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.     

Difference between human and guinea pig Hageman factors in activation by bacterial proteinases: cleavage site shift due to local amino acid substitutions may determine the activation efficiency of serine proteinase zymogens

Human and guinea pig Hageman factors have been subjected to the action of pseudomonal elastase and serratial E15 proteinase. The pseudomonal elastase cleaved 22-24% of the human molecule at Arg353-Val354, and the remainder at Gly357-Leu358 resulting in the generation of about 20% of potential activity as activated Hageman factor, compared with trypsin activation, while it hydrolyzed Arg340-Ile341 bond in guinea pig molecule and generated about 75% of activity as activated Hageman factor. The serratial proteinase did not hydrolyze the essential cleavage site (Arg353-Val354) of the human zymogen but Gly356-Gly357 (30%) and Gly357-Leu358 (70%) bonds. Both products showed no activity. The guinea pig zymogen, in contrast, was cleaved mostly at Arg340-Ile341 (70%) and less abundantly at Gly344-Leu345 (30%), generating about 85% of the whole potential activity as activated Hageman factor. From the high correspondence between the proportions of activation and of hydrolysis at the essential cleavage site in activation, it was concluded that hydrolysis of the bonds different from the essential bond did not cause activation, even when the spatial separation was only 3 or 4 residues. Considering the amino acid differences between human and guinea pig Hageman factors, -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val-Ala360- and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val-Ala347-, respectively, it was realized that even the minor amino acid substitutions caused the cleavage site shift which resulted in significant differences in activation efficiency of the proteinase zymogens.     

Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction

Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose scrub typhus and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Serotype-specific primers were used in the second PCR amplification. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, were identified by nested PCR. In addition, the serotype identified by PCR with DNA from blood clots was the same as that of the strain isolated from five patients with scrub typhus. These findings indicate that this method is useful for diagnosis and identification of the rickettsial serotype in infected patients.     

a-Si alloy three-stacked solar cells with high stabilized-efficiency

a-Si alloy three-stacked solar cells have been studied to improve the stabilized efficiency of a-Si: H based solar cells. Based on the analysis by the individual characterization method of the component cells in stacked type cells, the a-Si :H middle cell was replaced with an a-SiGe :H cell. Furthermore, the optical confinement technology was improved to obtain a high-output current with thin i-layer thickness in the a-SiGe :H bottom cell. By this device design, the initial conversion efficiency was improved up to 12.4% and more than a 10% stabilized efficiency was obtained in a-SiC :H/a-SiGe :H/a-SiGe :H three-stacked cells. These cell characteristics were confirmed by measurements at the JQA Organization (the former JMI Institute).     

Decreasing the number of test specimens by utilizing both fracture stress and fracture location for the estimation of Weibull parameter

In a bending load test for brittle materials, such as ceramics for spacecraft and aircraft, decreasing the number of test specimens required is a crucial problem. This paper discusses the effectiveness of using the information of both fracture stress and fracture location to decrease the number of specimens required to obtain the same precision as the Weibull estimator. The following results were obtained: It was found that by adding the fracture location information, the precision of the Weibull parameter estimation under the optimal design became 1.5–1.9 times better compared with the case of using only the fracture stresses. This means the number of samples necessary to attain the same precision becomes 1/1.5–1/1.9. Tables and figures which give information on the number of samples necessary to attain the required precision are given.     

Effects of Surface Concentration on Drying Behavior of Carbohydrate Solutions

As the surface properties of the drying materials are very important not only for the drying rate but also for the quality change during drying, the effects of surface concentration on the drying behavior of liquid foods (sugar solutions) were investigated by isothermal drying experiments and by numerical calculation experiments. The isothermal drying experiments with gelled sugar solution systems (sucrose and maltodextrin) were carried out at various relative humidity (RH) values (RH = 0 to 84%). Separate experiments were carried out for determination of the desorption isotherms.

The isothermal drying curves of sugar solutions at RH = 0 to 51% were very similar. Numerical simulations also showed that the drying curves of these sugars at the surface concentration = 0 and 0.1 are almost the same, although the concentration distributions are different.

When a small amount of gelatin was added to sugar solutions, the drying rate decreased remarkably as the gelatin might form a thin film (skin) near the surface, and consequently the retention of ethanol increased.     

Influence of protein and lipid concentration of the test lubricant on the wear of ultra high molecular weight polyethylene

To gain a better understanding of the ultra-high molecular weight polyethylene (UHMWPE) wear mechanism in the physiological environment, the effects of protein and lipid constituents of synovial fluid on the specific wear rate of UHMWPE were examined experimentally. The multidirectional sliding pin-on-plate wear tester was employed to simulate the simplified sliding condition of hip joint prostheses. Bovine serum γ-globulin and synthetic l--DPPC were used as model protein and lipid constituents of synovia, respectively. Results of the wear test indicated that the UHMWPE wear rate primarily depended on the protein concentration of the test lubricant. Lipids acted as a boundary lubricant and reduced polyethylene wear in the low protein lubricants. However, the polyethylene wear rate increased with increasing lipid concentrations if the protein concentration was within the physiological level. Increased interactions between protein and lipid molecules and lipid diffusion to polyethylene surface might be responsible for the increased wear.     

Thermal stability of reinforced Nb3Sn composite superconductor under cryocooled conditions

Several types of reinforced Nb₃Sn wires have been developed to prevent reduction of superconducting properties by applying a strong electromagnetic force. To fabricate a cryocooled magnet using those reinforced wires, we experimentally measured the minimum quench energy (MQE) under cryocooled conditions of some reinforced Nb₃Sn wires. As a result, it became clear that thermal stability expressed as MQE was controlled by the temperature margin between the temperature of the operating condition and the transition temperature from superconductivity to normal. Using the FEM analysis, it was realized that the cause of the decline in thermal stability for the reinforced wires was the low thermal conductivity of the reinforced materials.