AIM: To determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G2/M arrest in human gastric cancer MGC803 cells.METHODS: MGC803 cell growth inhibition was measured by MTT...AIM: To determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G2/M arrest in human gastric cancer MGC803 cells.METHODS: MGC803 cell growth inhibition was measured by MTT assay. Phase distribution of cell cyde was analyzed by flow cytometry. Expression of Cdc25C, p38, phosphorylation of p38 (pp38) were determined by Western blotting.RESULTS: MT1- assay showed that SB203580, a specific p38 MAPK inhibitor blocked DADS-induced growth inhibition.Flow cytometry analysis revealed that treatment of MGC803 cells with 30 mg/L DADS increased the percentage of cells in the G2/M phase from 9.3% to 39.4% (P<0.05), whereas inhibition of p38 activity by SB203580 abolished induction of G2/M arrest by DADS. Western blotting showed that phosphorylation of p38 was increased 3.52-fold following treatment of MGC803 cells with 30 mg/L DADS for 20 min(P<0.05), whereas Cdc25C was decreased 68% following treatment of MGCS03 cells with 30 mg/L DADS for 24 h(P<0.05). Decreased Cdc25C protein expression by DADS was attenuated by SB203580 (P<0.05).CONCLUSION: DADS-induced G2/M arrest of MGC803 cells involves activation of p38 MAP kinase pathways.Decreased Cdc25C protein expression by p38 MAPK played a crucial role in G2/M arrest after treatment with DADS.展开更多
OBJECTIVE To analyze the whole transcriptome of zoanthid Protopalythoa variabilis(P.variabilis),a cnidarian,and discover the potential toxic substances in P.variabilis.METHODS The P.variabilis RNA deep sequencing was ...OBJECTIVE To analyze the whole transcriptome of zoanthid Protopalythoa variabilis(P.variabilis),a cnidarian,and discover the potential toxic substances in P.variabilis.METHODS The P.variabilis RNA deep sequencing was performed using the HiSeq 2500 automatic sequencing platform.All the unigenes generated from the assembly process were functionally annotated based on the similarity with databases.The multiple alignments of translated toxin-related sequences were performed with Clustalw2,and amino acid identity and similarity highlighted by using BoxShade tool.Three different methods including ITASSER,PEP-FOLD and MODELLER were applied to predict tri-dimensional models of toxin-related polypeptides from translated transcript sequences of P.variabilis.The toxicity of one of the putative toxins,namely ShK/Aurelin-like peptide,was evaluated using zebrafish model.RESULTS A total of 67,549,914 pairs of quality-filtered,90-base-pair Illumina reads from an mRNA sample were obtained.The de novo assemblies yielded 276,526 contigs.The sequence comparison of 130,121 unigenes with entries in Toxin database showed that 1542 unigenes were potential peptide toxins at which 11 unigenes were related to Stichodactyla toxin(ShK)domain(Pfam ID:PF01549).ShK is a 35 residues peptide sequence that was firstly discovered from the sea anemone Stichodactyla helianthus.Here,we found out one ShK-like peptide that processed a relatively higher sequence similarity with known ShK(Uniprot ID:P29186)of Bunodosoma granuliferum(red warty sea anemone).The Protopalythoa Shk-like peptide was submitted to Probis server to detect probable binding site and found to match with a protein AURELIN(PDB id:2lg4,UniProt id:Q0MWV8)which possesses structural homology with previously identified antimicrobial peptides and K+-channel-blocking toxins.Our results showed that the ShK/Aurelin-like peptide was lethal to zebrafish embryos at concentrations above 30-μmol·L1,and could induce zebrafish locomotor deficit at 10μmol·L-1.CONCLUSION This study,for the first time,presented the whole transcriptome profile and a potential toxic peptide of P.variabilis.展开更多
基金Supported by the Natural Science Foundation of Hunan Province,No.02JJY2026,01JJY2146 and the Foundation of Hunan ProvinceEducation Department,No.01A016
文摘AIM: To determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G2/M arrest in human gastric cancer MGC803 cells.METHODS: MGC803 cell growth inhibition was measured by MTT assay. Phase distribution of cell cyde was analyzed by flow cytometry. Expression of Cdc25C, p38, phosphorylation of p38 (pp38) were determined by Western blotting.RESULTS: MT1- assay showed that SB203580, a specific p38 MAPK inhibitor blocked DADS-induced growth inhibition.Flow cytometry analysis revealed that treatment of MGC803 cells with 30 mg/L DADS increased the percentage of cells in the G2/M phase from 9.3% to 39.4% (P<0.05), whereas inhibition of p38 activity by SB203580 abolished induction of G2/M arrest by DADS. Western blotting showed that phosphorylation of p38 was increased 3.52-fold following treatment of MGC803 cells with 30 mg/L DADS for 20 min(P<0.05), whereas Cdc25C was decreased 68% following treatment of MGCS03 cells with 30 mg/L DADS for 24 h(P<0.05). Decreased Cdc25C protein expression by DADS was attenuated by SB203580 (P<0.05).CONCLUSION: DADS-induced G2/M arrest of MGC803 cells involves activation of p38 MAP kinase pathways.Decreased Cdc25C protein expression by p38 MAPK played a crucial role in G2/M arrest after treatment with DADS.
基金The project supported by grants from the Science and Technology Development Fund of Macao,China(058/2009and 078/2011/A3)Research Committee,University of Macao〔MYRG138(Y1-Y4)-ICMS12-LMY and MYRG139(Y1-Y4)-ICMS-LMY〕
文摘OBJECTIVE To analyze the whole transcriptome of zoanthid Protopalythoa variabilis(P.variabilis),a cnidarian,and discover the potential toxic substances in P.variabilis.METHODS The P.variabilis RNA deep sequencing was performed using the HiSeq 2500 automatic sequencing platform.All the unigenes generated from the assembly process were functionally annotated based on the similarity with databases.The multiple alignments of translated toxin-related sequences were performed with Clustalw2,and amino acid identity and similarity highlighted by using BoxShade tool.Three different methods including ITASSER,PEP-FOLD and MODELLER were applied to predict tri-dimensional models of toxin-related polypeptides from translated transcript sequences of P.variabilis.The toxicity of one of the putative toxins,namely ShK/Aurelin-like peptide,was evaluated using zebrafish model.RESULTS A total of 67,549,914 pairs of quality-filtered,90-base-pair Illumina reads from an mRNA sample were obtained.The de novo assemblies yielded 276,526 contigs.The sequence comparison of 130,121 unigenes with entries in Toxin database showed that 1542 unigenes were potential peptide toxins at which 11 unigenes were related to Stichodactyla toxin(ShK)domain(Pfam ID:PF01549).ShK is a 35 residues peptide sequence that was firstly discovered from the sea anemone Stichodactyla helianthus.Here,we found out one ShK-like peptide that processed a relatively higher sequence similarity with known ShK(Uniprot ID:P29186)of Bunodosoma granuliferum(red warty sea anemone).The Protopalythoa Shk-like peptide was submitted to Probis server to detect probable binding site and found to match with a protein AURELIN(PDB id:2lg4,UniProt id:Q0MWV8)which possesses structural homology with previously identified antimicrobial peptides and K+-channel-blocking toxins.Our results showed that the ShK/Aurelin-like peptide was lethal to zebrafish embryos at concentrations above 30-μmol·L1,and could induce zebrafish locomotor deficit at 10μmol·L-1.CONCLUSION This study,for the first time,presented the whole transcriptome profile and a potential toxic peptide of P.variabilis.
