Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases

cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.     

Control of polymerization processes of 10,12-pentacosadiynoic acid LB films

Studies have been carried out on KrF excimer laser light (EX), X-ray or electron beam (EB) induced polymerization of 10,12-pentacosadiynoic acid (PDA) Langmuir-Blodgett (LB) films in relation to molecular density or molecular arrangement of the films using X-ray diffraction analysis, infrared (IR) spectroscopy and Raman spectroscopy. The molecular arrangement or density of the PDA LB films was controlled by subphase conditions when the films were built up, such as pH, temperature of a subphase or salt concentration in the subphase. Polymerization sensitivity of the PDA LB film was affected by the arrangement or molecular density. On low density (A type) films the polymerization occurred by irradiation with EX, X-ray or EB, but on high density (B type) films the polymerization occurred only when the irradiation was carried out by high energy beams such as X-ray or EB. Decomposition of polymerized films was observed further by excessive irradiation of EX or X-ray, but not on the B type films. It was revealed by X-ray diffraction analysis that in the A type film, the PDA molecules bent to a larger extent than those in the B type film and the polymerization proceeded topochemically, that is, the thickness decreased little after EB irradiation in a helium atmosphere. On the other hand, in the B type film, the thickness decreased by about 10% as a result of EB irradiation. By IR reflection-absorption (RA) and Raman measurements, it was confirmed that conjugated diacetylenic bonds disappeared and conjugated double and new conjugated triple bonds appeared after high energy beam irradiations. These results support the supposition that 1,4-polymerization, i.e. polydiacetylene type polymerization, occurs easily in the A type film and 1,2- or 3,4-polymerization, i.e. polyacetylene type polymerization, occurs in the B type film, and that the polymerized A type film was decomposed at the polydiacetylenic bond when the irradiation continued further. It was also shown that the polyacetylene type polymer was obtained only when the B type film was irradiated with the high energy beam.     

High performance 35 nm gate length CMOS with NO oxynitride gate dielectric and Ni salicide

The 35 nm gate length CMOS devices with oxynitride gate dielectric and Ni salicide have been fabricated to study the feasibility of higher performance operation. Nitrogen concentration in gate oxynitride was optimized to reduce gate current I/sub g/ and to prevent boron penetration in the pFET. The thermal budget in the middle of the line (MOL) process was reduced enough to realize shallower junction depth in the S/D extension regions and to suppress gate poly-Si depletion. Finally, the current drives of 676 /spl mu/A//spl mu/m in nFET and 272 /spl mu/A//spl mu/m in pFET at V/sub dd/=0.85 V (at I/sub off/=100 nA//spl mu/m) were achieved and they are the best values for 35 nm gate length CMOS reported to date.     

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC₅₀ at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.     

Preparation and properties of graft and block copolymers of poly(p-phenylene terephthalamide) with polybutadiene

Graft copolymers of polybutadiene (PBD) onto poly(p-phenylene terephthalamide) (PPTA) were prepared by the nucleophilic substitution of N-metalated PPTA with telechelic PBD having bromide end groups. Block copolymers were synthesized by the condensation reaction of telechelic PBD having acid chloride end groups with amino-group-terminated PPTA. The structure of these copolymers was identified by IR spectra. Graft and block copolymers contained PBD segments up to 85 wt % and 45 wt %, respectively. Thermomechanical analyses (TMA) proved the existence of distinctive primary absorption peak corresponding with T_g of PBD for both graft and block copolymers. The T_g's of both types of the copolymers were further ascertained by the DSC curves. TMA curves suggested that the microphase separation occurred between PPTA and PBD. The incorporation of PPTA segments into PBD increased the decomposition temperature compared with the blend polymer composed of PPTA and PBD with the same composition.     

