Purification and biochemical characterization of a novel transglutaminase from Mythimna separata larvae (Noctuidae, Lepidoptera).

A novel transglutaminase (MsTGase) from Mythimna separata larvae was separated and purified; its biochemical property and enzymatic catalytic activities were investigated. MsTGase was obtained chromatographically by the precipitation of Sephadex G-100 gel and DEAE-Cellulose-52 ion-exchange column with 48-fold purification and a reproducible yield of approximately 12%. Molecular weight of the MsTGase was 63.5 KDa and its N-terminal amino acid sequence was GKIEEG-LVI. Michaelis constant of the MsTGase for the substrate N-CBZ-Gln-Gly was 12.83mM with a Vmax of 7.99U/mL. Optimum conditions for MsTGase activity were at 42°C and pH7.5. The enzyme didn't possess metal ion at its catalytic active site; its activity could be significantly inhibited by Mg2+, but activated by Ca2+. Chlorpyrifos and spinosad showed a strong potential to increase MsTGase activity, supporting the view that MsTGase was a novel target. Moreover, the formation of intermolecular cross-links of casein and bovine serum albumin polymerized by MsTGase in the presence of DTT was observed. These findings pave the way for future studies on the physiological role of MsTGase and the potential impact of its regulation on MsTGase-associated pest management.

Emamectin benzoate induces ROS-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cells.

Emamectin benzoate (EMB), a novel macrocyclic lactone insecticide, possesses high efficacy and beneficial selective toxicity in agriculture, but so far the EMB-induced cytotoxic action in arthropod insect remains unclear. The present studies were carried out to characterize the property of EMB on the induction of reactive oxygen species (ROS)-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cell model. Following the exposure to EMB at 2.5, 5, 10 or 15 µM, the cells changed to be round, suspended and aggregated, and the decline of cell proliferating ability and cell viability was positively related with the exposure time. Median inhibitory concentration (IC50) of EMB on cell viability was 3.72 µM during 72 h exposure. Apoptosis was induced in 29.8% (24 h) and 39.5% (48 h) of the cells by EMB at 15 µM, showing chromatin condensation in nuclei. The content of ROS in the cells increased rapidly as the concentration of EMB increased, and the pre-incubation of the cells with vitamin E significantly reduced the ROS accumulation. In the treatment of 15 µM EMB, the migrated cell nucleus with DNA strand breaks appeared a teardrop, pear-shaped, or large fan-like tail, and 63.1% of γH2AX-positive cells contained more than four foci, accompanying with high expression level of caspase-3 in time-dependent manner, which consequently led to cell apoptotic death. These evidences in ROS-mediated DNA damage and cell apoptosis induced by EMB may be helpful for deep understanding the cytotoxic action of EMB based on cell model.

Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10µM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10µM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca2+]i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia.

Antioxidative cellular response of lepidopteran ovarian cells to photoactivated alpha-terthienyl.

Photodynamic sensitizers as useful alternative agents have been used for population control against insect pests, and the response of insect ovarian cells towards the photosensitizers is gaining attention because of the next reproduction. In this paper, antioxidative responses of lepidopteran ovarian Tn5B1-4 and Sf-21 cells to photoactivated alpha-terthienyl (PAT) are investigated. PAT shows positive inhibitory cytotoxicity on the two ovarian cells, and its inhibition on cell viability is enhanced as the concentrations are increased and the irradiation time is extended. Median inhibitory concentrations (IC50) are 3.36µg/ml to Tn5B1-4 cells, and 3.15µg/ml to Sf-21 cells at 15min-UV-A irradiation 2h-dark incubation. Under 10.0µg/ml PAT exposure, 15min-UV-A irradiation excites higher ROS production than 5min-UV-A irradiation does in the ovarian cells, the maximum ROS content is about 7.1 times in Tn5B1-4 cells and 4.3 times in Sf-21 cells, and the maximum malondialdehyde levels in Tn5B1-4 and Sf-21 cells are about 1.47- and 1.36-fold higher than the control groups, respectively. Oxidative stress generated by PAT strongly decreases the activities of POD, SOD and CAT, and induces an accumulation of Tn5B1-4 cells in S phase and Sf-21 cells in G2/M phase in a concentration-dependent fashion. Apoptosis accumulation of Tn5B1-4 cells and the persistent post-irradiation cytotoxicity are further observed, indicating different antioxidative tolerance and arrest pattern of the two ovarian cells towards the cytotoxicity of PAT.

A comparative assessment of cytotoxicity of commonly used agricultural insecticides to human and insect cells.

The cytotoxic potential of 13 commonly used agricultural insecticides was examined using cell-based systems with three human HepG2, Hek293, HeLa cells and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells. Data showed that (1) an enhancement of some insecticides (e.g. pyrethroids) on cells proliferation; (2) an inhibition of some insecticides on cells viability; (3) various levels of susceptibility of different cells to the same insecticide; and (4) the cell type dependent sensitivity to different insecticides. The degree of cytotoxicity of insecticides on human cells was significantly lower than that on insect cells (P<0.05). Methomyl, even 20µg/ml, showed little cytotoxicity at 24h exposure whereas emamectin benzoate possessed the strongest cytotoxic potential in a dose-dependent fashion. The results revealed comparable cytotoxic property of agricultural insecticides against intact cells.