Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain.

Vascular endothelial growth factor A165 (VEGF-A165) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A165 receptor-binding domain (VEGFA165-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA165-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA165-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA165-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA165-RBD. 25 ng sKDR1-3 and 20 ng VEGFA165-RBD showed strong binding. sKDR1-3 bound to VEGFA165-RBD and VEGF-A165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA165-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.

Clodronic Acid has Strong Inhibitory Interactions with the Urease Enzyme of Helicobacter Pylori: Computer-aided Design and in vitro Confirmation.

BACKGROUND: Helicobacter Pylori (HP) infection could lead to various gastrointestinal diseases. Urease is the most important virulence factor of HP. It protects the bacterium against gastric acid. OBJECTIVE: Therefore, we aimed to design urease inhibitors as drugs against HP infection. METHODS: The DrugBank-approved library was assigned with 3D conformations and the structure of the urease was prepared. Using a re-docking strategy, the proper settings were determined for docking by PyRx and GOLD software. Virtual screening was performed to select the best inhibitory drugs based on binding affinity, FitnessScore, and binding orientation to critical amino acids of the active site. The best inhibitory drug was then evaluated by IC50 and the diameter of the zone of inhibition for bacterial growth. RESULTS: The structures of prepared drugs were screened against urease structure using the determined settings. Clodronic acid was determined to be the best-identified drug, due to higher PyRx binding energy, better GOLD FitnessScore, and interaction with critical amino acids of urease. In vitro results were also in line with the computational data. IC50 values of Clodronic acid and Acetohydroxamic Acid (AHA) were 29.78 ± 1.13 and 47.29 ± 2.06 µg/ml, respectively. Diameters of the zones of inhibition were 18 and 15 mm for Clodronic acid and AHA, respectively. CONCLUSION: Clodronic acid has better HP urease inhibition potential than AHA. Given its approved status, the development of a repurposed drug based on Clodronic acid would require less time and cost. Further, in vivo studies would unveil the efficacy of Clodronic acid as a urease inhibitor.

Streptomyces strains can improve the quality properties and antifungal bioactivities of tomato fruits by impacting WRKY70 transcription factor gene and nitrate accumulation.

The current study evaluated the effect of plant growth-promoting (PGP) strains of Streptomyces on yield, quality, and nitrate content of fruits, plant-microbe responses, and antifungal effect against blight disease caused by fungus pathogen Alternaria solani on tomato fruits in commercial greenhouse conditions. Greenhouse trials were done with four treatments including strains Y28, IC10, IT25, and commercial bio-fertilizer (Barvar NPK®) on tomato plants. In PGP treatments, the number of infected fruits significantly reduced (60%) compared to Barvar and control. Strain Y28 improved the quality of tomatoes more than other treatments. All three PGP treatments contained a higher level of total sugar concentration and antioxidant enzyme activities than Barvar and control. In contrast, PGP strains, especially Y28, significantly reduced nitrate accumulation (25%) compared to Barvar and control tomatoes. Streptomyces treatments induced more than a 20-fold increase in UDP and WRKY70 transcription factor gene expression relative to the control (P < 0.01). Based on the results, microbe-dependent plant defense induced by these strains is positively correlated to WRKY70 expression and nitrate reduction in commercial greenhouse conditions. These findings suggest that the commercial application of specific strains not only can illustrate an eco-friendly solution to induce resistance against fungal pathogens but also improve the quality properties of food plants with lower nitrate content.

Data on cell survival, apoptosis, ceramide metabolism and oxidative stress in A-494 renal cell carcinoma cell line treated with hesperetin and hesperetin-7-O-acetate.

Ceramide pathway is a key regulator in cell proliferation and apoptosis and oxidative stress up-regulate ceramides. Acid ceramidase (ACDase), neutral sphingomyelinase (NSMase) and glucosylceramide synthase (GCS) are critical enzymatic systems in ceramide metabolism. Our data represent the comparative assessment of Hesperetin (Hst) and hesperetin-7-O-acetate (HTA) effects on A-494 renal carcinoma cells include cell survival, caspase-3 and 9 activities, total cellular ceramide and the activities of ACDase, NSMase, GCS and superoxide dismutase (SOD). Data reveals potentiating effects of both HTA and Hst on ceramide pathway and may offer a novel tool in human renal cell carcinoma therapy.

