Erratum: [Corrigendum] Expression of porcine protegrin­1 in Pichia pastoris and its anticancer activity in vitro.

[This corrects the article DOI: 10.3892/etm.2015.2202.].

Immune Efficacy of different immunization doses of divalent combination DNA vaccine pOPRL+pOPRF of Pseudomonas aeruginosa.

At present, there is no vaccine available against Pseudomonas aeruginosa, a common zoonotic pathogenic bacterium. In a previous study, the authors prepared a divalent combination DNA vaccine, pOPRL+pOPRF, which exhibited good protective efficacy. To explore the optimal immunization dose of this divalent combination DNA vaccine, in the present study, chickens were vaccinated with 25, 50, 100, and 200 µg doses. The levels of serum antibody, interferon-γ (IFN-γ), and interleukin-2 (IL-2) were determined, and lymphocyte proliferation assays were performed. After challenge with virulent P. aeruginosa, the protective efficacy was evaluated. Following vaccination, the serum antibodies, stimulation index values, and concentrations of IFN-γ and IL-2 were significantly higher in chickens vaccinated with 100 and 200 µg vaccines than in those vaccinated with 25 and 50 µg doses (P<0.05). IFN-γ and IL-2 concentrations in chickens immunized with 100 µg vaccine were slightly higher than those in chickens immunized with 200 µg vaccine, although the difference was not statistically significant. The protective rates were 55%, 65%, 85%, and 85% with 25, 50, 100, and 200 µg of the pOPRL+pOPRF DNA vaccine, respectively. Thus, the immune efficacy of the pOPRL+pOPRF DNA vaccine increased with an increase in immunization dose, but this does not imply that a higher dose necessarily achieves a better outcome. The optimal immunization dose of pOPRL+pOPRF DNA vaccine in chickens was 100 µg.

Expression of porcine protegrin-1 in Pichia pastoris and its anticancer activity in vitro.

Protegrin-1 (PG-1), a ß-hairpin antimicrobial peptide (AMP), is amongst the shortest AMPs in sequence length while remaining active against a variety of microorganisms. The aim of this study was produce recombinant PG-1 and investigate its anticancer activity. A DNA sequence encoding the mature PG-1, fused with a 6His-tag, was cloned into the pPICZα-A vector and transformed into Pichia pastoris. Expression was induced following culture for ~96 h with 1% methanol at 28°C, and ~15.6 mg PG-1 was expressed in 100 ml culture medium. Following purification using a Ni-chelating Sepharose column, ~20 mg pure active PG-1 was obtained from 500 ml culture broth supernatant. The expressed PG-1/6His exhibited strong dose- and time-dependent anticancer activity against HepG2 cells in vitro.

Molecularly imprinted polymer for specific extraction of hypericin from Hypericum perforatum L. herbal extract.

The molecularly imprinted polymers (MIPs) were prepared by an oxidation-reduction polymerization system using a non-covalent molecularly imprinting strategy with hypericin as the template, acrylamide as the functional monomer and pentaerythritol triacrylate as the cross-linker in the porogen of acetone. The UV spectrum revealed that a cooperative hydrogen-bonding complex between hypericin and acrylamide might be formed at the ratio of 1:6 in the prepolymerized system. Two classes of the binding sites were produced in the resulting hypericin-imprinted polymer with the dissociation constants of 16.61µgL(-1) and 69.35µgL(-1), and the affinity binding sites of 456.53µgg(-1) and 603.06µgg(-1), respectively. The synthesized MIPs were characterized by scanning electron microscope, thermogravimetric and differential thermal analysis. High-performance liquid chromatography was used to investigate the adsorption and recognition properties of the MIPs. Selective binding of the template molecule was demonstrated in comparison to the analog pseudohypericin. After the Hypericum perforatum L. plant being air dried and finely ground, an extract was prepared by shaking the powder in a methanol-water solution (80:20, v/v), vacuum filtration though a Büchner funnel, liquid-liquid extraction with ethyl ether and ethyl acetate, and evaporating on a rotary evaporator until dry. With the sorbents of the optimized MIPs, a molecularly imprinted solid-phase extraction (MISPE) procedure was developed for enrichment and separation of hypericin from the Hypericum extract in the presence of interfering substances. The selective extraction of hypericin from herbal medicine was achieved with the recovery of 82.30%. The results showed that MISPE can be a useful tool for specific isolation and effective clean-up of target compounds from natural products.

Effects of T-2 toxin on testosterone biosynthesis in mouse Leydig cells.

OBJECTIVE: To investigate the effects of T-2 toxin on testosterone biosynthesis in mouse Leydig cells. METHODS: Leydig cells isolated from clean and healthy Kunming male mice, whose concentration was adjusted to 5 × 10(5)/mL and the purity identified by the modified 3ß-hydroxysteroid dehydrogenase staining method, were used to establish a primary Leydig cell culture model. Blank control group (treated with 0 ng/mL human chorionic gonadotropin (hCG) and 0 mol/L T-2 toxin), inductive control group (treated with 10 ng/mL hCG and 0 mol/L T-2 toxin), low-dose T-2-toxin-exposure group (treated with 10 ng/mL hCG and 10(-9) mol/L T-2 toxin), middle-dose T-2 toxin-exposure group (treated with 10 ng/mL hCG and 10(-8) mol/L T-2 toxin) and high-dose T-2-toxin-exposure group (treated with 10 ng/mL hCG and 10(-7) mol/L T-2 toxin) were designed. The testosterone level was measured after 24 h incubation. RESULTS: After 24 h culture in liquid medium containing serum, the fresh isolated Leydig cells grew well and the purity exceeded 90%. By inducing 10 ng/mL hCG, the testosterone level of Leydig cells increased significantly and the difference compared with the blank control was of statistical sense. Compared with the inductive control group, the testosterone level of Leydig cells decreased, and the difference was of statistical sense in all T-2-toxin-exposure groups. Furthermore, the decrease was due to the increase in the dosage of T-2 toxin. CONCLUSIONS: T-2 toxin can directly decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis.

