensional (2D) extended network constructed by NH...Cl- hydrogen bonds between Cl- and the imidazole NH groups of three neighboring [Fe(II)H(3)L(Me)]2+ ions, while the anion X exists as an isolated counter anion and occupies the space between the 2D sheets. Magnetic susceptibilities and Mössbauer spectra have revealed a variety of SC behaviors depending on the counter anion, including a one-step HS<==>(HS + LS)/2 (1, X = PF(6) (-)), a two-step HS<==>(HS + LS)/2<==>LS with a slow thermal relaxation (2, X = AsF(6) (-)), a gradual one-step HS<==>LS (3, X = SbF(6) (-)), and a steep one-step HS<==>LS with hysteresis (4, X = CF(3)SO(3) (-)). The complexes assume the space group P2(1)/n in the HS state, P2(1) in the HS + LS state, and P2(1)/n in the LS state. The Fe-N bond lengths and the N-Fe-N bond angles are indicative of the HS, HS + LS, and LS states. The molecular volumes, V, of the counter anions have been evaluated by quantum-chemical calculations as follows: 53.4 A(3) (BF(4) (-)), 54.4 A(3) (ClO(4) (-)), 73.0 A(3) (PF(6) (-)), 78.5 A(3) (AsF(6) (-)), 88.7 A(3) (SbF(6) (-)), and 86.9 A(3) (CF(3)SO(3) (-)). The size and shape of the counter anion affects the flexible 2D network structure constructed by the hydrogen bonds, leading to modifications of the SC behavior. These estimated relative sizes of the counter anions correlate well with the observed SC behaviors.

We encountered 3 male patients with spontaneous pneumomediastinum. The patients were a 16-year old and a 17-year old and a 24-year old. Predisposing episodes for the development of spontaneous pneumomediastinum could be identified in all 3 patients: throwing a ball during a baseball game in 1, lifting a heavy load during work in 2. However, they were healthy and suddenly developed symptoms in the absence of any underlying disease. The presenting complaint was chest pain in all 3 patients. Chest X-ray films and chest CT images revealed pneumomediastinum. A diagnosis of spontaneous pneumomediastinum was made based on chest X-ray films and chest CT images. After conservative treatment, all 3 patients recovered.

We have already found that silver (I) cation specifically binds to C:C mismatch base pair in heteroduplex, which increases the melting temperature of heteroduplex involving C:C mismatch base pair by about 4 degrees C. This result shows that addition of the metal cation is a promising strategy for the mismatch base pair detection in heteroduplex, but UV melting to determine the melting temperature is time-consuming. In the present study, to develop a more convenient way for the mismatch base pair detection, we examined the fluorescence spectral change of fluorescent-labelled duplex upon addition of the metal cation. Addition of silver (I) cation to the heteroduplex involving the C:C mismatch base pair significantly changed the fluorescence intensity, but no significant change in the fluorescence intensity was observed for the duplexes involving other base pairs. Our results certainly support the idea that the fluorescence spectral change upon the addition of the metal cation could be a convenient strategy for the mismatch base pair detection by the heteroduplex analysis, and may eventually lead to progress in SNP genotyping.

We have already found that a single silver (I) cation specifically binds to a single C:C mismatch base pair in heteroduplex, which increases the melting temperature (T(m)) of heteroduplex involving a single C:C mismatch base pair by about 4 degrees C. Here, to examine the properties of the interaction between two C:C mismatch base pairs and silver (I) cations, we analyzed the effect of silver (I) cations on the thermal stability of heteroduplexes involving two C:C mismatch base pairs. The positions of the two C:C mismatch base pairs in heteroduplex did not significantly affect the magnitude of the increase in T(m) upon addition of silver (I) cation, suggesting that the binding affinity of the first silver (I) cation with one of the two C:C mismatch base pairs and that of the second silver (I) cation with the other of the two mismatch base pairs may be independent of the positions of the two mismatch base pairs. The larger magnitude of the increase in T(m) for the second silver (I) cation binding than that observed for the first silver (I) cation binding suggests that the binding affinity for the second silver (I) cation may be significantly higher than that for the first silver (I) cation. Our results certainly support the idea that addition of the metal cation is a promising strategy for the mismatch base pair detection in the heteroduplex analysis and may eventually lead to progress in SNP genotyping.

We have already found that mercury (II) cation specifically binds to T:T mismatch base pair in heteroduplex, which increases the melting temperature of heteroduplex involving T:T mismatch base pair by about 4 degrees C. This result shows that addition of the metal cation is a promising strategy for the mismatch base pair detection in heteroduplex, but UV melting to determine the melting temperature is time-consuming. In the present study, to develop a more convenient way for the mismatch base pair detection, we examined the fluorescence spectral change of fluorescent-labelled duplex upon addition of the metal cation. Addition of mercury (II) cation to the heteroduplex involving the T:T mismatch base pair significantly changed the fluorescence intensity, but no significant change in the fluorescence intensity was observed for the duplexes involving other base pairs. Our results certainly support the idea that the fluorescence spectral change upon the addition of the metal cation could be a convenient strategy for the mismatch base pair detection by the heteroduplex analysis, and may eventually lead to progress in SNP genotyping.

