ABSTRACT
Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial L-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 µg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 µg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane.
Subject(s)
Anti-Bacterial Agents/metabolism , Escherichia coli/drug effects , L-Amino Acid Oxidase/metabolism , Staphylococcus/drug effects , Yersinia/drug effects , Animals , Blotting, Western , Escherichia coli/growth & development , Flounder/genetics , Gene Expression , L-Amino Acid Oxidase/genetics , Microbial Sensitivity Tests , Microscopy, Electron, Scanning , Pichia/genetics , Pichia/metabolism , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staphylococcus/cytology , Staphylococcus/growth & development , Yersinia/growth & developmentABSTRACT
BACKGROUND: We evaluated neuropsychological function before and after surgery in patients with unruptured cerebral aneurysms. METHODS: Neuropsychological functions in 43 patients with unruptured cerebral aneurysms were evaluated before and 1 month after surgery. The neuropsychological examination included the Mini-Mental State examination, "Kana-hiroi" test, Kohs Block Design test, and Miyake's Memory test. Then, if scores of even a single test were decreased 1 month after surgery, the tests were performed again 5 months later. In 24 of the 43 subjects, cerebral blood flow (CBF) was measured before and 1 month after surgery by single-photon emission tomography. RESULTS: The outcome in all patients was evaluated as good according to the Glasgow Outcome Score (GOS). In 17 (40%) of the 43 patients, neuropsychological function had deteriorated 1 month after surgery. The most sensitive test applied was the Miyake's Memory test. Of 14 patients with neuropsychological deterioration 1 month after surgery, 6 showed complete recovery, 5 showed partial recovery, and 3 still showed cognitive deterioration 6 months after surgery. Patients over 65 years old, those with anterior communicating artery aneurysms, those operated by interhemispheric approach, or those with systemic diseases showed a greater tendency toward a decline in postoperative neuropsychological function than the other patients. The postoperative CBF and vascular response in the frontal lobe of affected-side was decreased in cases showing cognitive deterioration. CONCLUSIONS: These results suggested that the neuropsychological outcomes after surgery for unruptured cerebral aneurysms were not satisfactory.
