Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.     

A note on upwinding and anisotropic balancing dissipation in finite element approximations to convective diffusion problems

In one dimension, Petrov—Galerkin nonsymmetric weighting for the convective diffusion equation can be interpreted as an added dissipation. The addition of an appropriate amount of dissipation can therefore give the same oscillation-free solutions as the ‘unwinding’, Petrov—Galerkin, finite element methods. The ‘balancing dissipation’ is optimally chosen so that excessive dissipation does not occur. A scheme is presented for extending this approach to two-dimensional problems, and numerical examples show that the new method can be used with improved computational efficiency.     

Determination of benzimidazole anthelmintics in livestock foods by HPLC

A simple and rapid liquid chromatographic method was developed for the simultaneous determination of twelve benzimidazole anthelmintics in livestock foods using reversed-phase high-performance liquid chromatography with photodiode array detection (PDA). A sample was homogenized with acetonitrile and n-hexane, and centrifuged. The acetonitrile phase was isolated and evaporated. The residue was dissolved in 0.1 mol/L carbonate buffer solution (pH = 9.1), sonicated, and then subjected to clean-up on a Bond Elut LRC-C18 cartridge. The benzimidazole compounds were separated isocratically on a Capcell Pak C18 UG 120 (5 microns, 150 x 4.6 mm i.d.) column and detected by PDA at 295 and 313 nm. Mixtures of acetonitrile and 0.05 mol/L ammonium acetate in mixing ratios of (20:80) and (40:60) were used as the mobile phase, and the flow-rate was 1.0 mL/min at 40 degrees C. The mean recoveries (n = 3) from 0.1-0.5 microgram/g added samples were 72.6-97.2% with coefficients of variation of 0.3-8.5%. The detection limits were 0.01-0.05 microgram/g.     

Cautionary observations on preparing and interpreting brain images using molecular biology-based staining techniques

Though molecular biology-based visualization techniques such as antibody staining, in situ hybridization, and induction of reporter gene expression have become routine procedures for analyzing the structures of the brain, precautions to prevent misinterpretation have not always been taken when preparing and interpreting images. For example, sigmoidal development of the chemical processes in staining might exaggerate the specificity of a label. Or, adjustment of exposure for bright fluorescent signals might result in overlooking weak signals. Furthermore, documentation of a staining pattern is affected easily by recognized organized features in the image while other parts interpreted as "disorganized" may be ignored or discounted. Also, a higher intensity of a label per cell can often be confused with a higher percentage of labeled cells among a population. The quality, and hence interpretability, of the three-dimensional reconstruction with confocal microscopy can be affected by the attenuation of fluorescence during the scan, the refraction between the immersion and mounting media, and the choice of the reconstruction algorithm. Additionally, visualization of neurons with the induced expression of reporter genes can suffer because of the low specificity and low ubiquity of the expression drivers. The morphology and even the number of labeled cells can differ considerably depending on the reporters and antibodies used for detection. These aspects might affect the reliability of the experiments that involves induced expression of effector genes to perturb cellular functions. Examples of these potential pitfalls are discussed here using staining of Drosophila brain.     

10-GHz 8.7-ps Pulse Generation From a Single-Mode Gain-Switched AlGaAs VCSEL at 850 nm

We have successfully generated a 10-GHz 8.7-ps pulse train at 850 nm by using a gain switching technique with a high-speed AlGaAs-based vertical-cavity surface-emitting laser (VCSEL). The VCSEL, which operates in a single transverse mode by employing an aluminum oxide aperture, has a modulation bandwidth of 6.3 GHz and a relaxation oscillation frequency at around 11.7 GHz. An error-free 10-Gb/s return-to-zero data modulation was demonstrated by using the generated 10-GHz pulse train.     

Ultraviolet bonding of perfluoropolyethers to carbon surfaces investigated using quantum chemical methods

For improving the tribological performance of hard disk drives, nanometer-thick perfluoropolyether (PFPE) lubricant films are generally treated with ultraviolet (UV) irradiation to bond them to the carbon overcoats of the disks. By modeling UV irradiation as an electron emission and attachment process, we investigate the UV bonding of nonfunctional PFPE Z and functional PFPE Zdol to hydrogenated and nitrogenated carbon surfaces with quantum chemical methods. Our calculation results show that, upon electron attachment, Z dissociates at its main chain to two fragments terminated by CF₂CF₂ and CF₂O groups, whereas Zdol dissociates to a hydrogen fluoride and a fragment. The perfluoromethoxy oxygen in one of the Z fragments and the carbon radical and the hydrogen-truncated end group in the Zdol fragment interact strongly with sp² and oxidized sites on carbon surfaces. Imine moieties on the CNx surface also contribute considerably to the UV bonding of Zdol.     

Determination of bisphenol A in canned vegetables and fruit by high performance liquid chromatography

A high performance liquid chromatograph y (HPLC) method was developed for the determination of bisphenol A (BPA) that had migrated into canned fruit and vegetables. BPA was extracted with acetonitrile from the solid portion of canned food, and with an OASIS HLB cartridge from the aqueous portion, respectively. Both extracts were cleaned up on a Florisil cartridge. The HPLC separation was carried out on a Wakosil II 3C18 RS column (4.6 × 150mm) with acetonitrile-water (40:60, v/v) as a mobile phase with a flow rate of 0.8 ml/min. BPA was detectable by UV detector at 228 nm and determined with the similarity of chromatographic peak spectrum by multiwavelength detector (similarity index was 0.99 or above). The quantification limits were 10 ng/g for the solid portion and 5 ng/ml for the aqueous portion, respectively. BPA was mainly detected in the solid portion of canned food and found at the maximum level of 11 μg per can. To verify migration into the solid portion of canned food, a partitioning experiment was carried out.     

Study on environmental effect on fatigue and creep-fatigue strength of 316FR stainless steel in sodium at elevated temperature

Low-carbon 316 stainless steel with medium-nitrogen (316FR) is considered as the principal structural material for next generation fast breeder reactor (FBR) plants in Japan. The material strength standard and the creep-fatigue life evaluation method for 316FR have been developed. However, they are based on the results of material tests in air, while actual structural material will be used mainly in liquid sodium environment in the plants. In order to clarify the environmental effect, cyclic bending tests were carried out with and without hold time in sodium. Tested materials were 316FR and conventional 304 and 316 stainless steels. Weld metal of 316FR was also tested. As a result, it was found that fatigue and creep-fatigue lives of 316FR in sodium were larger than those in air and no explicit consideration of the environmental effect is necessary in design. It was also found that the life evaluation method based on the ductility exhaustion concept is applicable to creep-fatigue life assessment in sodium.     

40 Gbit/s return-to-zero transmission over 500 km of standard fibreusing chirped fibre Bragg gratings with small group delay ripples

A 40 Gbit/s return-to-zero transmission has been successfully achieved over 500 km using standard fibres (non-dispersion shifted fibre, singlemode fibre) and chirped fibre Bragg gratings (CFBG). CFBGs were fabricated with small group delay ripples, and error free transmission (BER <1×10^-12) was achieved using these CFBGs for dispersion compensation