Three FeII complexes, [Fe(HLR)2](BPh4)2.solvent (R=H, Me, Ph), were synthesized, where BPh4-=tetraphenylborate and HLR=2-substituted-imidazol-4-yl-methylideneamino-2-ethylpyridine. The magnetic susceptibility measurements in 5-300 K revealed that [Fe(HLH)2](BPh4)2.H2O, [Fe(HLMe)2](BPh4)2.2CH3CN, and [Fe(HLPh)2](BPh4)2.CH3CN are low-spin (LS), spin-crossover (SC), and high-spin (HS) FeII complexes, respectively, indicating that the spin state can be effectively tuned by the bulkiness of the substituent. Complex shows a steep SC around 250 K, where it assumes a cyclic structure of {[Fe(HLMe)2]BPh4}2 constructed by four NH/pi bonds between the imidazole group and the phenyl ring of BPh4- in the HS state and a deformed structure with NH/pi bonds and linear CH3CN...HN hydrogen bonds at the terminals in the LS state.

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.

It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.

Synergic stabilization of DNA triplexes by oligo-N3'-->P5' phosphoramidate (PN) modification and additions of comb-type cationic copolymers was demonstrated. The combination of the copolymer and the PN modification increased triplex K(a) about 4 orders of magnitude. Kinetic analysis revealed that observed stabilization resulted from kinetic complimentarity between increased association rates by the copolymer and decreased dissociation rates by the PN modification of triplex forming oligonucleotides. No countering interference between these stabilizing effects was observed. We propose that kinetic analyses of stabilizing effects permit selection of a rational combination of stabilizing methods for successful synergy in stabilizing complex formation.

Ab initio calculations were carried out to characterize the structure and energetic of silver cation (Ag (I)) complex with cytosine (C:Ag:C) and mercury cation (Hg (II)) complex with thymine (T:Hg:T) systems. These metal-modified mismatch base pairs have been optimized using Hatree-Fock method without any symmetry constrains. Using above methods, the models of Ag (I) in a crosslink between O2 carbonyl oxygen of cytosine, O2(C):Ag:O2(C), and Hg (II) in a crosslink between N3 nitrogen atom of thymine, N3(T):Hg:N3(T) were obtained. Furthermore, the interaction energies of C:Ag:C and T:Hg:T models were estimated. The result showed that the coordination silver (I) cation with cytosine is more stable than hydrate state.

Triplex formation-based artificial transcription factor to recognize any upstream sequence of desired genes was developed to regulate expression of any target genes. The reporter beta-galactosidase activity in yeast was increased 1.5-2 times by the introduction of the artificial transcription factor. The novel factor may be a useful tool to regulate expression of any target genes and reveal unknown function of the target genes.

We compared the structure of fission yeast telomeric DNA, 4G4: d(GGGGTTAC)4, and nontelomeric DNA, 4T4: d(TTTTTTAC)4, and examined their interaction with telomeric DNA binding domain of telomeric DNA binding protein Pot1 (Pot1DBD). 4T4 did not form any higher-order structure, but 4G4 formed intramolecular folded tetraplex structure in the presence of Na+. Although Pot1DBD did not induce any significant structural change of 4T4, the intramolecular folded tetraplex structure of 4G4 was unfolded by the interaction with Pot1DBD. Pot1 may facilitate telomere elongation of telomerase by disrupting the tetraplex structure of the telomeric DNA.

We examined the effect of silver (I) cation on the thermal stability of heteroduplex and homoduplex. Addition of silver (I) cation increased the melting temperature of heteroduplex containing C:C mismatch base pair by about 4 degrees C. The thermal stability of homoduplex and heteroduplexes containing other kinds of mismatch base pairs was not significantly changed by the addition of silver (I) cation. Isothermal titration calorimetric study demonstrated that silver (I) cation directly bound to C:C mismatch base pair in heteroduplex at a molar ratio of 1:1. The binding constant and the enthalpy change for the binding of silver (I) cation to C:C mismatch base pair was approximately 10(6) M(-1) and -3 kcal mol(-1), respectively. We conclude that silver (I) cation directly binds to C:C mismatch base pair in heteroduplex with high affinity and specificity. Our results certainly support the idea that the addition of silver (I) cation to C:C mismatch base pair in heteroduplex could be a convenient strategy for heteroduplex analysis and may eventually lead to progress in single nucleotide polymorphism genotyping.

