Biotin-protein ratios and stability of biotinylated immunoglobulins as standards for the quantitation of biotin-binding immunoglobulins.

Biotin-binding IgG in human sera was quantitated using F(ab')(2)anti-human IgG-coated multiwell microplates (Muratsugu, M. et al. 2003, Biol. Pharm. Bull., 26, 1605-1608). The biotin-protein ratio of biotinylated IgG, which was used as standard in the assay, was very important to quantitate the level of biotin-binding IgG. We investigated a synthesis method of biotinylated human immunoglobulins, how to determine the biotin-protein ratio of the biotinylated proteins, and their stability to prepare standards for measuring biotin-binding IgG, IgA, and IgM.

Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing.

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.

Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound.

Recently, we have reported that ultrasound (US)-induced apoptosis is due to inertial cavitation and that extracellular reactive oxygen species (ROS) generated by inertial cavitation are not directly correlated with the apoptosis (Honda et al. 2002). The molecular mechanism of apoptosis induced by US is not yet sufficiently clear. Here, we examine the role of intracellular calcium ions and the intracellular ROS on apoptosis induced by US. Human myelomonocytic lymphoma U937 cells were exposed to continuous 1-MHz US at an intensity of 4.9 W/cm(2) (I(SPTA)) in the presence of air, and changes of intracellular calcium ion concentration ([Ca(2+)]i) in individual cells by digital imaging, various flow cytometric analyses of endpoints of apoptosis (early apoptosis, secondary necrosis, loss of mitochondria membrane potential, superoxide formation, caspase-3 activation) and DNA fragmentation were explored. Furthermore, the effects of an intracellular calcium ion chelator (BAPTA-AM), an antioxidant (N-acetyl-L-cysteine, NAC), a calcium channel blocker (verapamil), Ca(2+)-free buffer and Levovist were also investigated. These results indicate that: 1. the mitochondria-caspase pathway and the Ca(2+)-dependent pathway play cardinal roles in apoptosis induced by US because BAPTA-AM partially inhibited DNA fragmentation, loss of mitochondria membrane potential and caspase-3 activation; 2. intracellular ROS generated from mitochondria, rather than extracellular ROS (which were directly produced by inertial cavitation in the medium), are involved in the regulation of apoptosis induced by US because addition of NAC after sonication showed effective suppression of the apoptosis; and 3. increase of [Ca(2+)]i appears to be due to nonspecific influx from outside the cells because verapamil is not effective and no increase of [Ca(2+)]i due to sonication could be observed in the Ca(2+)-free buffer.

Effects of dissolved gases and an echo contrast agent on ultrasound mediated in vitro gene transfection.

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.

Effects of dissolved gases and an echo contrast agent on apoptosis induced by ultrasound and its mechanism via the mitochondria-caspase pathway.

Human histiocytic lymphoma U937 cells were exposed to continuous 1-MHz ultrasound (US) for therapeutic use, (0 approximately 6.5 W/cm(2) (I(SPTA)). Apoptosis and its related end points were examined by flow cytometry. Fraction of cells with low mitochondria membrane potential were observed after sonication and significant superoxide and peroxide formation, increased activity of caspase-3, and DNA fragmentation revealed biochemically, were also found. The fraction of early apoptosis and secondary necrosis increased with the incubation time after sonication. Early apoptosis observed at 6 h after sonication reached its maximum at 2 min of sonication and gradually decreased. On the other hand, secondary necrosis increased with the duration of sonication. When the effects of dissolved gases, Ar, N(2), O(2), air, N(2)O and CO(2), on free radical formation due to inertial cavitation were investigated by electron spin resonance (ESR) spin trapping, formation of hydroxyl radicals and hydrogen atoms was found in solutions saturated with Ar, N(2), O(2) and air, but not with N(2)O and CO(2). Apoptosis induced by US was also dependent on the dissolved gases in the order Ar = N(2) = O(2) = air >> N(2)O = CO(2) approximately 0. These results suggest that US-induced apoptosis, which is mitochondria-caspase dependent, was linked to inertial cavitation. However, quantities of free radicals did not influence the fraction of early apoptosis and secondary necrosis. When the cells were sonicated in the presence of an echo contrast agent, Levovist; synergistic enhancement of secondary necrosis induced by US was observed at concentrations of more than 20 mg/mL. In contrast, an additive increase of early apoptosis was observed in the combined treatments. These results suggest that Levovist; acting as cavitation nuclei, enhances secondary necrosis induced by US due to an increase in the membrane damage.