Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation

Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.     

Stress intensity factors for cracks initiated from a center-holed plate with unsymmetrical lengths under tension

The values of stress intensity factors of unsymmetrical cracks initiated from hole edges under tension were calculated in a center-holed plate, that is a plate having a hole in the center of a front face. The correction factor for stress intensity in the case of the holed plate with cracks was compared with that of a center-cracked plate whose crack length was equal to the total of the hole diameter and the lengths of the cracks in the case of the holed plate. In the present study, to understand the variation of the stress intensity factor, counterplots of the stress intensity factor K_I and the correction factor F_I are employed. Also, the variation of ratio R_F of correction factors between the holed plate with crack and the center-cracked plate were investigated. Where non-dimensional hole diameter a/W is smaller than 0.2, the ratio R_F is less than 1.1 after crack growth length c/W reaches 0.1. Where, 2a, 2c and 2W are hole diameter, the growth length of the crack from hole edge, and plate width, respectively. Therefore, the stress intensity factor for cracks initiated from a holed plate can be approximated within 10% error to that of a center-cracked plate where a/W is smaller than 0.2, though the unsymmetrical cracks are initiated from the edges. Where a/W is larger than 0.3, R_F becomes larger than 1.1 in the wide range of c/W.     

High-dose rate intracavitary irradiation for carcinoma of the uterine cervix. The adverse effect of treatment prolongation

AIM: To investigate the adverse effect of treatment prolongation on the local control and survival of the cervical carcinoma of the uterus. PATIENTS AND METHOD: Two hundred and sixteen patients with stage IIB and III cervical carcinoma treated with a combination of external radiation and high-dose rate (HDR) intracavitary irradiation between 1978 and 1989 were retrospectively studied. A multivariate analysis was used to determine the effect of treatment time on local control and survival. RESULTS: Overall treatment time was the most highly significant factors for local control in the multivariate analysis (p = 0.0005). The 5-year cumulative relapse rates were significantly different with the treatment times 35 to 42 days: 9% versus 43 to 49 days: 19% versus 50 to 62 days: 42% (p = 0.001). The second most significant parameter was stage classification (p = 0.02). Concerning relapse-free survival, stage classification (p = 0.0001), overall treatment time (p = 0.0035) and hemoglobin level (p = 0.0174) were the 3 most important prognostic factors, although there was no relationship between treatment time and late complications. CONCLUSION: These results suggest that prolongation of treatment time is associated with decreased local control and survival in patients treated with external radiation and HDR intracavitary irradiation.     

Calculation method for strand current distributions in armature winding of a turbine generator

We have developed a calculation method for the strand current distributions in the turbine generator armature windings. These distributions are calculated by using a circuit equation and a 3D finite element method. In the circuit equation, the strand current is solved by introducing a circulating current at each strand of the armature windings. The induced voltage in the circuit equation is produced by leakage flux of the coil end region. The leakage flux is obtained by the 3D finite element method. The calculated strand current distributions had good agreement with the measurements of 360°, 450°, and 540° transposed model coils. © 2003 Wiley Periodicals, Inc. Electr Eng Jpn, 143(2): 50–58, 2003; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/eej.10131     

Role of endothelin-1 in astrocyte responses after acute brain damage

The mucosal injury of active ulcerative colitis (UC) could involve enhanced migration and activation of neutrophils (PMNs). Because, in vitro, PMNs from patients with UC appear normal and are not therefore a likely cause for the enhancements, we hypothesized an abnormal colonic milieu. We previously found that factors in the UC colonic milieu markedly increase production of reactive oxygen species (ROS) by control PMNs. We now hypothesize that these factors also regulate PMN surface integrins, that regulation of UC PMNs is different than that of control PMNs, and that the integrin regulation is consistent with the ROS regulation. Using rectal dialysis, we sampled the colonic milieu in patients with active UC, in patients with inactive UC, and in control subjects. We monitored a key PMN adhesion molecule, CD11b. When control PMNs were tested, active UC rectal dialysate was almost as effective (+115%) as N-formyl-methionyl-leucyl-phenylalanine (+132%) in up-regulating CD11b. When inactive UC PMNs were tested, baseline CD11b was 50% higher than that for control PMNs. In contrast, rectal dialysates failed to up-regulate CD11b of inactive UC PMNs and in fact down-regulated CD11b. Preincubating control PMNs with UC rectal dialysates converted their CD11b response to PMN activators from up-regulation to down-regulation, mimicking inactive UC PMNs. Changes in intracellular calcium levels paralleled these changes in CD11b. Rectal dialysate-induced changes in both CD11b and calcium paralleled our previous findings on rectal dialysate-induced changes in ROS production. Thus the net overall effect of factors in the colonic milieu is a consistent and predictable regulation of PMN function--proinflammatory in UC, anti-inflammatory in control subjects. These factors may be a critical part of the pathophysiology of UC.     

