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Background: Cytoglobin (Cygb) is a relatively newly identified globin protein that acts as an oxygen transporter in tissues like hemoglobin (Hb) in erythrocytes and myoglobin (Mb) in muscles. The natural oxidation of the Fe2+ ion in its heme group into metglobin (globin-Fe3+) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb5r3. However, metCygb reductase had not been previously identified. This study aims to analyze the reducing activity of bovine diaphorase on metCygb. Methods: Diaphorase was isolated from bovine erythrocyte and purified using gel filtration and cationicexchanger chromatography. Its purity was verified by SDS-PAGE and western blot (WB). The metCygb was obtained from Cygb oxidation with potassium ferrocyanide and its reducing activity was determined by spectroscopy. Results: The diaphorase (MW=30.09 kDa) was purified 10.77-fold from crude enzyme with specific activity against metHb 8.479 U/mg. The purity was confirmed by WB using primary antibody anti-cyb5r3. The purified enzyme reduced metCygb at 0.785 µgmin-1, which was 13.7 times less than the Vmax of metHb. Conclusions: In conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells. [ABSTRACT FROM AUTHOR]

Hemolytic activity of nonionic surfactants, polyoxyethylene cholesteryl ethers, CHO(CHCHO)H (Chol-E, n=, 25, 30, 50) and polyoxyethylene dihydrocholeseryl ethers, CHO(CHCHO)H (DHChol-E, n=15, 30 50) were measured, changing the concentration of surfactant and erythrocyte at 37 °C. Maximum hemolytic activity was observed in these cholesteryl derivatives with 25-30 oxyethylene units. The time course of hemolysis was also measured as a function of the concentrations of surfactant and erythrocyte. Hemolysis started after a certain induction period, τ, and then apparently proceeded as a first-order reaction with respect to the erythrocyte concentration. The surfactant inducing 50% hemolysis at low concentration had a small τ value and large rate constant. The maximum amount of adsorption without inducing hemolysis, a, decreased with increasing polyoxyethylene chain length. Chol-E has the maximum activity for the solubilization of egg yolk lecithin at 37 °C. Based on these results, the mechanism of hemolysis by these surfactants was quantitatively discussed. [ABSTRACT FROM AUTHOR]

Purpose. Ultrasound has been shown to transiently permeabilize biological membranes, thereby facilitating delivery of large compounds such as proteins and DNA into cells and across tissues such as skin. In this study, we sought to quantitatively determine the dependence of cell membrane permeabilization on ultrasound parameters and to identify acoustic signals which correlate with observed membrane permeabilization. Methods. Bovine red blood cells were exposed to ultrasound at 24 kHz over a range of controlled conditions. The degree of membrane permeabilization was measured by release of hemoglobin and was determined as a function of ultrasound parameters and measured acoustic signals. Results. These studies showed that permeabilization increased with incident ultrasound pressure, increased with total exposure time above a threshold of approximately 100 msec, showed a weak dependence on pulse length with a small maximum at 3 msec, and did not depend on duty cycle under the conditions examined. Using measured acoustic spectra we found that red blood cell membrane permeabilization correlated best with the pressure measured at half the driving frequency (f/ 2=12 kHz) and its ultraharmonics, less strongly with the broadband noise pressure measured between peaks, and least strongly with pressure measured at the driving frequency and its higher harmonics. Permeabilization caused by ultrasound applied at any set of conditions tested in this study could be well predicted by the parameter τ⋅Pf /2, which characterizes the total cavitational exposure. Conclusions. This study provides a quantitative guide to designing ultrasound protocols useful for drug delivery. The acoustic measurements support the hypothesis that ultrasonic cavitation is the mechanism by which membranes are permeabilized. They also suggest that measurable acoustic signals can provide noninvasive, real-time feedback about membrane permeabilization and drug delivery. [ABSTRACT FROM AUTHOR]

Apical membrane antigen 1 is a microneme protein which plays an indispensable role during Apicomplexa parasite invasion. The detailed mechanism of AMA-1 molecular interaction with its receptor on bovine erythrocytes has not been completely defined in Babesia bovis. This study was focused on identifying the minimum B. bovis AMA-1-derived regions governing specific and high-affinity binding to its target cells. Different approaches were used for detecting ama-1 locus genetic variability and natural selection signatures. The binding properties of twelve highly conserved 20-residue-long peptides were evaluated using a sensitive and specific binding assay based on radio-iodination. B. bovis AMA-1 ectodomain structure was modelled and refined using molecular modelling software. NetMHCIIpan software was used for calculating B- and T-cell epitopes. The B. bovis ama-1 gene had regions under functional constraint, having the highest negative selective pressure intensity in the Domain I encoding region. Interestingly, B. bovis AMA-1-DI (100YMQKFDIPRNHGSGIYVDLG119 and 120GYESVGSKSYRMPVGKCPVV139) and DII (302CPMHPVRDAIFGKWSGGSCV321)-derived peptides had high specificity interaction with erythrocytes and bound to a chymotrypsin and neuraminidase-treatment sensitive receptor. DI-derived peptides appear to be exposed on the protein's surface and contain predicted B- and T-cell epitopes. These findings provide data (for the first-time) concerning B. bovis AMA-1 functional subunits which are important for establishing receptor-ligand interactions which could be used in synthetic vaccine development. [ABSTRACT FROM AUTHOR]