Synthesis, structures and photophysical properties of hexacoordinated organosilicon compounds with 2-(2-pyridyl)phenyl groups.

We synthesised novel hexacoordinated organosilicon compounds with two 2-(2-pyridyl)phenyl groups. Single-crystal X-ray structure analyses indicated that Lewis acid-base interactions exist between the silicon atom and two nitrogen atoms of the pyridine rings, and that hexacoordinated organosilicon compounds have slightly distorted octahedral structures in the solid state. The hexacoordinated organosilicon compounds are stable in air, water, heat, acids, and bases. The fluorescent quantum yield increased dramatically and a significant red-shift of the maximum fluorescence wavelength was observed with the introduction of amino groups on the 2-(2-pyridyl)phenyl aromatic rings. The fluorescence colours of a hexacoordinated organosilicon compound with two amino groups can be switched by protonation and deprotonation (neutralisation) of the amino groups.

Control of Multicolor and White Emission by Adjusting the Equilibrium between Fluorophores, Lewis Acids, and Their Complexes in Polymers.

Multicolor emissive materials consisting of a single luminophore, a Lewis acid, and their complex were developed. The emission colors can be tuned by changing the concentration of the solution and the ratio of mixed solvents. Various emission colors in the solid state were observed when the complexes were added to polymers in different amounts. The color change is due to equilibrium disruption between the single luminophore, the Lewis acid, and the complex thereof. White emission was observed by appropriately controlling the equilibrium by changing the amount of the complex in the polymer.

A single-molecule assessment of the protective effect of DMSO against DNA double-strand breaks induced by photo-and γ-ray-irradiation, and freezing.

Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant for organs, tissues, and cell suspension in storage. In addition, DMSO is known to be a useful free radical scavenger and a radio-protectant. To date, many in vitro assays using cultured cells have been performed for analysing the protective effect of DMSO against genomic DNA damage; however, currently it has been rather difficult to detect DNA double strand breaks (DSBs) in a quantitative manner. In the present study, we aimed to observe the extent of DNA damage by use of single molecular observation with a fluorescence microscope to evaluate DSBs induced by photo- and γ-ray-irradiation, or freeze/thawing in variable concentrations of DMSO. As a result, we found that 2% DMSO conferred the maximum protective effect against all of the injury sources tested, and these effects were maintained at higher concentrations. Further, DMSO showed a significantly higher protective effect against freezing-induced damage than against photo- and γ-ray-irradiation-induced damage. Our study provides significant data for the optimization of DNA cryopreservation with DMSO, as well as for the usage of DNA as the protective agent against the injuries caused by active oxygen and radiations.

Chromatin compaction protects genomic DNA from radiation damage.

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5-50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.

Probability of double-strand breaks in genome-sized DNA by γ-ray decreases markedly as the DNA concentration increases.

By use of the single-molecule observation, we count the number of DNA double-strand breaks caused by γ-ray irradiation with genome-sized DNA molecules (166 kbp). We find that P1, the number of double-strand breaks (DSBs) per base pair per unit Gy, is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. The inverse relationship implies that the total number of DSBs remains essentially constant. We give a theoretical interpretation of our experimental results in terms of attack of reactive species upon DNA molecules, indicating the significance of the characteristics of genome-sized giant DNA as semiflexible polymers for the efficiency of DSBs.

Single-molecular enzymatic elongation of hyaluronan polymers visualized by high-speed atomic force microscopy.

Using high-speed scanning atomic force microscopy, we directly observed single-molecular enzymatic elongation of hyaluronan polymer chains at intervals of 10 s on a mica or lipid bilayer surface, on which Pasteurella multocida hyaluronic acid synthase (pmHAS) was immobilized. The reaction was started by the addition of both UDP-glucuronic acid and UDP-N-acetylglucosamine monomers. The average catalytic elongation rate constant (k(cat)) was found to be 1.8 mer s(-1) from one active enzyme physically adsorbed on a mica surface. When pmHAS was immobilized by inserting its hydrophobic tail part into lipid bilayers, most of the enzymes retained their activity, and the k(cat) values were found to be in the range 1-10 mer s(-1) for 29 enzymes (average was k(cat) = 2-4 mer s(-1)). These k(cat) values were lowest level of k(cat) = 1-100 s(-1) obtained in bulk solution by radioisotope methods.

Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27†MHz quartz-crystal microbalance.

Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 µM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.

Single-molecule force spectroscopy for studying kinetics of enzymatic dextran elongations.

Catalytic elongation of dextran by a single molecule of dextransucrase (DSase) was directly monitored by observing the movements of the positions of a rupture peak, which represented the adhesive force between an isomaltoheptaose (dextran 7-mer)-immobilized probe and a DSase-immobilized mica surface. This was initiated with the addition of sucrose monomers. From the histograms of the rupture peaks after elongation reactions on each individual enzyme and the continuous peak shift of certain single enzymes, the catalytic elongation rate constant (k(cat)) was ascertained to be 1.2-2.7 s(-1).

Kinetic studies of dextransucrase enzyme reactions on a substrate- or enzyme-immobilized 27 MHz quartz crystal microbalance.

Catalytic elongation by dextransucrase (DSase) was monitored directly on a dextran-acceptor- or DSase-immobilized 27 MHz quartz crystal microbalance (QCM). Kinetic parameters for the binding of the enzyme to the dextran acceptor (k(on), k(off), and K(d)) and enzymatic elongation in the presence of a sucrose monomer (K(m) for sucrose and k(cat)) were determined. The kinetic parameters obtained by both methods were consistent.

Changes in the images of teaching, teachers, and children expressed by student teachers before and after student teaching.

The purpose of this study was to investigate how education majors' images of teaching, teachers, and children change before and after student teaching, with special attention to the grade level (Grades 1-2, 3-4, 5-6) taught by the student teachers at primary school in Japan. A total of 126 student teachers from an education faculty (49 men, 77 women) participated in this study using metaphor-questionnaires before and after student teaching. For images of teaching, responses to the factors Dull Event and Live Event changed, suggesting that students started to develop more positive, active, and clear images of teaching. For images of teachers, responses on the factor Performer changed, suggesting that students started to develop more active images of teachers. For images of children, responses on the factors Critic and Pure-minded Person changed, suggesting that student teachers started to develop more realistic images of children. However, grade level taught had no significant effect.

Kinetic analyses of bindings of Shiga-like toxin to clustered and dispersed Gb3 glyco-arrays on a quartz-crystal microbalance.

One-, two-, four-, and eight-branched globotriaosyl saccharides (Gb(3): Gal-alpha1,4-Gal-beta1,4-Glc), whose reducing ends were biotinylated, were prepared (1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, and 8Gb(3)-bio, respectively). They are dispersively immobilized as a glyco-array in the matrix of biotinylated maltotriose (Glc(3)-bio) on a streptavidin-covered 27 MHz quartz-crystal microbalance (QCM). The binding kinetics of the verotoxin B subunit (VTB) to various branched Gb(3)-bio ligands in the Glc(3)-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. VTB can recognize the Gb(3) unit but not the Glc(3) unit, where VTB is a pentamer having five binding sites for one Gb(3) unit per each B subunit (having a total of 15 binding sites for Gb(3)). By changing the Gb(3) multivalency, the Gb(3) packing density, and the Gb(3) cluster size in the Glc(3) matrix, association constants (K(a)), maximum amounts bound (Delta m(max)), and binding and dissociation rate constants (k(on) and k(off)) were obtained. When 15 sites of VTB were recognized by 16 Gb(3) units, K(a) was 100 times larger than that when 15 sites of VTB were recognized by only 2 Gb(3) units, with a 6-fold-larger k(on) and a 25-fold-smaller k(off). When the Gb(3) multivalency was changed by covering with two 1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, or 8Gb(3)-bio ligands on two pockets of one streptavidin, the K(a) values increased with increasing branch number from one to eight. When the Gb(3) cluster size was changed from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit in the matrix, the K(a) values increased but the Delta m(max) values decreased with increasing cluster size from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit. This is the first example of systematically obtaining all kinetic parameters of sugar-binding proteins to sugars on a glyco-array by changing the sugar multivalency, the sugar packing density, and the sugar cluster size in the matrix.

