Stabilizing osmolytes' effects on the structure, stability and function of tc-tenecteplase: A one peptide bond digested form of tenecteplase.

Organic osmolytes, as major cellular compounds, cause protein stabilization in the native form. In the present study, the possible chaperone effects of the three naturally occurring osmolytes on the two-chain form of tenecteplase (tc-TNK), a recombinant, genetically engineered mutant tissue plasminogen activator, have been explored by using circular dichroism, steady-state fluorescence, UV-Visible spectroscopy, and in silico experiments. The tc-TNK is derived from the one-chain protein upon disruption of one peptide bond. Thermal denaturation experiments showed a slightly more stabilizing effect of the three co-solvents on the single-chain TNK (sc-TNK) in comparison to that on tc-TNK. Unlike single-chain tenecteplase, the two-chain form undergoes reversible denaturation which is somehow perturbed in some cases as the result of the presence of osmolytes. Very minor changes in the secondary structure and the tertiary structure were observed. The molecular dynamics simulations and comparative structural analysis of catalytic domain of the protein in the single-chain and two-chain forms in pure water, mannitol/water, trehalose/water, and sucrose/water showed that while the stabilizing effect of the three osmolytes on tc-TNK might be induced by preferential accumulation of these molecules around the nonpolar and aromatic residues, that is to say, fewer water-hydrophobic residues' interactions in tc-TNK, sc-TNK is stabilized by preferential exclusion effect.

A comparative study of structure, stability and function of sc-tenecteplase in the presence of stabilizing osmolytes.

The aim of the present study was to investigate the effect of three routine drug excipients, as osmolytes, in three different concentrations, on structure, thermal stability and the activity of single-chain (sc-) tenecteplase. To see the influence of trehalose, mannitol, and sucrose on the structure, stability and function of sc-tenecteplase, thermal stability, fluorescence, circular dichroism (CD) and enzyme kinetic measurements and molecular docking studies were carried out. To measure the effect of osmolytes on stability of sc-tenecteplase, thermodynamic parameters (transition temperature (Tm), standard enthalpy change (ΔH°), standard entropy change (ΔS°) and ΔG°, the standard Gibbs free energy change, were determined from heat-induced transition curves of the protein in absence and presence of each osmolyte. It was observed that all three osmolytes acted as an enhancer for the sc-tenecteplase stability, with varying efficacies and efficiencies. The results of the kinetic study showed that the activity of sc-tenecteplase is increased in the presence of osmolytes. The near-UV and far-UV CD studies showed transfer of Trp, Phe and Tyr residues to a more flexible environment in the presence of osmolytes. The sc-tenecteplase fluorescence quenching suggested the more polar location of Trp residues. Molecular docking studies revealed that (i) Gibbs free energy of interaction between the osmolyte and sc-tenecteplase is negative, and (ii) hydrogen bond and hydrophobic interactions dominate within the interaction sites.

Display of B. pumilus chitinase on the surface of B. subtilis spore as a potential biopesticide.

BACKGROUND: Chitinases can inhibit the growth of many fungal diseases which are a great threat for global agricultural production. Biological control of pathogens like fungi, is believed to be one of the best ways to eliminate the adverse effects of plant pathogens. To this end, we expressed and displayed a chitinase from Bacillus pumilus (ChiS) on the surface of Bacillus subtilis spores, as a biocontrol agent. RESULT: ChiS enzyme from B. pumilus was expressed on the spores of B. subtilis using CotG as a carrier protein. Immunofluorescence microscopy confirmed the expression of ChiS on the surface of the spores. Enzyme activity assay showed that the surface displayed ChiS was active and was also able to inhibit the growth of Rhizoctonia solani and Trichoderma harzianum fungi. Western blot analysis also indicated that CotG-ChiS is partially processed after display. Molecular dynamics simulation showed that the stability of the heterologous protein was decreased after fusion. CONCLUSION: ChiS was successfully displayed on the surface of Bacillus spores by fusion to the CotG, one of the main spore coat proteins. In-vitro experiments showed that the displayed enzyme was effective in growth inhibition of R. solani and T. harzianum fungi.

Evaluation of Iron Oxide Nanoparticles Toxicity on Liver Cells of BALB/c Rats.

BACKGROUND: Because of their unique magnetic properties, Fe3O4 nanoparticles (Fe3O4-NPs) have extensive applications in various biomedical aspects. Investigation of the possible adverse aspects of these particles has lagged far behind their fast growing application. OBJECTIVES: The current study aimed to evaluate the toxicity of Fe3O4-NPs in the liver of mice. MATERIALS AND METHODS: In the present clinical trial, 90 BALB/c mice were randomly divided in 15 groups. Five control groups were fed by usual water and food. Five placebo groups were gavaged with physiological serum in doses of 25, 50, 75, 150, and 300 micrograms per gram of body weight (µg/gr). Five experimental groups were gavaged with Fe3O4-NPs, in doses of 25, 50, 75, 150, and 300 µg/gr. This pattern was repeated every other day, for 3 days. Then, the levels of liver enzymes [alanine transaminase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP)] were compared between these groups. The histological alterations of livers were examined, as well. For statistical analysis, Kruskal-Wallis and Mann-Whitney, with type I Bonferroni correction, as post-hoc, have been used. RESULTS: The administration of 150 and 300 µg/gr doses of Fe3O4-NPs were associated with significant elevation in liver enzymes, compared to controls (P < 0.0001). Furthermore, the histopathological effects were observed in the liver tissue of these groups. However, in groups treated with lower doses of Fe3O4-NPs, no significant adverse effect was observed. CONCLUSIONS: Based on our results, the administration of Fe3O4-NPs causes dose dependent adverse effects on liver.