In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.     

Tribology of silicon-thin-film-coated SiC ceramics and the effects of high energy ion irradiation

The sliding friction coefficients and specific wear of SiC ceramics coated with a silicon thin film (Si/SiC) with and without subsequent Ar⁺ irradiation against a diamond pin were measured with a pin-on-disk tester at room temperature in laboratory air of approximately 50% relative humidity without oil lubrication for 40 h. The friction coefficient of Ar⁺-irradiated Si/SiC was about 0.05 with a normal load of 9.8 N and remained almost unchanged during the 40 h test, while that of SiC increased from 0.04 to 0.12 during the test. The silicon deposition also reduced the specific wear of SiC to less than one tenth of that of the uncoated SiC. Effectively no wear was detected in Si/SiC irradiated to doses of over2×10¹⁶ions cm⁻².     

Effects of phase changes on reflection and their wavelength dependence in optical profilometry

The method as well as an appropriate instrumentation for measuring phase changes of reflected light is described. The phase changes on samples of Au, Al, Ag, and Cr evaporated films are measured for five wavelengths (lambda) from 442 to 633 nm, with respect to the phase change at the glass-air interface, where it should be zero. The measured results for the Au film are in fairly good agreement with values calculated by use of optical constants from a handbook or the complex refractive index measured by an ellipsometer. The phase changes for Al and Ag films are different from calculated values by ~5 degrees or a shift length of 4.4 nm at lambda = 633 nm, while those of the Cr film show large shifts as high as 16 degrees or a shift length of 9.8 nm at lambda = 442 nm.     

Infrequent presence of anti-c-Myc antibodies and absence of c-Myc oncoprotein in sera from lung cancer patients

To clarify the host immune response and explore a new serological marker of lung cancer, we examined serum c-Myc antigens and auto-antibodies against c-Myc in 68 lung cancer patients and 30 healthy volunteers using bacterially synthesized glutathione S-transferase c-Myc fusion proteins and immunoblotting. The detection rate of anti-c-Myc antibodies was 13.2% (9/68) in lung cancer patients and 3.3% (1/30) in healthy volunteers. These anti-c-Myc antibodies were directed toward exon 2 alone (4/68), exon 3 alone (1/68), and both exon 2 and exon 3 (4/68) of c-Myc. Circulating c-Myc antigen was not detected in any individuals with lung cancer and normal controls. Age, sex, performance status, histology, stage, smoking history, and prior treatment of the patients with and without anti-c-Myc antibodies were not significantly different. The low incidence of anti-c-Myc antibodies and c-Myc antigens in peripheral blood suggests that these examinations are not useful in the serological diagnosis of lung cancer.     

Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains

BACKGROUND: Overexpression of the 70 kDa heat shock protein (Hsp70) protects myocytes and neural cells from hypoxic injury. In contrast, Hsp70 induction in the kidney after ischemic or thermal preconditioning does not correlate well with protection from hypoxic injury. Herein, we directly tested if Hsp70 overexpression protects LLC-PK1 porcine tubular epithelial cells from hypoxic or thermal injury. METHODS: LLC-PK1 cells were either cotransfected with an Hsp70 and a luciferase expression vector or singly transfected with the luciferase expression vector. Loss of intracellular luciferase activity was used to assess injury after exposure to hypoxia or hyperthermia and after recovery under normal growth conditions. RESULTS: Overexpression of Hsp70 decreased loss of and improved restoration of intracellular luciferase activity in LLC-PK1 cells exposed to hyperthermia. In contrast, Hsp70 overexpression did not decrease the loss of or improve restoration of luciferase activity in cells exposed to hypoxia. CONCLUSIONS: These results suggest that Hsp70 overexpression is sufficient to protect LLC-PK1 proximal tubular cells from hyperthermia but is not sufficient for protection from hypoxia.     

Effects of defocus and algorithm on optical step height calibration

Defocus effects on step height measurements by interferometric microscopy are estimated using different algorithms to calculate the step height. The interferometric microscope here is a Mirau-type with a 20x objective and a numerical aperture (NA) of 0.4. Although the focus is adjusted within the range of the depth of focus, a defocus corresponding to 4 fringes (1.3 μm) distorts the measured profile into a curve with a radius of curvature of about 24 m. The effect of this distortion on step height determination by a basic ISO step height algorithm is estimated to be 0.11 nm/fringe. An algorithm developed by NIST and a second-order fitted ISO step-height algorithm show good robustness against defocus errors. Because of sample imperfection, a discrepancy of about 1 % exists among step-height values determined by the different algorithms at the optimum fringe contrast position. It has been pointed out theoretically that the NA correction factor varies with the difference of optical path length between the sample and the reference surface. However, in our case, the changes of optical path length do not change the NA correction factor by more than 0.3 %.     

A new class of transparent polymeric materials. III. Miscible blends of poly(N-methylmaleimide-alt-isobutene) with poly(acrylonitrile-co-styrene) and the properties of the blends

The blend miscibility of poly(N-methylmaleimide-alt-isobutene) [poly-(MeMI-IB)] with poly(acrylonitrile-co-styrene) (SAN) was investigated by means of measurement of the glass transition temperature of the blends. Poly(MeMI-IB) was found to be miscible with SAN of a specific range of acrylonitrile (AN) contents in the copolymer to produce transparent moldings. The refractive index changed from 1.58 to 1.53 and the dispersion decreased with increasing the amount of poly(MeMI-IB) in the blends. The stress optical coefficient of poly(MeMI-IB) was found to be reduced by the blending of SAN. The glass transition temperature, flexural modulus, and surface hardness of the blends increased with an increase in the amount of poly(MeMI-IB) in the blend. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 925–929, 1997     

Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Microbial synthesis of copolymers of [R]-3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB), P(3HB-co-4HB), by Alcaligenes eutrophus, Alcaligenes latus, and Comamonas acidovorans from various carbon sources has been studied. The copolyester compositions varied from 0 to 100 mol% 4HB, depending on the microorganism and the combination of carbon substrates supplied. The thermal and physical properties of compositions with 0–100 mol% 4HB were investigated. The copolyesters represented a wide variety of polymeric materials, from hard crystalline plastic to very elastic rubbers, depending on composition. The copolyester films with high 4HB fractions (64–100 mol% 4HB) exhibited the characteristics of a thermoplastic elastomer, and the tensile strength increased from 17 to 104 MPa as the 4HB fraction increased. The enzymatic degradation of P(3HB-co-4HB) films was studied in an aqueous solution of extracellular polyhydroxybutyrate (PHB) depolymerase from Alcaligenes faecalis or lipase from Rhizopus delemer. The erosion rate of P(3HB-co-4HB) films was strongly dependent on the copolymer composition. In addition, environmental degradation of P(3HB-co-4HB) films in sea water was investigated.