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance.

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K(a)) and binding and dissociation rate constants (k(on) and k(off)) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K(a)=(4+/-1)x10(6) M(-1), k(on)=(4+/-1)x10(4) M(-1) s(-1), and k(off)=(12+/-2)x10(-3) s(-1). On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K(a)=(14+/-2)x10(6) M(-1), k(on)=(14+/-2)x10(4) M(-1) s(-1), and k(off)=(7+/-2)x10(-3) s(-1). Thus, K(a) for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k(on)) and a three times smaller dissociation rate constant (k(off)). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.

Nanometer-scale immobilization of polysaccharides on hydrophobic polymer plates in supercritical fluoroform/water emulsions.

Hydrophilic polysaccharides such as dextran and hyaluronan were immobilized on a hydrophobic polystyrene (PSt) plate by a nanometer-scale surface penetration method in the emulsion of aqueous solutions in supercritical fluoroform (scCHF3). Since a supercritical fluid has high diffusiveness, water emulsions of polysaccharides can penetrate into the polymer surface. Dextran was surface-penetrated by two different methods: (1) the penetration of sucrose as a glucose donor and then the enzymatic polymerization to dextran near the surface catalyzed by dextransucrase, and (2) the direct penetration of dextran polymer into the PSt plate. The contact angle for water of the dextran-penetrated PSt plate was decreased to 78 degrees from 95 degrees of the untreated plate. The surface coverage and the penetration depth of polysaccharides could be obtained to be 10-30% and 10-20 nm, respectively, by X-ray photoelectron spectroscopy. These values could be controlled by the pressure of scCHF3. The transparency of the PSt dish did not change after the dextran penetration. Dextrans on the PSt plate could be elongated enzymatically by dextransucrase in the presence of sucrose as a glucose donor, and be detected by the enzyme-linked biotin-avidin assay. When anionic hyaluronan was surface-penetrated on the PSt plate instead of the neutral dextran, the plate showed the specific adhesion for human T-cells having hyaluronan receptors.

Analysis of 8-hydroxy-2'-deoxyguanosine in urine using high-performance liquid chromatography-electrospray tandem mass spectrometry.

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC-MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mmx120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n=246) varied from 0.16 to 16.48 microg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 microg of 8-OHdG/g creatinine. The correlation between the developed HPLC-MS/MS method and the existing HPLC-EC method was good with an R2 value of 0.8707.

Overexpression of pituitary tumor transforming gene 1 in HCC is associated with angiogenesis and poor prognosis.

The pituitary tumor transforming gene 1 (PTTG1) protein is cell-cycle regulated and is identified as a human securin that inhibits sister chromatid separation and is involved in transformation and tumorigenesis. PTTG1 has very low or undetectable expression in most normal human tissues, but it is abundantly expressed in malignant cell lines and pituitary tumors. In this study, we investigated human PTTG1 expression in 62 hepatocellular carcinoma (HCC) specimens using quantitative real-time reverse transcription polymerase chain reaction analysis. We found that, compared with corresponding noncancerous liver tissues, PTTG1 was remarkably overexpressed in HCCs (PTTG1/beta-actin; 0.443 +/- 0.073 vs. 0.068 +/- 0.007; P < .0001). Furthermore, we found a significant correlation between PTTG1 expression and serum alpha-fetoprotein level (P < .001). Univariate and multivariate analyses revealed that the PTTG1 messenger RNA (mRNA) expression was an independent prognostic factor for disease-free (odds ratio 2.70; P = .037) and overall (odds ratio 5.35; P = .007) survival. Moreover, we discovered a significant relationship between PTTG1 expression and intratumoral microvessel density. Our data supported an important role for PTTG1-mediated upregulation of fibroblast growth factor (FGF)-2, one of angiogenesis and modulation of tumor progression, in hepatocarcinogenesis. In conclusion, PTTG1 might be critically involved in the development of HCCs through the promotion of angiogenesis. PTTG1 is overexpressed in HCC and our results suggest that PTTG1 mRNA expression has prognostic significance for the survival of postoperative patients with HCC.