Plasmin digest of κ-casein as a source of antibacterial peptides.

This study investigated the antibacterial properties of plasmin, the plasmin hydrolysis of bovine κ-casein and the fractions (named κC1, κC2, κC3, κC4, and κC5) liberated from it using RP-HPLC. The target bacteria were Escherichia coli, Staphylococcus aureus (pathogenic), Lactobacillus casei and Lactobacillus acidophilus (probiotic). Three peptides (kC1, kC3, and kC4) were found to have antibacterial activity, with κC3 peptide being the most active. The plasmin digest of bovine κ-casein proved to be stronger than any of its fractions in terms of antibacterial potential. Measurement of the minimum inhibitory concentration (MIC) showed that Gram-positive bacteria are generally more sensitive to antibacterial activity than Gram-negative bacteria. The MIC of nisin, as a bacteriocin peptide, was also measured. The three antibacterial peptides were identified using LC-Mass. The molecular mass of kC1, kC3, and kC4 corresponded to the f(17-21), f(22-24), and f(1-3) of bovine κ-casein, respectively. It was also found that the positive charge and hydrophobicity of a peptide are not key factors in antibacterial activity. On the whole, the present study demonstrated that the plasmin digest of κ-casein has a high antibacterial potential and can be considered as a natural antibacterial agent in the food chain.

Longitudinal follow-up of oxidative stress and DNA damage parameters in detergent workers.

BACKGROUND: The aim of this study was the follow-up of work place enzyme and detergent dust exposure effects and smoking habit on DNA damage parameters of workers and the evaluation of their antioxidant enzyme activities and lipid peroxidation with regard to bag-filter installation in the work place. MATERIAL AND METHODS: All investigated parameters were studied in a group of 153 workers of enzyme-free detergent production plant (E-free) and a group of 138 workers of enzyme-plus detergent plant (E-plus) and compared with 45 controls 7.2 years before and 3.1 years after filter system installation. The following methods were used: antioxidant enzymes by an ultraviolet-visibles spectrophotometer, malondialdehyde (MDA), 8-hydroxy-2'deoxyguanosine (8OH-2'dG) by high-performance liquid chromatography, trace elements by atomic absorption spectroscopy, and comet assay by single cell gel electrophoresis. RESULTS: Compared with controls, significant increases were observed in both detergent-exposed groups with respect to the levels of MDA, antioxidant enzyme activities, and DNA damage parameters, including 8OH-2'dG, endonuclease III-sensitive sites, and DNA strand breaks, with enhancement effect of smoking before filter system installation. After filter installation, besides significant decrease in the detergent and enzyme dust of airborne and oxidative stress indicators, there was improvement in all DNA damage investigated parameters at the end of this study. The levels of cumulative exposure index of detergent dusts decreased significantly after airborne improvement and showed positive correlation with internal biochemical parameters. CONCLUSIONS: We concluded that high levels of enzyme and detergent contents of work place dusts had a cumulative effect and smoking had a synergistic effect on the imbalance of antioxidant status and lipid peroxidation, suggesting that oxidation stress is important in the occurrence and progression of DNA damage over this study. Detergent and enzyme contents in respirable and total dust had the main role and sufficient potential in their genotoxicity.

Level of oxidative stress markers among physicians in a medical residency program.

The authors investigated the effect of engaging in a medical residency program, as a stressful situation, on blood and urine levels of oxidative stress markers. Newly admitted medical residents participated in the study, along with a control group of (nonmedical) students. The authors assessed superoxide dismutase, glutathione peroxidase, catalase, malondialdehyde, micronuclei test, sister chromatid exchange, and 8-hydroxy-2'-deoxyguanosine level. All the biomarkers declined after entrance to the residency program, and the parameters were strongly higher in residents than in the control group. There was no significant relationship between demographic factors and levels of stress biomarkers. The greater extent of oxidative stress may be due to higher tension before entrance to a supposedly critical new position, and the declined levels of biomarkers seen after several months in the program could be attributed to an appropriate adjustment of the residents to the new situation.