Pharmacologic evidence for involvement of phospholipase C in pemphigus IgG-induced inositol 1,4,5-trisphosphate generation, intracellular calcium increase, and plasminogen activator secretion in DJM-1 cells, a squamous cell carcinoma line

The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca++]i) and inositol 1,4,5-trisphosphate (IP3) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca++]i, IP3, PA secretion, and cell-cell detachment in DJM-1 cells. [Ca+2]i and IP3 contents were determined with or without 30-min pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxymethylester and a specific IP3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell-cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca++]i and IP3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 microM), but not with U73343 (10 microM), dramatically reduced the pemphigus IgG-induced increases in [Ca++]i, IP3, and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 caused no effects on cell viability and IgG binding to the cell surface. These results suggest that phospholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.     

A localized surface plasmon resonance based immunosensor for the detection of casein in milk

In this research, a localized surface plasmon resonance (LSPR) immunosensor based on gold-capped nanoparticle substrate for detecting casein, one of the most potent allergens in milk, was developed. The fabrication of the gold-capped nanoparticle substrate involved a surface-modified silica nanoparticle layer (core) on the slide glass substrate between bottom and top gold layers (shell). The absorbance peak of the gold-capped nanoparticle substrate was observed at ～520 nm. In addition, the atomic force microscopy (AFM) images demonstrated that the nanoparticles formed a monolayer on the slide glass. After immobilizing anti-casein antibody on the surface, our device, casein immunosensor, could be applied easily for the detection of casein in the raw milk sample without a difficult pretreatment. Under the optimum conditions, the detection limit of the casein immunosensor was determined as 10 ng/mL. Our device brings several advantages to the existing LSPR-based biosensors with its easy fabrication, simple handling, low-cost, and high sensitivity.     

杆状支护系统的耦合模型及应用

锚杆/锚索等杆状支护系统在土木工程界的应用很广。但到目前为止，杆状支护物与被支护体之间的耦合机理却尚未十分清晰，有关支护效果的可靠合理的评价方法也并未成熟。基于Shear-Lag理论，提出一种改良的计算分析方法来描述杆状支护材料、交界面和被支护体之间的耦合作用机理。分析锚杆拉拔试验中由耦合到破坏的整个过程，提出一个计算交界面其实粘结强度的新方法。普通拉拔试验中，交界面的平均剪切强度低于其真实强度。二者的差别受支护杆的锚固长度和试件材料的物理力学性质的影响。若忽略试件材料的变形模量，可能导致试验的计算值与其实强度之间出现很大的偏差。分析不同变形模量试件体的拉拔试验过程，对于软岩支护中金属体的室内拉拔试验，建议试件材料的变形模量和相应的锚固长度。圆形隧道周边锚杆的受力状态分析表明，锚杆受力后存在拉拔区、中性点和锚固区。中性点的位置不但和隧道的几何尺寸有关，还与岩石的物理力学性质有关．计算结果和实测结果进行了对比，二者较为接近。     

Label-free detection of melittin binding to a membrane using electrochemical-localized surface plasmon resonance

Localized surface plasmon resonance (LSPR) and electrochemistry measurements connecting to core-shell structure nanoparticle are successfully exploited in a simultaneous detectable scheme. In this work, the surface plasmon band characterizations of this nanostructure type are initially examined by controlling the core size of the silica nanoparticle and shell thickness of the deposited gold. These results clearly show that when the shell thickness is increased, keeping the core size constant, the peak wavelength of the LSPR spectra is shifted to a shorter wavelength and the maximum of peak intensity is achieved at a particular shell thickness. On the basis of this structure, we present a membrane-based nanosensor for optically detecting the binding of peptide toxin melittin to hybrid bilayer membrane (HBM) and electrochemically assessing its membrane-disturbing properties as a function of concentrations. It will open up the way to detect functionally similar protein toxins and other membrane-targeting peptides with the intension of integrating this chip into a microfluid and expanding it into multiarray format.     

Hydrothermal synthesis of LiFePO<Subscript>4</Subscript> as a cathode material for lithium batteries

LiFePO₄ (space group: Pnma) was prepared by hydrothermal method at 170 °C. LiFePO₄ was prepared from precursor solutions consisting of FeSO₄ · 7H₂O, (NH₄)₂HPO₄, and three kinds of Li sources. LiCl, Li(CH₃COO), and LiOH · H₂O were used as Li sources. The pH of the precursor solution varied depending on Li source. The particle size, particle shape, and crystal texture of the obtained LiFePO₄ changed depending on pH. The electrochemical properties of the prepared LiFePO₄ were characterized as a cathode material for lithium batteries in an organic electrolyte at room temperature. The LiFePO₄ particle prepared from the precursor solution with Li(CH₃COO) was flake-like crystal (particle size: 1–2 μm) and had a preferred crystal orientation with a (020) texture. This LiFePO₄ exhibited a discharge capacity of 147 mA h g⁻¹, which was 85% of the theoretical capacity 170 mA h g⁻¹.     

Influence of gas adsorption on optical transition energies of single-walled carbon nanotubes

The photoluminescence (PL) spectra of suspended single-walled carbon nanotubes (SWNTs) were measured in an ethanol gas atmosphere. When the gas pressure was decreased, the PL peaks were initially blue-shifted to a small extent before a rapid blue-shift took place at a transition pressure that depended on the temperature and diameter of the SWNT being measured. This pressure dependence is due to the adsorption of ethanol molecules on the SWNT surface. The optical transition energies measured below the transition pressure are intrinsic to the SWNT.     

Characteristics of Strength for Hydraulic Fracturing of Buffer Material

The fundamental characteristics of strength for the hydraulic fracturing of highly compacted bentonite were studied. Firstly, the constant pressurize rate tests were carried out for the material having various specifications. Secondly, the cyclic pressurize test was carried out to examine the self-sealing function as a buffer material. Thirdly, the constant pressure test was carried out to observe the change in strength during seepage. The observed phenomena were analytically examined. As a result, it was found that the strength for hydraulic fracturing of the buffer material increased with the increase of initial dry density, decrease of sand-mixture ratio and decrease of water content. The swelling pressure of the buffer material worked as a constraint stress for the strength for hydraulic fracturing. The fracture made by hydraulic fracturing was fixed through the supply of water. However a long period of low-pressure supply was needed to recover the strength at the failed parts. While the tensile failure was dominant, the specimen having a low dry density might be failed initially by the shear failure. When the water content became large during seepage, the strength for hydraulic fracturing reduced.     

PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.