Analysis of 8-hydroxydeoxyguanosine among workers exposed to diesel particulate exhaust: comparison with urinary metabolites and PAH air monitoring.

Oxidative DNA damage and repair, as measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine and DNA samples were studied in association with work-related diesel exhaust exposure among garage and waste collection workers. Seasonal variations of the urinary 8-OHdG levels in pre- and two post-workshift urine samples of 29 exposed workers and 36 control persons were evaluated. The mean+/-SE levels of post-workshift 8-OHdG (mumol/mol crea) were 1.52+/-0.44 in winter and 1.61+/-0.33 in summer for the exposed workers, and 1.56+/-0.61 in winter and 1.43+/-0.49 in summer for the controls, respectively. No significant difference in the urinary 8-OHdG levels between exposed workers and control subjects in winter (p=0.923) and summer (p=0.350) was observed. A linear mixed model, adjusted for years of employment, age, ex/non-smoking and BMI, indicated no significant dose exposure-relationships between the urinary 8-OHdG and 15 PAH air concentrations nor between the 8-OHdG and 7 PAH monohydroxy-metabolites analyzed in the same workers. 8-OHdG was also analyzed in the mononuclear cell DNA of 19 exposed and 18 control subjects. The mean value of 8-OHdG/non-modified 2'-deoxyguanosine (8-OHdG/105 dG+/-SE) were 4.89+/-0.17 for the exposed and 4.11+/-0.16 for the control persons, which showed no correlation with the urinary 8-OHdG levels (r=0.01, n=28, P=0.96). The PAH exposure at workplaces was mainly composed of volatile compounds, particularly naphthalene, suggesting low exposure through the respiratory tract and a low effect of PAH in ROS induction.

Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance.

A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.

Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma.

BACKGROUND/AIMS: Alteration in transforming growth factor-beta signaling pathway is one of the main causes of hepatocellular carcinoma (HCC). The human runt-related transcription factor 3 gene (RUNX3) is an important component of this pathway. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including HCC. Therefore, we examined genetic and epigenetic alterations of RUNX3 in human HCC. METHODS: Five HCC cell lines and 41 patients with HCC were investigated in this study. We examined the expression of RUNX3 mRNA, methylation status of RUNX3 promoter region, loss of heterozygosity (LOH) at 1p36, and mutation analysis. These results were compared with clinicopathological data. RESULTS: Promoter hypermethylation was detected in four (80%) of five HCC cell lines and 31 (75.6%) of 41 HCC tissues, confirmed by sequence of bisulfite-treated DNA. LOH was detected in 14 (37.8%) of 37 HCC. By comparison with clinicopathological data, hypermethylation was more common in hepatitis C virus antibody and formation of capsule-positive cases, and decrease of expression was correlated strongly with advanced stage and LOH-detected cases. CONCLUSION: Hypermethylation and LOH appear to be common mechanisms for inactivation of RUNX3 in HCC. Therefore, RUNX3 may be an important tumor suppressor gene related to hepatocarcinogenesis.

Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance.

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.

Enzymatic preparation of biotinylated naturally-occurring sialylglycan and its molecular recognition on a quartz-crystal microbalance.

A biotinylated sialylglycan was prepared enzymatically by endo-M, and binding behavior of an SSA lectin was studied on a different coverage of a sialylglycan-immobilized 27 MHz quartz-crystal microbalance (QCM).