The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.
b S Supporting Information ' EXPERIMENTAL SECTION Spherical Mesoporous Silicas. Spherical mesopores with diameters ranging from 6.6 to 28.6 nm were prepared using three different synthesis procedures in order to access such sizes.
BACKGROUND: Recent observational studies of nebulizers placed on the wet side of the humidifier suggest that, after some time, considerable condensation can form, which triggers an occlusion alarm. In the current study, an inline breath-enhanced jet nebulizer was tested and compared in vitro with a vibrating mesh nebulizer on the humidifier dry-inlet side of the ventilator circuit. METHODS: Two duty cycle breathing patterns were tested during continuous infusion (5 or 10 mL/h) with and without dynamic changes in infusion flow and duty cycle, or bolus delivery (3 or 6 mL) of radiolabeled saline solution. Inhaled mass (IM) was measured by a realtime ratemeter (lCi/min) and analyzed by multiple linear regression. RESULTS: During simple continuous infusion, IM increased linearly for both nebulizer types. IM variability was attributable to the duty cycle (P < .001) (34%) and infusion flow (P < .001) (32%) but independent of nebulizer technology (P 5 .38) (7%). Dynamic continuous infusion studies that simulate clinical scenarios with ventilator and pump flow changes demonstrated a linear increase in the rate of aerosol that was dependent on pump flow (P < .001) (63%) and minimally dependent on the duty cycle (P 5 .003) (8%). During bolus treatments, IM increased linearly to plateau. IM variability was attributable to the duty cycle (P < .001) (40%) and residual radioactivity in the nebulizer (P < .001) (20%). Separate analysis revealed that the vibrating mesh nebulizer residual volume contributed 16% of the variability and inline breath-enhanced jet nebulizer contributed 5%. IM variability was independent of bolus volume (P 5 .82) (1%). System losses were similar (the inline breath-enhanced jet nebulizer: 32% residual in nebulizer; the vibrating mesh nebulizer: 34% in circuitry). CONCLUSIONS: Aerosol delivery during continuous infusion and bolus delivery was comparable between the inline breath-enhanced jet nebulizer and the vibrating mesh nebulizer, and was determined by pump flow and initial ventilator settings. Further adjustments in ventilator settings did not significantly affect drug delivery. Expiratory losses predicted by the duty cycle were reduced with placement of the nebulizer near the ventilator outlet.
Background: A new real-time method for assessing factors determining aerosol delivery is described. Methods: A breath-enhanced jet nebulizer operated in a ventilator/heated humidifier system was tested during bolus and continuous infusion aerosol delivery. 99m Tc (technetium)/saline was either injected (3 or 6 mL) or infused over time into the nebulizer. A shielded gamma ratemeter was oriented to count radioaerosol accumulating on an inhaled mass (IM) filter at the airway opening of a test lung. Radioactivity measured at 2-10-minute intervals was expressed as % nebulizer charge (bolus) or % syringe activity per minute infused. All circuit parts were measured and imaged by gamma camera to determine mass balance. Results: Ratemeter activity quantitatively reflected immediate changes in IM: 3 and 6 mL bolus IM% ¼ 16.1 and 18.8% in 6 and 14 minutes, respectively; infusion IM% ¼ 0.64 + 0.13 (run time, min), R 2 0.999. Effect of nebulizer priming and system anomalies were readily detected in real time. Mass balance (basis ¼ dose infused in 90 minutes): IM 39.2%, breath-enhanced jet nebulizer residual 35.5%, circuit parts including humidifier 23.4%, and total recovery 98.1%. Visual analysis of circuit component images identified sites of increased deposition. Conclusion:Real-time ratemeter measurement with gamma camera imaging provides operational feedback during in vitro testing procedures and yields a detailed analysis of the parameters influencing drug delivery during mechanical ventilation. This method of analysis facilitates assessment of device function and influence of circuit parameters on drug delivery.
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