Structural significance of the alkyl substituent at the C-4 of (+)-trans-verbenyl acetate in sex pheromonal activity of the American cockroach

The significance of the alkyl group at the C-4 of (+)-trans-verbenyl acetate, which is the sex pheromone mimic of the American cockroach, was investigated. Seven alcohols possessing an ethyl, propyl, or dimethyl group at this position of the 6,6-dimethylbicyclo[3.1.1]heptane skeleton were synthesized and evaluated by behavioral assay. All of the alcohols were inactive, while three of four acetates of the 2-alcohols induced sexual behavior in male cockroaches at the 0.02 or 0.5 mg dosage level, either of which is many orders of magnitude higher than the threshold level of the natural sex pheromones (10^–8 mg). Among the acetates, the compounds with a methyl group or an -oriented ethyl group at C-4 showed the highest activity. The results are discussed in terms of spatial requirements of the molecules for interactions with the receptor.Studies on the sex pheromone mimic of the American cockroach, (+)-trans-verbenyl acetate. Part VIII. For Part VII, seeComp. Biochem. Physiol.,70A: 229–234 (1981).     

Preparation of poly(p-phenylene terephthalamide)/poly(vinyl chloride) molecular composite and its thermal and mechanical properties

Poly(p-phenylene terephthalamide) (PPTA) was blended with poly(vinyl chloride) (PVC) by solution-blending method. PPTA was metalated for dissolving in dimethyl sulfoxide. Dimethyl sulfoxide was used as a common solvent. In PPTA/PVC composite, PPTA accelerated the thermal degradation of PVC. PPTA molecules are aggregated as microfibrillar form in PVC matrix. Such microfibrils are dispersed homogeneously in PVC matrix, according to polarizing microscopic observation. The average diameter of the microfibrils becomes smaller in the composite with lower content of PPTA. In the surface region of PPTA microfibrils the intermolecular hydrogen bonds between C? Cl of PVC and N? H of PPTA are formed. Young's modulus and the yield stress at room temperature were higher in the composites than those in PVC. The modulus of the composites was higher, especially at the high temperatures above their glass transition temperatures, than that in PVC. The temperature dependence of modulus can be calculated by using the mechanical model equivalent to the quasi-3-dimensional microfibrillar model which will be approximately applied to the composite structure. It becomes apparent that the modulus of the PPTA microfibrils evaluated by using the mechanical model is higher in the higher molecular weight PPTA.     

Note: detection of micro-cracks in the interlayer dielectric film by the stress based light scattering method

A phenomenon was discovered wherein light scattering strength from cracks increases when tensile stress is applied to micro-cracks produced in the interlayer dielectric film by chemical mechanical polishing treatment. It is likely that the change in light scattering intensity occurs because a region of high stress concentration (region with high variation in index of refraction) is produced near the crack tip due to stress, thus forming a type of scatterer. With this method, it is possible to detect only scatterers which respond to stress, and thus, it is possible to classify and separately detect cracks and particles.     

Two-stage nuclear demagnetization refrigerator

Two-stage nuclear demagnetization has been performed using PrCu₆ and Cu as coolants. The Cu nuclear stage reached temperatures as low as 10 K with a rate of temperature rise of less than a few K/h. The corresponding conduction electron temperature is estimated to be less than 50 K. A nuclear orientation thermometer of Al ⁵⁴Mn has been successfully used to measure temperatures down to 10 K.Supported by the Japan Society for the Promotion of Science.     

Solving a Nonlinear Problem in Magneto-Rheological Fluids Using the Immersed Interface Method

Many applications lead to a nonlinear elliptic interface problem in which the discontinuous coefficient depends on the solution and the material properties. A finite difference method based on Cartesian grids and the maximum principle preserving immersed interface method is proposed for the nonlinear elliptic interface problems discussed in this paper. Numerical experiments against the exact solutions reveal that our method is nearly second order accurate in the infinity norm. The method is applied to study the magneto-rheological field-responsive fluids that contain iron particles. Numerical experiments are performed against the results from the literature.