siRNA-mediated Knockdown of the Heme Synthesis and Degradation Pathways: Modulation of Treatment Effect of 5-Aminolevulinic Acid-based Photodynamic Therapy in Urothelial Cancer Cell Lines

Photodynamic therapy mediated by 5-aminolevulinic acid (ALA-PDT) has been developed as a therapeutic modality for refractory superficial bladder cancers. Here, in experiments using urothelial cancer cell lines, we investigated the effects of siRNA modulating heme-synthetic and degradation pathways for ALA-PDT. Targeted knockdown of ferrochelatase (FECH) suppressed heme synthesis and significantly increased intracellular protoporphyrin IX (PpIX) accumulation, leading to enhanced phototoxicity in four of five cell lines. Heme oxygenase-1 (HO-1) is recognized as important for cytoprotection against oxidative stress such as PDT. Targeted knockdown of HO-1 leads to decreased intracellular PpIX accumulation, resulting in a failure to enhance ALA-PDT effect in four cell lines. Knockdown of HO-1 caused marked growth inhibition in UM-UC-2 overexpressing HO-1, whereas no inhibitory effect was observed in UM-UC-3 lacking HO-1 expression. Moreover, HO-1 protein levels and (GT) _n repeat polymorphism of the HO-1 gene promoter region were examined with the implication that the constitutive expressions of HO-1 protein were associated with a shorter (GT) _n repeat. Our results suggested that (1) FECH siRNA improved the phototoxicity of ALA-PDT, (2) overexpression of HO-1 was associated with shorter (GT) _n repeat of the promoter region, and (3) siRNA-mediated knockdown of HO-1 could suppress the growth of bladder cancer cells overexpressing HO-1.     

Histidine-tagged shiga toxin B subunit binding assay: simple and specific determination of gb3 content in mammalian cells

A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 microg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37 degrees C; and its binding could be visualized by the following applications of HisProbe-HRP (8 microg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1.     

Self-regulated Ni cluster formation on the TiO2(1 1 0) terrace studied using scanning tunneling microscopy

We have studied the growth mode and morphology of Ni clusters on a TiO₂(1 1 0) surface with a wide terrace using scanning tunneling microscopy (STM) at a low coverage (less than 3 atoms nm⁻²). The Ni clusters are formed on the terrace at the low coverage of 0.2 atoms nm⁻². Their average dimensions are constant in three directions up to 1 atoms nm⁻². The Ni clusters have an oval shape with average sizes of 1.8 nm (along [0 0 1]) × 1.4 nm (along (in the [1 1 0] directions). Above the coverage of 1.0 atoms nm⁻², an increase in the cluster height occurs, retaining an almost constant lateral size. It is proposed that the interaction of the Ni cluster and the support surface regulates the Ni cluster size.     

Evidence of Nonelectrochemical Shift Reaction on a CO-Tolerant High-Entropy State Pt-Ru Anode Catalyst for Reliable and Efficient Residential Fuel Cell Systems

A randomly mixed monodispersed nanosized Pt-Ru catalyst, an ultimate catalyst for CO oxidation reaction, was prepared by the rapid quenching method. The mechanism of CO oxidation reaction on the Pt-Ru anode catalyst was elucidated by investigating the relation between the rate of CO oxidation reaction and the current density. The rate of CO oxidation reaction increased with an increase in unoccupied sites kinetically formed by hydrogen oxidation reaction, and the rate was independent of anode potential. Results of extended X-ray absorption fine structure spectroscopy showed the combination of N(Pt-Ru)/(N(Pt-Ru) + N(Pt-Pt)) ? M(Ru)/(M(Pt) + M(Ru)) and N(Ru-Pt)/(N(Ru-Pt) + N(Ru-Ru)) ? M(Pt)/(M(Ru) + M(Pt)), where N(Pt-Ru)(N(Ru-Pt)), N(Pt-Pt)(N(Ru-Ru)), M(Pt), and M(Ru) are the coordination numbers from Pt(Ru) to Ru(Pt) and Pt (Ru) to Pt (Ru) and the molar ratios of Pt and Ru, respectively. This indicates that Pt and Ru were mixed with a completely random distribution. A high-entropy state of dispersion of Pt and Ru could be maintained by rapid quenching from a high temperature. It is concluded that a nonelectrochemical shift reaction on a randomly mixed Pt-Ru catalyst is important to enhance the efficiency of residential fuel cell systems under operation conditions.     