Automated homogeneous liposome-based assay system for total complement activity

We developed an automated homogeneous immunoassay, based on immune lysis of dinitrophenyl (DNP)-labeled liposomes, for measuring total complement activity. Liposome lysis caused by complement activity was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. Complement activity in human sera was quantified by comparison with a calibration curve. For ease of application to fully automated routine clinical analyzers, we adopted a two-reagent system, one reagent containing a homogeneous population of small DNP-labeled liposomes and one containing antibody/substrate. This system required calibration only once a week. Within-run and between-run CVs were 0.4-1.3% (n = 10) and 1.8-4.7% (n = 10), respectively. Serum results were linear upon dilution (with saline) over a twofold range. Bilirubin, hemoglobin, Intrafat, and serum proteins such as rheumatoid factor, M protein, IgG, and IgA did not affect the assay results. The results (y) correlated well with those from a hemolytic complement activity test (x): y = 1.05x - 1.14, r = 0.92, on 66 samples in the range < 10- > 50 kU/L. This method should therefore be of great use for the determination of complement activity.     

The subunit structure of myosin from skeletal muscle of the early chick embryo

The subunit composition and immunological properties of two types of myosins, the 3S and 6S myosin components, from skeletal muscle of early chick embryos were studied by SDS-acrylamide gel electrophoresis and immunodiffusion techniques. It was shown that the 6S myosin in the early embryonic stage was composed of two heavy chains and three kinds of light chains, as is well-known in the complete myosin molecule, having the same molecular weights and the same antigenicities as corresponding subunits of the myosin from adult chicken skeletal muscle. The heavy chain of 6S myosin was also reactive with the antibody against the heavy chain of cardiac myosin. The embryonic 3S myosin was shown to be composed of a heavy chain which was roughly the same in molecular weight but not the same in antigenicity as those of adult or embryonic 6S myosin. No light chains were detected either electrophoretically or immunologically in the 3S myosin component.     

Application of ADINA to nonlinear analysis of reinforced concrete shear walls with openings

This paper is concerned with application of ADINA to elasto-plastic analysis of the shear walls with openings. The authors analyzed the types of structures. One is the shear wall with many openings (the model of a secondary shield wall in nuclear power plant), on which scale model experiments were made. The other is the shear wall with openings in concrete rigid frame (the model of a shear wall in a building), on which parametric study was made.

In both cases, concrete is modeled using 8 nodes isoparametric 2 dimensional plane stress elements, reinforcing steels are modeled as truss elements. Concrete and elasto-plastic models are adopted for non-linear material model of concrete and reinforcing steel, respectively. The total numbers of nodes are 248–308, and that of 2D elements are 66–80.

Both analytical results are satisfactory from the view point of structural design. Close agreement to experimental results in the cracking load, crack extension, elasto-plastic stiffness and total strength was verified.     

Chromium oxides as cathodes for lithium cells

The performances of organic electrolyte lithium cells with the chromium oxides, Cr₂O₃, CrO₂, Cr₅O₁₂, Cr₂O₅, Cr₆O₁₅, and Cr₃O₈, as cathodes, have been examined. The discharge behaviors of the couples Li/Cr₂O₅, Li/Cr₆O₁₅, and Li/Cr₃O₈, gave satisfactory results, especially in terms of energy density. The highest calculated energy density, based on the weights of the chromium oxides and to a 2.0 V cutoff at 0.5 mA/cm², was 1210 W h/kg for the Li/Cr₃O₈ couple. Further, the performance of a coin-type cell with a Cr₃O₈ cathode was demonstrated, and it was found that the cell exhibited good rechargeability.