ABSTRACT
Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Toxins/antagonists & inhibitors , Cross Infection/drug therapy , Pore Forming Cytotoxic Proteins/antagonists & inhibitors , Pseudomonas Infections/drug therapy , Single-Chain Antibodies/pharmacology , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/therapeutic use , Antigens, Bacterial/metabolism , Bacterial Toxins/metabolism , Cell Line, Tumor , Cloning, Molecular , Computer Simulation , Cross Infection/immunology , Cross Infection/microbiology , Half-Life , Humans , Molecular Docking Simulation , Pore Forming Cytotoxic Proteins/metabolism , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/immunology , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Single-Chain Antibodies/genetics , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/therapeutic useABSTRACT
BACKGROUND: The anti-aging property of ß-D-mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory and immunosuppressive agent was investigated on several determinants relative to the oxidative stress in an animal model. METHODS: Sprague-Dawley rats were used for evaluating the safety and efficacy properties of M2000 on some oxidative stress enzymes, including the following: mitochondrial superoxide dismutase (SOD2), catalase (CAT), glutathione peroxidase (GPX1), glutathione S-transferase (GST), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS) gene expression by real-time PCR. Malondialdehyde (MDA), carbonyl protein (PCO) (the lipid and protein oxidation marker, respectively), and total antioxidant capacity (TAC) were tested in serum by biochemical analysis. In addition, cortisol as a steroid hormone was surveyed by chemiluminescence immunoassay after 12 weeks of M2000 consumption. The rats were sacrificed 3 months after daily oral administration of M2000. RESULTS: Our findings revealed the favorable effects of M2000 on several antioxidant enzyme and gene expression, including SOD2, CAT, GPX1, and GST; however, our results were not statistically significant. Moreover, there was no significant difference in MDA and PCO as lipid and protein oxidation markers, TAC, and cortisol compared with the control group following M2000 consumption. A slight weight increase in the M2000-treated group was also observed. CONCLUSIONS: Our data showed the anti-aging property of M2000 as a novel designed non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive property on various oxidative stress determinants.
Subject(s)
Hexuronic Acids/pharmacology , Immunosuppressive Agents/pharmacology , Oxidative Stress/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/pharmacology , Catalase/metabolism , Disease Models, Animal , Female , Glutathione Transferase/metabolism , Male , Malondialdehyde/metabolism , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolismABSTRACT
BACKGROUND: G2013 molecule is a novel non-steroidal anti-inflammatory agent with immunosuppressive property, which was investigated on determinants relative to the oxidative stress in animal model. MATERIALS AND METHODS: The Sprague-Dawley rats were used for evaluating properties of G2013 on some oxidative stress enzymes including: Myeloperoxidase (MPO), Glutathione peroxidase (GPX1), mitochondrial Superoxide dismutase (SOD2), Catalase (CAT), Glutathione S-Transferase (GST), and inducible nitric oxide synthase (iNOS) genes expression by Real Time PCR. The rats were sacrificed 3 months after daily oral administration of G2013. Moreover, Malondialdehyde (MDA), Carbonyl protein (PCO), the lipid and protein oxidation markers respectively and total anti-oxidant capacity (TAC) were tested in serum by biochemical analysis. Also cortisol as a steroid hormone was evaluated by chemiluminescence immunoassay after 12 weeks consumption of G2013 solution. RESULT: Our findings revealed a significant decrease in MPO in G2013 treated group, indicating its favorable effects but has no significant effects on genes expression of another antioxidant enzymes, including: SOD2, CAT, GPX1, and GST. Also, there were no significant differences in PCO, TAC and cortisol compared to control group following G2013 consumption. While an enhancement in serum MDA level was observed in the treatment group. In addition, G2013 therapy did not show any weight loss. CONCLUSIONS: Our data showed the safety and efficacy of G2013 as a novel designed NSAID on various oxidative stress determinants.
