Evolution of phosphoenolpyruvate carboxylase activity and lipid content during seed maturation of two spring rapeseed cultivars (Brassica napus L.)

Phosphoenolpyruvate carboxylase (PEPc: EC 4.1.1.31) activity was monitored during seed maturation of two varieties (Hybridol and Pactol) of rapeseed (Brassica napus L.), widely cultivated in Tunisia. In the Hybridol variety, PEPc activity did not exceed 5 micromol h(-1) per gram of fresh weight (FW) during the first stages of maturation. It then highly increased to reach more than 30 micromol h(-1) g(-1)/FW. On the contrary, in the Pactol variety, the evolution of PEPc activity showed a classical curve, i.e. an increase during the most active phase of lipid accumulation in maturating seeds, followed by a rapid decrease until the end of seed maturation. In both varieties, the seed oil was characterised by a high content of oleic acid (C(18:1)), linoleic (C(18:2)) and linolenic acids (C(18:3)). Saturated fatty acids were also present, although decreasing with maturation course. The analysis of the triacylglycerols (TAG) showed that trioleoylglycerol (OOO) and dioleoyllinoleoylglycerol (OOL) were the major species (ca. 35% and ca. 25% of the total respectively). The evolution pattern of fatty acids and TAG contents was similar to that of PEPc activity. Taken together, our findings suggest that PEPc may be involved in fatty acid and triacylglycerol biosynthesis during seed maturation of both rapeseed varieties.     

Toxicological Study and Oxidative Stress Evaluation for Safety Assessment of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Vip3Aa16 Toxin in Adult Mice

Vegetative insecticidal proteins were produced by some Bacillus thuringiensis strains and were successfully used in biological control against different agricultural pests such as Lepidoptera. To assess the safety of Vip3Aa16 toxins in mammalian organisms, we evaluated their toxicity using histological, hematological, and oxidative stress parameters on albino Swiss mice. The animals were orally treated with 2500, 5000, 7500 milligrams (mg) of the toxin/kilogram (kg) of body weight for 14 days. Then samples of blood, kidney and hepatic tissues were collected at the end of the treatment. Hematological parameters were monitored by RBC, WBC, hemoglobin, hematocrit, MCV, MCH, and MCHC. Liver and kidney MDA, SOD, vitamin C and H₂O₂ were analyzed to assess oxidative damage. Hepatotoxicity was monitored by analysis of the plasma enzymes ALT and AST and bilirubin levels. Renal toxicity was tested by urea, uric acid and creatinine evaluation. The histopathology of kidney and liver tissues was also investigated. The results of the toxicological study revealed that the Vip3AaA16 has no lethal effect since no mortality was observed at any dose. Moreover, body weight, hematological, histological, biochemical and oxidative findings showed no significant differences between treated and control groups. All these findings confirmed that this toxin is highly safe and doesn’t represent any risk on animal health and subsequently, Vip3Aa16 toxin can be safely used in biological programs to control Lepidopteran pests attacking crops around the world.     

A molecular approach to optimize hIFN α2b expression and secretion in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): pre–proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre–proLIP2 signal secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular secretion of small heterologous proteins in Yarrowia lipolytica.     

Evaluation of Bacillus subtilis SPB1 Lipopeptide Biosurfactant Toxicity Towards Mice

The in vivo potential toxicity of SPB1 lipopeptide biosurfactant towards male mice was evaluated. An LD50 value (defined as the dose required to kill half the members of a tested population) was determined to be about 475 mg/kg. Results show that daily administration of SPB1 biosurfactant did not show any death cases at any dose. Also, no unusual changes in behavior and no intoxication were observed during the 28 days period of treatment. Analysis proved that there were no significant differences in the serum glucose concentration levels, plasma total cholesterol, aspartate aminotransferase activity and bilirubin concentration among the control and experimental groups. In contrast, a little enhancement of alanine aminotransferase activities was observed for mice treated by the highest dose of the biosurfactant corresponding to 47.5 mg/kg of body weight which indicated the necrosis of hepatocyte. A daily intake of doses lower than 47.5 mg/kg of body weight had no significant adverse effect on hematological parameters and serum biochemical data. These results proved that SPB1 biosurfactant could be of a great interest as an additive in food, cosmetic and pharmaceuticals fields.