Multiple ligand transfer reaction between [CpRu(L)(AN)2][PF6] (L=AN, CO, P(OMe)3; AN=acetonitrile) and CpFe(CO)L′X (L′=CO, PMe3, PMe2Ph, PMePh2, PPh3, P(OPh)3; X=Cl, Br, I)

Treatment of ruthenium complexes [CpRu(AN)₃][PF₆] (1a) (AN=acetonitrile) with iron complexes CpFe(CO)₂X (2a–2c) (X=Cl, Br, I) and CpFe(CO)L′X (6a–6g) (L′=PMe₃, PMe₂Ph, PMePh₂, PPh₃, P(OPh)₃; X=Cl, Br, I) in refluxing CH₂Cl₂ for 3 h results in a triple ligand transfer reaction from iron to ruthenium to give stable ruthenium complexes CpRu(CO)₂X (3a–3c) (X=Cl, Br, I) and CpRu(CO)L′X (7a–7g) (L′=PMe₃, PMe₂Ph, PMePh₂, PPh₃, P(OPh)₃; X=Br, I), respectively. Similar reaction of [CpRu(L)(AN)₂][PF₆] (1b: L=CO, 1c: P(OMe)₃) causes double ligand transfer to yield complexes 3a–3c and 7a–7h. Halide on iron, CO on iron or ruthenium, and two acetonitrile ligands on ruthenium are essential for the present ligand transfer reaction. The dinuclear ruthenium complex 11a [CpRu(CO)(μ-I)]₂ was isolated from the reaction of 1a with 6a at 0°C. Complex 11a slowly decomposes in CH₂Cl₂ at room temperature to give 3a, and transforms into 7a by the reaction with PMe₃.     

Finite element analysis of density flow using the velocity correction method

A finite element method is proposed for the analysis of density flow which is induced by a difference of density. The method employs the idea that density variation can be pursued by using markers distributed in the flow field. For the numerical integration scheme, the velocity correction method is successfully used, introducing a potential for the correction of velocity. This method is useful because one can use linear interpolation functions for velocity, pressure and potential based on the triangular finite element. The final equations can be formulated using the quasi-explicit finite element method. A flume in a tank with sloping bottom has been analysed by the present method. The computed results show extremely good agreement with the experimental observations.     

Coumarins with an unprecedented tetracyclic skeleton and coumarin dimers from chemically engineered extracts of a marine-derived fungus

The unprecedented tetracyclic coumarin derivatives 1 and 2 and the coumarin dimers 3–5 were isolated from chemically engineered extracts (coumarin dimerization of natural extract) of the marine-derived fungus Eurotium rubrum. The structures of these compounds were established using NMR, MS and IR methods. The absolute configuration of 1 was determined by ECD calculations. The unprecedented tetracyclic coumarin skeleton was generated by domino-Knoevenagel-Diels-Alder reactions. Compounds 1–5 showed tyrosinase inhibitory activity (IC₅₀?=?1.7, 1.2, 4.9, 1.8 and 2.9?μM, respectively). The isolated coumarin derivatives 1–5 were not observed by HPLC analysis in crude extracts of E. rubrum, suggesting that chemically engineered extract generated these new coumarin derivatives with tyrosinase inhibitory activity.     

Aerobic Oxidation of 5‐(Hydroxymethyl)furfural Cyclic Acetal Enables Selective Furan‐2,5‐dicarboxylic Acid Formation with CeO2‐Supported Gold Catalyst

The utilization of 5‐(hydroxymethyl)furfural (HMF) for the large‐scale production of essential chemicals has been largely limited by the formation of solid humin as a byproduct, which prevents the operation of stepwise batch‐type and continuous flow‐type processes. The reaction of HMF with 1,3‐propanediol produces an HMF acetal derivative that exhibits excellent thermal stability. Aerobic oxidation of the HMF acetal with a CeO₂‐supported Au catalyst and Na₂CO₃ in water gives a 90–95 % yield of furan 2,5‐dicarboxylic acid, an increasingly important commodity chemical for the biorenewables industry, from concentrated solutions (10–20 wt %) without humin formation. The six‐membered acetal ring suppresses thermal decomposition and self‐polymerization of HMF in concentrated solutions. Kinetic studies supported by DFT calculations identify two crucial steps in the reaction mechanism, that is, the partial hydrolysis of the acetal into 5‐formyl‐2‐furan carboxylic acid involving OH^? and Lewis acid sites on CeO₂, and subsequent oxidative dehydrogenation of the in situ generated hemiacetal involving Au nanoparticles. These results represent a significant advance over the current state of the art, overcoming an inherent limitation of the oxidation of HMF to an important monomer for biopolymer production.     

A theorem on the first heteroclinic tangency in two-dimensional maps. Orientation-preserving cases

Using the properties of the Jordan curve, the following theorem on the heteroclinic tangency in orientation-preserving two-dimensional maps is proved: LetT :R ² R ² be a one-parameter family ofC ¹ diffeomorphisms andJ=DetDT be such that 0<J1 or 1J<. LetW _u ⁿ be the unstable manifold of a hyperbolicn-cycle andW _s ^m the stable manifold of a hyperbolicm-cycle. Suppose that for< _c ,W _u ⁿ andW _s ^m have no common points, and that for> _c ,W _u ⁿ andW s/m have a transversal heteroclinic point. Then at= _c ,W _u ⁿ andW _s ^m are in the first asymptotic heteroclinic tangency except for the following three cases: (1)n=m; both cycles are without reflection. (2)m=2n; then- andm-cycles are with and without reflection, respectively; (3)n=2m; then- andm-cycles are without and with reflection, respectively.