Bacillus thuringiensis cry toxin triggers autophagy activity that may enhance cell death.

Although it is well known that Bacillus thuringiensis Cry toxins kill insect pest by disrupting midgut cells of susceptible larvae through their pore formation activity, it is not clear what intracellular events are triggered after pore formation on the cell membrane of the target cells. Here we analyzed the role of Cry toxins on autophagy activation using several cell lines as models as well as in Helicoverpa armigera larvae. The selected insect cell lines (Hi5, Sl-HP and Sf9) were susceptible to activated Cry1Ca toxin, but only Sl-HP cells were also susceptible to activated Cry1Ac toxin. In contrast, the mammalian cell line 293 T was not susceptible to Cry1Ac or to Cry1Ca. Results show that Cry toxins induced autophagy only in the susceptible cell lines as shown by the analysis of the changes in the ratio of Atg8-PE to Atg8 and by formation of autophagosome dots containing Atg8-PE. The Cry1Ac enhanced autophagy in the midgut tissue of H. armigera larvae. Silencing expression of specific genes by RNAi assays confirmed that the autophagy induced by activated Cry toxins was dependent on AMPK and JNK pathways. Finally, inhibition of autophagy in the cell lines by specific inhibitors or RNAi assays resulted in delayed cell death triggered by Cry toxins, suggesting that the increased autophagy activity observed after toxin intoxication may contribute to cell death.

FOXA transcriptional factor modulates insect susceptibility to Bacillus thuringiensis Cry1Ac toxin by regulating the expression of toxin-receptor ABCC2 and ABCC3 genes.

Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase, aminopeptidase or cadherin. Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae.

[Expression and characterization of Coprinus cinereus peroxidase].

OBJECTIVE: The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently. METHODS: We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115. RESULTS: We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol. CONCLUSION: The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.

Exhaust and evaporative emissions from motorcycles fueled with ethanol gasoline blends.

The emission characteristics of motorcycles using gasoline and E10 (90% gasoline and 10% ethanol by volume) were investigated in this article. Exhaust and evaporative emissions of three motorcycles were investigated on the chassis dynamometer over the Urban Driving Cycle (UDC) and in the Sealed Housing for Evaporative Determination (SHED) including regulated and unregulated emissions. The regulated emissions were detected by an exhaust gas analyzer directly. The unregulated emissions including carbonyls and volatile organic compounds (VOCs) were sampled through battery-operated air pumps using tubes coated with 2,4-dinitrophenylhydrazine (DNPH) and Tenax TA, respectively. The experimental results showed that the emission factors of total hydrocarbons (THC) and carbon monoxide (CO) from E10 fueling motorcycles decreased by 26%-45% and 63%-73%, while the emission factor of NOx increased by 36%-54% compared with those from gasoline fueling motorcycles. For unregulated emissions, the emission amount of VOCs from motorcycles fueled with E10 decreased by 18%-31% while total carbonyls were 2.6-4.5 times higher than those for gasoline. For evaporative emissions of THC and VOCs, for gasoline or E10, the diurnal breathing loss (DBL) was higher than hot soak loss (HSL). Using E10 as a fuel does not make much difference in the amount of evaporative THC, while resulted in a slightly growth of 14%-17% for evaporative BETX (benzene, toluene, ethylbenzene, xylene).

Electroacupuncture pretreatment prevents cognitive impairment induced by limb ischemia-reperfusion via inhibition of microglial activation and attenuation of oxidative stress in rats.

Limb ischemia-reperfusion (LI/R) is associated with high morbidity and mortality. Furthermore, critical trauma survivors can present cognitive impairment. Cognitive function, survival rate, oxidative stress and neuronal health were examined to elucidate (1) the magnitude of cognitive effects of prolonged reperfusion, (2) potential players in the mechanistic pathway mediating such effects, and (3) possible benefits of electroacupuncture (EA) pretreatment at Baihui (GV20), Yanglingquan (GB34), Taichong (LR3), Zusanli (ST36) and Xuehai (SP10) acupoints. LI/R was induced in rats by placing a rubber tourniquet on each hind limb for 3h, and the animals were evaluated periodically for 7d after LI/R. Rats subjected to LI/R had significantly lower survival rates, and displayed evidence of brain injury and cognitive dysfunction (as determined by the Morris water maze test) 1d and 3d after reperfusion compared to sham-operated controls. LI/R also resulted in higher levels of reactive oxygen species (ROS) and malondialdehyde (MDA), microglial activation, and decreased superoxide dismutase (SOD) activity within Cornu Ammonis area 1 (CA1) of the hippocampus. Depressed survival rates, microglial activation, oxidative damage, and histological changes, as well as cognitive dysfunction were partially or fully attenuated in rats that received 14d of EA prior to LI/R. These findings indicate that LI/R can result in cognitive dysfunction related to activated microglia and elevated oxidative stress, and that EA has neuroprotective potential mediated, at least in part, by inhibition of microglial activation and attenuation of oxidative stress.