No abstract

Although enhancement of ultrasound-induced cell killing by photodynamic reagents has been shown, the sonochemical mechanism in detail is still not clear. Here, comparison between sonodynamic effect and photodynamic effect with photosensitizers at a concentration of 10 microM on free radical formation and cell killing was made. When electron paramagnetic-resonance spectroscopy (EPR) was used to detect 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) after photo-irradiation or sonication with 2,2,6,6-tetramethyl-4-piperidone (TMPD), the order of TAN formation in the photo-irradiated samples was as follows: rhodamine 6G (R6) > sulforhodamine B (SR) > hematoporphyrin (Hp) > rhodamine 123 (R123) > rose bengal (RB)>erythrosine B (Er) = 0; although there was time-dependent TAN formation when the samples were sonicated, no significant difference among these agents were observed. All these agents suppressed ultrasound-induced OH radical formation detected by EPR-spin trapping. Sensitizer-derived free radicals were markedly observed in SR, RB and Er, while trace level of radicals derived from R6 and R123 were observed. Enhancement of ultrasound-induced decrease of survival in human lymphoma U937 cells was observed at 1.5 W/cm(2) (less than inertial cavitation threshold) for R6, R123, SR and Er, and at 2.3 W/cm(2) for R6, R123, Er, RB and SR. On the other hand, photo-induced decrease of survival was observed for R6, Hp and RB at the same concentration (10 microM). These comparative results suggest that (1) (1)O(2) is not involved in the enhancement of ultrasound-induced loss of cell survival, (2) OH radicals and sensitizer-derived free radicals do not take part in the enhancement, and (3) the mechanism is mainly due to certain mechanical stress such as augmentation of physical disruption of cellular membrane by sensitizers in the close vicinity of cells and/or cavitation bubbles.

No abstract

The effects of acoustic cavitation on in vitro transfection by ultrasound were investigated. HeLa cells were exposed to 1.0 MHz continuous ultrasound in culture media containing the luciferase gene. Transfection efficiency was elevated when an echo contrast agent, Levovist was added or air was dissolved in the medium. When cells were sonicated in medium saturated with Ar, N2 or N2O which have different gamma values (Cp/Cv), or were saturated with He, Ar or Ne with different thermal conductivities, the effectiveness for the dissolved gases in the ultrasound mediated transfection was Ar > N2 > N2O or Ar > Ne > He, respectively. When free radical formation in water by ultrasound was monitored as a measure of inertial cavitation, it was similarly affected by dissolved gases. These results indicate that the efficiency of ultrasound mediated transfection was significantly affected either by occurrence of or by modification of inertial cavitation due to various gases.

Immunoglobulins to which biotin was covalently linked were found in human sera for the first time in 1993.1) The prevalence of biotin-binding immunoglobulin was higher in patients with atopic dermatitis, other dermatitis, allergic disorders, and autoimmune disease. [1][2][3] Recently, biotin-binding IgG was detected in a multiwell microplate format for the first time. 4) We developed a quantitative assay for biotinbinding IgG without any purification of samples using F(ab') 2 anti-human IgG-coated multiwell microplates. 5) We showed that the ratio of biotin molecules to protein molecules (biotin-protein ratio) in biotinylated IgG in human sera affected the detection limit in this assay.5) The slope of doseresponse curve of the assay decreased as the biotin-protein ratio of standards decreased and the observed values of biotinylated IgG in human sera depended on the biotin-protein ratio of standards. Thus it was very important to measure the biotin-protein ratio of biotinylatd immunoglobulins which were used as standards in the assay.In this study, we investigated a synthesis method for biotinylated human immunoglobulins, how to determine the biotin-protein ratio of the biotinylated proteins, and their stability. MATERIALS AND METHODS MaterialsThe following materials were obtained from the sources as indicated: flat-bottomed multiwell microplates (Immulon I) from Dynatech Laboratories, Inc., U.S.A.; N-hydroxysuccinimidobiotin (NHS-biotin) and BCA Protein Assay Reagent kit from Pierce Chemical Co., Rockford, U.S.A.; biotin, horseradish peroxidase (HRP)-labeled streptavidin, dimethyl sulfoxide (DMSO), human IgG, human IgA, human IgM, and BSA from Sigma Chemical Co., St. Louis, U.S.A.; ELISA Color Reagent kit (Type O) from Sumitomo Bakelite Co., Ltd., Tokyo, Japan; Biotin Assay Medium ® from Nissui Pharmaceutical Co., Ltd., Tokyo, Japan; Microcon YM-3 from Millipore Co., Beldford, U.S.A. All other chemicals were of reagent grade or better. The water used was 17-Mohm grade.Biotinylation of IgA, IgM, IgG, and BSA Biotinylation of BSA was also examined, since biotinylated BSA was used for the quantitation of biotin in this study. Twenty-five microliters of NHS-biotin DMSO solution at various concentrations was added to 1 ml of 2 mg ml Ϫ1 IgA, IgM, IgG, or BSA in 50 mmol l Ϫ1 sodium bicarbonate/HCl buffer (pH 8.6). The mixture was gently stirred at room temperature for a fixed time. The reaction solution was dialyzed against 0.15 mol l Ϫ1NaCl and finally against Dulbecco's phosphate buffered saline without Ca 2ϩ and Mg 2ϩ (PBS). The amounts of biotinylated IgA, IgM, IgG, and BSA were expressed as the amounts of IgA, IgM, IgG, and BSA, respectively, which were determined using a BCA Protein Assay kit with human IgG and BSA as standards for biotinylated IgA, IgM, and IgG and for BSA, respectively. Quantitation of Biotin Using Biotinylated BSA-Coated Multiwell MicroplateThe principle of biotin quantitation was the same as has been previously reported. [6][7][8][9] We used the following procedure with a small amount of biot...