Expression, purification and identification of Pla a1 in a codon-optimized Platanus pollen allergen.

The present study aimed to express, purify and identify the major allergen gene, Pla a1, in Platanus pollen. According to previous studies, the major gene sequences of the Pla a1 allergen were obtained and codon optimization and synthesis of the genome were performed using DNAStar software. Following binding of the target gene fragment and the pET-44a vector, the JM109 cells were transfected to produce positive clones. The vectors were then transformed into Escherichia coli Rosetta cells to induce the expression of the target protein. The exogenous protein was purified using affinity chromatography and was identified by western blot analysis. Pla a1, the major allergen protein in Platanus pollen, was successfully isolated and this exogenous protein was purified using affinity chromatography. The present study was the first, to the best of our knowledge, to obtain expression of the allergen recombinant protein, Pla a1, fused with a Strep-TagII via codon optimization and provided the basis for the preparation of allergens with high purity, recombinant hypoallergenic allergens and allergen nucleic acid vaccines.

Effect of α-linolenic acid on endoplasmic reticulum stress-mediated apoptosis of palmitic acid lipotoxicity in primary rat hepatocytes.

BACKGROUND: Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of saturated and unsaturated fatty acids (FA) on apoptosis in primary rat hepatocytes. METHODS: The primary rat hepatocytes were treated with palmitic acid and/or α-linolenic acid in vitro. The expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels were detected after the treatment. RESULTS: The treatment with palmitic acid produced a significant increase in cell death. The unfolded protein response (UPR)-associated genes CHOP, GRP78, and GRP94 were induced to higher expression levels by palmitic acid. Co-treatment with α-linolenic acid reversed the apoptotic effect and levels of all three indicators of ER stress exerted by palmitic acid. Tunicamycin, which induces ER stress produced similar effects to those obtained using palmitic acid; its effects were also reversed by α-linolenic acid. CONCLUSIONS: α-Linolenic acid may provide a useful strategy to avoid the lipotoxicity of dietary palmitic acid and nutrient overload accompanied with obesity and NAFLD.

Protective effect of a coffee preparation (Nescafe pure) against carbon tetrachloride-induced liver fibrosis in rats.

BACKGROUND & AIMS: We examined the effects of a coffee preparation on liver fibrosis induced by carbon tetrachloride (CCl(4)) and explored the possible mechanisms. METHODS: Rats were divided randomly into four groups: control, CCl(4), and two coffee preparation groups. Except for the control group, liver fibrosis was induced in male Sprague-Dawley (SD) rats by subcutaneous injection with 40% CCl(4) twice a week for 8 weeks. At the same time, a coffee preparation (300 mg/kg and 150 mg/kg) was administered to the two coffee preparation groups intragastrically once daily. RESULTS: Upon pathological examination, a coffee preparation treatment significantly reduced liver damage and symptoms of liver fibrosis. The mRNA expression of collagen I, collagen III, bcl-2, vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-beta1) were markedly increased by CCl(4) treatment but suppressed by a coffee preparation treatment. Whereas compared with the CCl(4) group, the mRNA expression of Bax was increased in the coffee preparation group. The protein expression of Bax and bcl-2 were confirmed by western blot. Intragastric administration of a coffee preparation reduced the protein expression of alpha-smooth muscle actin (alpha-SMA) and the glucose-regulated proteins (GRP) 78 and 94 in rats increased by CCl(4). CONCLUSIONS: Our data indicate that a coffee preparation can efficiently inhibit CCl(4)-induced liver fibrosis in rats. The coffee preparation may therefore be a potential functional food for preventing liver fibrosis.

[Effect of genistein on hepatic stellate cell proliferation and lipid peroxidation in vitro].

OBJECTIVE: To investigate the effect of genistein on the proliferation and lipid peroxidation of hepatic stellate cells (HSC) in vitro and its the protective effect against hepatic fibrosis. METHODS: Rat hepatic stellate cells (HSC-T6 cells) were divided into 3 groups and incubated in the presence of 0.1 mol/L hydrogen dioxide followed by washing with PBS for 3 times. Genistein at different concentrations was added into the cell culture meclia, and after 48 h of incubation, the cell proliferation was assessed with MTT assay and the levels of superoxide dismutase (SOD), malonaldehyde (MDA), glutathione (GSH) and glutathione peroxidase (GSH-PX) in the supernatant of the cell culture were measured. RESULTS: Genistein at different concentrations inhibited the cell proliferation, showing a dose-effect relationship. Genistein significantly decreased the production of intracellular MDA and GSH and increased SOD and GSH PX activity. CONCLUSION: Genistein can prevent the formation of hepatic fibrosis probably by decreasing HSC proliferation and lipid peroxidation.