Transcriptomic profiling of Bursaphelenchus xylophilus reveals differentially expressed genes in response to ethanol.

Pinewood releases ethanol and other volatile compounds after Bursaphelenchus xylophilus infection. In the current study, we examined the influence of different ethanol concentrations on B. xylophilus reproduction. Low-concentrations of ethanol (8.5, 17, and 34 mM) increased egg production in B. xylophilus, whereas higher-concentrations (156 and 312 mM) reduced egg production. Transcriptome analysis was conducted to explore the molecular response of a low concentration of ethanol on the nematodes. The results suggest that the nematodes use ethanol as an energy source, which may promote survival. Ethanol induced changes in the expression of genes involved in the biosynthesis and metabolism of fatty acids and amino acids. Furthermore, ethanol promoted the expression of detoxification-related, cell wall-degrading, and reproduction-related genes. Such responses might contribute to the pathogenicity of B. xylophilus.

Transcriptomic analysis of Bursaphelenchus xylophilus treated by a potential phytonematicide, punicalagin.

Punicalagin showed significant nematotoxic activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, in the authors' previous research. The authors performed high-throughput transcriptomic sequencing of punicalagin-treated nematodes to generate clues for its nematotoxic mechanism of action. The authors identified 2,575 differentially expressed genes, 1,428 of which were up-regulated and 1,147 down-regulated. Based on a comprehensive functional in silico analysis, the authors speculate that PWN may respond to the stimulus of punicalagin through phagosome, endocytosis, peroxisome and MAPK signaling pathways. In addition, punicalagin could greatly affect PWN energy metabolism including oxidative phosphorylation. The genes encoding twitchin and a nematode cuticular collagen could be crucial regulation targets of punicalagin, which might contribute to its nematotoxic activity against PWN.Punicalagin showed significant nematotoxic activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, in the authors' previous research. The authors performed high-throughput transcriptomic sequencing of punicalagin-treated nematodes to generate clues for its nematotoxic mechanism of action. The authors identified 2,575 differentially expressed genes, 1,428 of which were up-regulated and 1,147 down-regulated. Based on a comprehensive functional in silico analysis, the authors speculate that PWN may respond to the stimulus of punicalagin through phagosome, endocytosis, peroxisome and MAPK signaling pathways. In addition, punicalagin could greatly affect PWN energy metabolism including oxidative phosphorylation. The genes encoding twitchin and a nematode cuticular collagen could be crucial regulation targets of punicalagin, which might contribute to its nematotoxic activity against PWN.

Reprogramming factors induce proliferation and inhibit apoptosis of melanoma cells by changing the expression of particular genes.

Uncontrolled proliferation and defective apoptosis are two major factors responsible for maintaining the malignant properties of melanoma cells. Our previous study demonstrated that induced expression of four reprogramming factors remodeled the phenotype of B16­F10 mouse melanoma cells into melanoma stem cells. The present study was conducted to investigate the effect of the four Yamanaka reprogramming factors, namely Oct4, Sox2, Klf4 and c­Myc (OSKM), on the proliferation and apoptosis of melanoma cells, and to identify the responsible molecular signals. The results identified that expression of the four reprogramming factors was highly induced by doxycycline treatment in the stable melanoma cell clone that was transfected with a plasmid expressing these factors, driven by the Tet­On element. It was further confirmed that induced expression of these factors enhanced the proliferation and suppressed the apoptosis of the melanoma cells. In addition, induced OSKM expression increased cell proliferation, accelerated the progression of the cell cycle, and upregulated the mRNA expression levels of Janus kinase 2 (JAK2) and Cyclin­B1. Induced expression of these factors also decreased the apoptosis, as well as upregulated B­cell lymphoma 2 (BCL­2) and downregulated BCL­2­associated X (BAX) mRNA expression levels. Taken together, the results suggested that upregulated JAK2 and Cyclin­B1 may be responsible for the enhanced proliferation of melanoma cells, and that BCL­2 upregulation and BAX downregulation may account for the suppressed apoptosis of these cells.

Nematotoxic coumarins from Angelica pubescens Maxim. f. biserrata Shan et Yuan roots and their physiological effects on Bursaphelenchus xylophilus.

