Secreted GRP78 activates EGFR-SRC-STAT3 signaling and confers the resistance to sorafeinib in HCC cells.

Acquired resistance is a common phenomenon for HCC patients who undergone sorafenib treatment, however the mechanism by which acquired resistance develops remains elusive. In this study, we found that GRP78 could be detected in the serum samples of HCC patients and the conditional medium of multiple HCC cell lines, suggesting that GRP78 is secreted by HCC cells. Further studies showed that secreted GRP78 facilitated the proliferation and inhibited the apoptosis induced by sorafenib both in HCC cell lines and in tumor xenografts. We further found that secreted GRP78 could interact physically with EGFR, therefore activates EGFR signaling pathway. knockdown of EGFR decreased secreted GRP78 induced phosphorylation of SRC and STAT3. By contrast, overexpression of EGFR further enhanced the phosphorylation of SRC and STAT3 induced by secreted GRP78, suggesting the critical role of EGFR in secreted GRP78 conferred resistance to sorafeinib. Moreover, inhibition of SRC by PP2 antagonized the resistance to sorafenib and inhibited the activation of STAT3 conferred by secreted GRP78. Taken together, our results showed that secreted GRP78 could interact with EGFR, activate EGFR-SRC-STAT3 signaling, conferring the resistance to sorafenib.

The expression of phenylalanine hydroxylase in the brain of ragworm Neanthes japonica (Polychaeta, Annelida) / 生物工程学报

Phenylalanine hydroxylase (PAH) is a member of aromatic amino acid hydroxylase (AAAHs) family, and catalyze phenylalanine (Phe) into tyrosine (Tyr). Using immunological and RT-PCR methods to prove the existence of phenylalanine hydroxylase (PAH) gene in the brain of Neanthes japonica in protein and nucleic acid level. Using Western blotting to detect the pah immunogenicity of Neanthes japonica. Making paraffin sections and using immunohistochemical technique to identify the presence and distribution of the phenylalanine hydroxylase gene in the brain of Neanthes japonica. Clone pah gene from the brain of Neanthes japonica by RT-PCR, constructing plasmid and transferring into E. coli to amplification, picking a single homogeneous colony, double digesting then making sequence and comparing homology. Western blotting results showed that the expression of the protein is present in Neanthes japonica brain, immunohistochemistry technique results showed that phenylalanine hydroxylase mainly expressed in abdominal of forebrain, dorsal and sides of midbrain. RT-PCR technique results showed that the phenylalanine hydroxylase exist in the brain of Neanthes japonica and has a high homology with others animals. PAH is present in the lower organisms Neanthes japonica, in protein and nucleic acid level. Which provide the foundation for further study the evolution of aromatic amino acid hydroxylase genes in invertebrate.

Screening of candidate molecules interacting with protein kinase Wee1B from human ovary cDNA library and its regulation effect / 吉林大学学报(医学版)

Objective:To screen the new candidate molecules interacting with protein kinase Wee1B by yeast two hybrid system, and to analyze their interaction with Wee1B in the early stage of mouse fertilized eggs by bioinformatics.Methods:The plasmid pcDNA3.1/V5-His-TOPO-Wee1B wild type encoding mouse Wee1B gene was used as template to construct bait plasmid pGBKT7 Wee1B and the bait plasmid pGBKT7-Wee1B was transformed into yeast competent cells at SD/Trp (SDO),SD/Trp/X-α-Gal (SDO/X)and SD/Trp/X α Gal/AbA plates (SDO/X/A)plates to detect the toxicity and self-activation ability of yeast and its expression in yeast using Western blotting method.The yeast cells containing pGBKT7-Wee1B were fused with human ovary cDNA library, the yeast plasmid transformation of Escherichia coli positive clones were sequenced after identified by yeast transformation.BLAST analysis was carried out in GenBank,and its effect on the development of mouse fertilized eggs was deduced according to the gene annotation.Results:The double enzyme digestion analysis and sequencing analysis results showed that the pGBKT7-Wee1B bait plasmid was successfully constructed.The plasmid was transformed into the yeast,and there were no clones in the SDO/X/A plates.The pGBKT7-Wee1B and pGBKT7 empty vectors were transformed into the yeast,the bacteria were inoculated on the SDO plates,and the clones were uniformly grown on the two SDO plates.The positive clones were picked and expanded in culture,the protein was extracted and Western blotting showed that pGBKT7 Wee1B was expressed in the yeast.The bait plasmids were fused with human ovary cDNA library and the positive clones inserted into the fragment were identified by PCR. Nine proteins which interacted with Wee1B protein kinase were screened out by sequencing and blast analysis,and the proteins which could be closely related to the development of mouse oocytes and the development of fertilized eggs were analyzed by bioinformatics analysis.Conclusion:Using the yeast two hybrid system from human ovary cDNA library,nine interacting proteins with Wee1B protein kinase are screened and these screened proteins may regulate mouse oocyte maturation and early embryo development through interacting with Wee1B.

Genetic characterization of VP1 gene of seven Sacbrood virus isolated from three provinces in northern China during the years 2008-2012.

The genetic diversity of seven northern China isolated Sacbrood virus strains (SBV) has been analyzed, and hypervariable regions of the VP1 gene of 7 SBV were sequenced and characterized, in order to obtain epidemiological and immunological information, and to suggest typing criteria for SBV. Sequence analysis of hypervariable regions of the VP1 gene in the genome of these isolates revealed a sequence homology of 91.0-99.3% among all seven local SBV isolates from Apis cerana from China, with a similarity of 93.3-100.0% in deduced amino acid sequences. These local isolates shared 87.4-92.8% sequence homology with six SBV reference strains in GenBank (including two SBV reference strains from Apis cerana from China), which represents a 91.8-97.6% similarity in deduced amino acid sequences. Genetic analysis also showed that five SBV strains from Apis cerana from China had a 13-amino-acid deletion at amino acid positions 287-299, and two SBV strains infecting the Korean honeybee had a 17-amino-acid deletion at amino acid positions 284-300 in comparison with other SBV. Phylogenetic analysis revealed two major groups (AC genotype SBV infecting Apis cerana and AM genotype SBV infecting Apis mellifera). The AC genotype could be further divided into subgroups. Based on the results of phylogenetic analysis, a similarity scan of SBV nucleotide sequences was carried out by using Simplot software and results in similar results. Our results suggest possible typing criteria for SBV based on the phylogenetic tree and sequence homology, and also that the virus has host specificity and regional variations.