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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]

B. bovis invasion of bovine erythrocytes requires tight junction formation involving AMA-1/RON2 complex interaction. RON2 has been considered a vaccine candidate since antibodies targeting the protein can inhibit parasite invasion of target cells; however, the mechanism controlling B. bovis RON2 interaction with red blood cells is not yet fully understood. This study was thus aimed at identifying B. bovis RON2 protein regions associated with interaction with bovine erythrocytes. Natural selection analysis of the ron2 gene identified predominantly negative selection signals in the C-terminal region. Interestingly, protein-cell and competition assays highlighted the RON2-C region's role in peptide 42918-mediated erythrocyte binding, probably to a sialoglycoprotein receptor. This peptide (1218SFIMVKPPALHCVLKPVETL1237) lies within an intrinsically disordered region of the RON2 secondary structure flanked by two helical residues. The study provides, for the first time, valuable insights into RON2's role in interaction with its target cells. Future studies are required for studying the peptide's potential as an anti- B. bovis vaccine component. [Display omitted] • B. bovis RON2 interaction mechanism with red blood cells is not yet fully understood. • ron2 gene contains predominantly negative selection signals in its C-terminal region. • Protein-cell and competition assays highlighted the RON2-C region's role in binding. • A peptide derived from the RON2-C region strongly binds to bovine erythrocytes. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Morella serrata L. is an indigenous medicinal plant to South and southern Africa with folkloric applications as aphrodisiac, laxative, antimicrobial, anti-inflammatory, analgesic, anti-hypertensive, antitussive and antidiabetic agent. Aim of the study This study evaluated the membrane stabilization and aphrodisiac potentials of its aqueous root extract in male Wistar rats. Materials and methods While the membrane stabilization of the extract was investigated against bovine erythrocytes (BE), the male rats for the aphrodisiac study were randomized into five groups with animals in group 1 given sterile placebo and served as control. The rats in group 2 were treated with 7.14 mg/kg body weight of PowMaxM, while animals in groups 3, 4 and 5 were administered with the extract (100, 200 and 400 mg/kg, respectively). All treatments (1 mL) were done once daily for 4 weeks via oral gavaging and their mating behavioural, testicular, spermatogenetic and antioxidant parameters were evaluated. Results With the exception of the mount, intromission and post ejaculatory latencies that were dose-dependently reduced by the extract, other mating parameters were significantly improved when compared with the control. Similar patterns of significant improvement were also observed on the testes-body weight ratio, quality and viability of sperm cells as well as testicular concentrations of proteins, cholesterol, glycogen, testosterone, follicle stimulating hormone, leuitenizing hormone and glutathione (reduced) subsequent to treatment with the extract. Although, administration of M. serrata had no significant (p>0.05) effect on the testicular activity of gamma glutamyl transferase, those of lactate dehydrogenase, phosphatases (alkaline and acid), superoxide dismutase and catalase were significantly (p<0.05) induced in the treated animals. The extract also conferred respective significant (p<0.05) membrane stabilization potential of 66.02% and 60.87% on the BE against hypotonic solution and heat-induced hemolysis relative to 62.14% and 40.19% for ibuprofen. The effect elicited by the extract at the tested doses could partly be attributed to its antioxidant and adaptogenic constituents. Conclusion The data presented in this study have enriched biochemical information on the root extract of M. serrata as a viable source of phytonutrients that could be potentially useful for the development of aphrodisiac drugs, and thus lending scientific credence to its much touted sex enhancing attributes by the Basothos of the eastern Free State Province of South Africa. [ABSTRACT FROM AUTHOR]

This paper describes effects of some antibiotics on glutathione reductase in vitro. Glutathione reductase was purified from bovine erythrocytes. Purification procedure consisted of four steps; preparation of hemolysate, ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtra­tion chromatography. As a result of these four consecutive procedures, the enzyme having the specific activity of 62.5 EU/mg proteins was purified 31 250-fold with a yield of 11.39%. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The effects of eight different antibiot­ics-streptomycin sulphate, gentamicin sulphate, netilmicin, teicoplanine, thiamphenicol, ampicillin, cefotaxime, and cefodizime- were investigated on the purified enzyme. Six of these antibiotics (streptomycin sulphate, gentamicin sulphate, netilmicin, teicoplanine, thiamphenicol, and ampicillin) increased the activity of glutathione reductase with increase in their concentrations while the two (cefotaxime and cefodizime) inhibited the enzyme activity. I50 values were 7.713 mM and 1.954 mM, and Ki constants were 11.011 mM and 8.956 mM for cefotaxime and cefodizime, respectively. Their inhibition types were competitive.

Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the full MSA-1 or its fragments. This research has thus been focused on identifying protein regions playing a role in erythrocyte attachment, based on genetic diversity and natural selection analysis. Two regions under functional constraint (nucleotides 134–428 and 464–629) having a preponderance of negatively-selected signals were identified in silico. Three non-overlapping peptides derived from functionally constraint regions (42422 (39PEGSFYDDMSKFYGAVGSFD58), 42424 (91NALIKNNPMIRPDLFNATIV110) and 42426 (150TDIVEEDREKAVEYFKKHVY169)) were able to specifically bind to a sialoglycoprotein located on the bovine erythrocyte surface as confirmed by sensitive and specific peptide-cell interaction competition assays using both enzymatically treated and untreated red blood cells. Interestingly, it was predicted that peptides 42422 and 42426 have a helical structure and conserved motifs in all strain/isolates. These findings provide evidence, for the first time, related to B. bovis MSA-1 short regions used by the parasite in erythrocyte binding which could be predicted using natural selection analysis. Future work focused on evaluating these peptides' antigenic ability during natural infection, and their ability to induce protection in immunisation assays are needed to confirm their usefulness as synthetic vaccine candidates. • Two functionally constraint regions were identified in B. bovis MSA-1. • B. bovis MSA-1 binds to bovine erythrocytes through 3 short protein fragments. • These MSA-1 peptides might be promising components of a B. bovis vaccine [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]