The ethanol extracts from the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan was toxic against the pine wood nematode Bursaphelenchus xylophilus. The ethyl acetate-soluble fraction derived from this extract increased its potency with a mortality of 95.25% in 72 hr at 1.0 mg/mL. Four nematotoxic coumarins were obtained from the ethyl acetate extract by bioassay-guided isolation. These were identified as osthole 1, columbianadin 2, bergapten 3 and xanthotoxin 4 by mass and nuclear magnetic resonance spectral data analysis. The LC50 values against B. xylophilus in 72 hr were 489.17, 406.74, 430.08, and 435.66 µM, respectively. These compounds also altered the smooth morphology of the B. xylophilus exoskeleton to a rough and pitted appearance as visualized by electron microscopy. The coumarins 1-4 possessed significant acetylcholinesterase inhibitory activities but had negligible effects on amylase and cellulase. This research provides additional clues to the nematotoxic mechanism of coumarins against the pine wood nematode B. xylophilus. This work will assist in the development of coumarin nematicides with enhanced activity using molecular modifications of the core coumarin structure.The ethanol extracts from the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan was toxic against the pine wood nematode Bursaphelenchus xylophilus. The ethyl acetate-soluble fraction derived from this extract increased its potency with a mortality of 95.25% in 72 hr at 1.0 mg/mL. Four nematotoxic coumarins were obtained from the ethyl acetate extract by bioassay-guided isolation. These were identified as osthole 1, columbianadin 2, bergapten 3 and xanthotoxin 4 by mass and nuclear magnetic resonance spectral data analysis. The LC50 values against B. xylophilus in 72 hr were 489.17, 406.74, 430.08, and 435.66 µM, respectively. These compounds also altered the smooth morphology of the B. xylophilus exoskeleton to a rough and pitted appearance as visualized by electron microscopy. The coumarins 1-4 possessed significant acetylcholinesterase inhibitory activities but had negligible effects on amylase and cellulase. This research provides additional clues to the nematotoxic mechanism of coumarins against the pine wood nematode B. xylophilus. This work will assist in the development of coumarin nematicides with enhanced activity using molecular modifications of the core coumarin structure.

Effects of copper-phenanthroline on pentachlorophenol-induced adaptation and cell death of Escherichia coli.

OBJECTIVE: To evaluate the effects of copper-phenanthroline (CuOP) on pentachlorophenol (PCP)-induced adaptation and cell death of Escherichia coli. METHODS: Bacterial growth and adaptation to PCP were monitored spectrophotometrically at 600 nm. Inactivation of bacterial cells was determined from colony count on agar dishes. Cellular ATP content and accumulation of PCP were assessed by chemiluminescence and HPLC analysis respectively. The formation of PCP-Cu-OP complex was shown by UV-visible spectra. RESULTS: Escherichia coli (E. coli) could adapt to PCP, a wood preservative and insecticide used in agriculture. The adaptation of E. coli to PCP prevented its death to the synergistic cytotoxicity of CuOP plus PCP and declined cellular accumulation and uncoupling of oxidative phosphorylation of PCP. Furthermore, CuOP and PCP neither produced reactive oxygen species (ROS) nor had a synergistic effect on uncoupling of oxidative phosphorylation in E. coli. The synergistic cytotoxicity of CuOP and PCP in E. coli might be due to the formation of lipophilic PCP-Cu-OP complex. CONCLUSION: Our data suggested that adaptation of E. coli to PCP decreased the synergistic effects of CuOP and PCP on prokaryotic cell death due to the formation of lipophilic PCP-Cu-OP complex, but it had no effect on the uncoupling of oxidative phosphorylation and production of reactive oxygen species in E. coli.

Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube.

The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.

[Expression and characterization of FNRD in cyanobacterium Synechococcus sp. PCC 7002].

The petHL genes under the control of Lac and Kan promoters were transformed into Synechococcus sp. PCC 7002, respectively. Both of the petHL genes are integrated into the cyanobacterium chromosomes, which is inferred from the results of Southern blot analysis. Western blot analysis results show that both petHL genes are expressed in the transformed cells, and Kan promoter is more effective than Lac promoter. The FNRD in vivo shows the same stability as that of FNR holoenzyme. Some FNRD molecules are probably acylated as judged by the result of Triton X-114 phase partition test. FNRD in vivo might act as a component in photosynthetic electron transport chain, which increases the photosynthetic oxygen evolution rate.

[Purification of a big defensin from Ruditapes philippinesis and its antibacterial activity].

A novel big defensin was isolated and characterized from the plasma of Ruditapes philippinesis. It was purified to homogeneity by means of precipitation with (NH(4))(2)SO(4), gel-exclusion chromatography, two kinds of cation-exchange chromatography and named RPD-1. Its relative molecular mass was 24.8 kD by means of SDS-PAGE. By means of ABI437 amino acid sequence analyser, its 11 NH(2)-terminal amino acid sequence is AVPDVAFNAYG. Two databank systems (NCBI and EBI/EMBL) was indexed, no sequence with homology to RPD-1 was found. Furthermore, it exhibited strong inhibition on the growth of Gram-negative and -positive bacteria. Minimal inhibitory concentration (MIC) of RPD-1 against Staphylococcus aureus, Bacillus subtilis, Micrococcus tetragenus, Escherichia coli, Vibrio parahaemolyticus, Vibrio anguillarum was 9.6 mg/L, 76.8 mg/L, 38.4 mg/L, 76.8 mg/L, 19.2 mg/L, 19.2 mg/L respectively.