The tulobuterol transdermal therapeutic system (TTS) is the world's first commercially available transdermal preparation of tulobuterol, a beta-2 stimulant, that can maintain effective blood tulobuterol levels for 24 hours when applied once daily. In the present study, a total of 36 adult patients with mildly persistent (Step 2) or moderately persistent (Step 3) bronchial asthma 19 who were using inhalational steroids and 17 who were not used tulobuterol TTS for one year and underwent measurement of peak expiratory flow (PEF) once daily. Peripheral eosinophil count, serum eosinophil cationic protein (ECP) level and airway responsiveness (Dmin) were evaluated at 6 months and 1 year after the start of the study. PEF exhibited significant improvements at 6 months and 1 year in patients treated with or without inhalational steroids, while serum ECP was improved significantly only in the patients on inhalational steroids. Patients not using inhalational steroids exhibited no significant exacerbation of Dmin at either 6 months or 1 year: One-year treatment with tulobuterol TTS did not appear to cause tachyphylaxis. The significant improvements in Dmin at 6 months and 1 year in the patients using inhalational steroids suggested that inhalational steroids offer beneficial effects in controlling airway inflammation. Tulobuterol TTS is considered quite beneficial in improving quality of life (QOL) in patients with bronchial asthma because its incidence of adverse effects including palpitations and shivering is significantly lower than those of oral preparations, because of its remarkable improvement of pulmonary function and symptoms of airway obstruction without increasing airway responsiveness even after repeated use, and because it is simple to use and offers excellent clinical efficacy.

We examined the effect of the combination of the two triplex-stabilizing factors, poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO), on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The combination of both stabilizing factors that was the triplex involving the 2',4'-BNA TFO in the presence of the copolymer synergistically increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the combination of the stabilizing factors can be a key method for triplex stabilization and may lead to progress in therapeutic applications of the antigene strategy in vivo.

Tulobuterol transdermal therapeutic system (TTS) is the world's first commercially available transdermal preparation of tulobuterol, a beta2-stimulant that can maintain effective blood tulobuterol (CAS 41570-61-0) levels for 24 h when applied once daily. In the present study, a total of 24 adult patients with mild persistent (Step 2) or moderate persistent (Step 3) bronchial asthma, consisting of 13 and 11 patients, who were or were not using inhalational steroids, respectively, used tulobuterol TTS (Hokunalin Tape) for one year and underwent measurement of peak expiratory flow (PEF) once daily. Peripheral eosinophil count, serum eosinophil cationic protein (ECP) level and airway responsiveness (Dmin) were evaluated at 6 months and 1 year, respectively, after the start of the study. PEF exhibited significant improvements after 6 months and 1 year in patients treated with or without inhalational steroids, while serum ECP was improved significantly only in the patients on inhalational steroids. Patients not using inhalational steroids exhibited significant exacerbation of Dmin at neither after 6 months nor after 1 year. One-year treatment with tulobuterol TTS did not appear to cause tachyphylaxis. The significant improvements in Dmin observed after 6 months and after 1 year in the patients using inhalational steroids suggested beneficial effects of inhalational steroids in controlling airway inflammation. Tulobuterol TTS is considered quite beneficial in improving quality of life (QOL) in patients with bronchial asthma because its incidence of adverse effects including palpitations and shivering is significantly lower than those of oral preparations because of its remarkable improvement of pulmonary function and symptoms of airway obstruction without increasing airway responsiveness even after repeated use, and because it is simple to use and ensures excellent clinical efficacy.

For the effective recognition of C x G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2'-O, 4'-C-methylene bridged nucleic acid with 1-isoquinolone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2'-O,4'-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.

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.

We analyzed the thermodynamics of purine motif triplex formations involving single mismatches of four different base triplets (A x A:T, T x A:T, A x T:A and T x T:A) by isothermal titration calorimetry. The T x A:T and A x A:T base triplets are the most stable and the A x T:A base triplet is the least stable among the four base triplets. The magnitude of the Gibbs free energy change varies within 1.0 kcal mol(-1) for the single mismatches of the base triplet. On the other hand, the magnitude of the enthalpy change exhibits very large sequence dependence with up to 14 kcal mol(-1) variation per the single mismatches among the four base triplets, which is 14 times larger than the sequence-dependent variation of the magnitude of the Gibbs free energy change. Thus, the single mismatches in the purine motif triplex do not have strong effects on the magnitude of the Gibbs free energy change, but the magnitude of the enthalpy change exhibits much larger sequence-dependent variations. The present results support the previously proposed nucleation-elongation mechanism of the triplex formation and offer some hints to design novel triplex-forming oligonucleotides with enhanced sequence specificity.

We examined the effect of morpholino (MOR) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with MOR-modified TFO was approximately 60 times larger than that observed with unmodified TFO. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the MOR backbone modification resulted mainly from the considerable increase in the association rate constant. The present results certainly support the idea that the MOR backbone modification of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.