Preparation, preliminary characterization and inhibitory effect on human colon cancer HT-29 cells of an acidic polysaccharide fraction from Stachys floridana Schuttl. ex Benth.

A polysaccharide fraction (SFPSA) was obtained from the rhizomes of Stachys floridana Schuttl. ex Benth. by hot water extraction and sequential purification of anion-exchange chromatography and gel-filtration chromatography. SFPSA was found to be an acidic polysaccharide fraction with an average molecular weight of 168kDa and composed of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar percentage of 7.75:1.65:14.92:1.87:33.17:40.64, respectively. SFPSA could inhibit the proliferation and growth of human colon cancer HT-29 cells in a time- and dose-dependent manner within 48h, induce the apoptosis and increase the accumulation in G2/M phase of HT-29 cells. Furthermore, it was found that SFPSA could decrease Bcl-2 mRNA level, and increase the mRNA expressions of Bax and p53 and the activity of caspase-3. These results indicated that SFPSA might induce apoptosis of HT-29 cells through regulation of apoptosis-associated gene expressions such as Bcl-2, Bax and p53 and the activation of caspase-3.

Immune responses and protective efficacy of a novel DNA vaccine encoding outer membrane protein of avian Pasteurella multocida.

Avian Pasteurella multocida is a causative agent of fowl cholera. Two proteins OmpH and OmpA are the major immunogenic antigens of avian P. multocida, which play an important role in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed DNA vaccines with the genes encoding the two antigens mentioned above. These DNA vaccines include monovalent (pcDNA-OMPH, pOMPH and pcDNA-OMPA, pOMPA), divalent combination (pcDNA-OMPH+pcDNA-OMPA, pOMPH+pOMPA) and fusion of two gene vaccines (pcDNA-OMPH/OMPA, pOMPHA). The immune responses to these DNA vaccines were evaluated by serum antibody titers, lymphocyte proliferation assay and titers of a cytokines, IFN-γ. The protective efficacy after challenging with a virulent avian P. multocida strain, CVCC474, was evaluated by survival rate. A significant increase in serum antibody levels was observed in chickens vaccinated with divalent combination and fusion DNA vaccines. Additionally, the lymphocyte proliferation (SI value) and the levels of IFN-γ were both higher in chickens immunized with divalent combination and fusion DNA vaccines than in those vaccinated with monovalent DNA vaccines (P<0.05). Furthermore, the protection provided by divalent combination and fusion DNA vaccines was superior to that provided by monovalent DNA vaccines after challenging with the avian P. multocida strain CVCC474. And the protective efficacy in chickens immunized three times with the fusion DNA vaccine was equivalent to the protective efficacy in chickens vaccinated once with the attenuated live vaccine. This suggests that divalent combination and fusion DNA vaccines represent a promising approach for the prevention of fowl cholera.

Expression of 4kD scorpion defensin and its in vitro synergistic activity with conventional antibiotics.

The 4kD scorpion defensin (SD) is a potent disulfide-linked peptide. In this study, we expressed it in methylotrophic yeast Pichia pastoris and purified it using Ni-NTA His Bind Resin. We investigated its in vitro antibacterial activity and effect in combination with several conventional antibiotics. We first examined its antibacterial activity towards several Gram-positive and Gram-negative bacteria. Then we used the broth microdilution method to test drugs alone and in combination and used the fractional inhibitory concentration (FIC index) to classify the drug interactions. Our study showed the expressed SD peptide has antibacterial activity against Salmonella typhimurium, E. coli and S. aureus etc. Synergy or additive interaction was observed between SD and Norfloxacin, Polymyxin B and Ampicillin. Cell growth tests showed that combination of SD and Norfloxacin can improve their activity against bacteria. This result maybe permit lower using of the conventional antibiotic agents more effectively and safely.

A simple iterative method to optimize protein-ligand-binding residue prediction.

This article describes an iterative method (IM) for improving protein-ligand-binding residue prediction. Through modifying the binding residue definition in every iteration, this method, step by step, increased the performance of the classifiers used. Using a balanced assessment index (BAI), the classifier optimized by the IM achieved a value of 80.4 that is bigger than the one (66.9) of the initial classifier. According to mean per-instance BAI scores, a direct comparison of methods has been carried out along with an analysis of statistical significance of the differences in performance. The results show that the iterative method (IM) does achieve a higher mean score than the threshold-altering method (TAM) used in our previous study and there is a statistically significant difference between the two methods. The IM has a significant advantage that it is independent of the concrete residue characterization models and learning algorithms, and more extensively applicable. These results indicate that optimizing the binding residue definition is also an effective approach to improve protein-ligand-binding residue prediction.