BACKGROUNDThe acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.

METHODSQualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.

RESULTSEYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.

CONCLUSIONSOur study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Chromatin Immunoprecipitation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; DNA Methylation ; genetics ; Epigenesis, Genetic ; genetics ; Gene Silencing ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; RUNX1 Translocation Partner 1 Protein ; Radioimmunoprecipitation Assay ; Trans-Activators ; genetics ; metabolism

To investigate the effect ofgene down-regulation on early hematopoietic development of zebrafish.Phosphorodiamidate morpholino oligomer (PMO) technology was used to downregulategene expression in Zebrafish. Zebrafish embryos injected phosphorodiamidate morpholino antisense oligonucleotide ofgene mRNA by microinjection at unicellular stage were taken as the experimental group, and those injected meaningless phosphorodiamidate morpholino antisense oligonucleotide were taken as the control. The embryos were collected at 18, 24, 30 and 36 hpf after the fertilization. The real-time fluorescent quantitative PCR (RT-PCR) and whole embryohybridization methods were used to detect the expression of myeloid hematopoietic transcription factorand erythroid hematopoietic transcription factorin zebrafish.RT-PCR showed that the expressions ofanddecreased in the experimental group compared with the control group (all<0.05). Whole embryohybridization showed that the blue-black positive hybridization signals ofandin experimental group were shallow than those in the control group.Myeloid hematopoietic and erythroid hematopoietic of zebrafish are blocked with the downregulation ofgene.
Animals ; Down-Regulation ; genetics ; Embryo, Nonmammalian ; physiopathology ; GATA1 Transcription Factor ; genetics ; metabolism ; Gene Knockdown Techniques ; Hematopoiesis ; In Situ Hybridization ; Lamin Type A ; genetics ; physiology ; Proto-Oncogene Proteins ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism ; Zebrafish ; embryology ; genetics

OBJECTIVETo explore the functions of phospholipase C (PLC)-independent protein kinase C signaling pathway (PTH/nonPLC/PKC) of parathyroid hormone (PTH) and its role in bone metabolism.

METHODSOsteoblasts isolated from the calvaria of 2- or 3-day-old C57BL mice, identified by alkaline phosphatase staining and Alizarin red staining, were treated for 4 h with 100 nmol/L [Gly(1), Arg(19)]hPTH(1-28) plus 10 nmol/L RP-cAMP, 10 nmol/L [Gly(1), Arg(19)]hPTH(1-34) plus 10 nmol/L RP-cAMP , 10 nmol/L PTH(1-34), or and 0.1% trifluoroacetic acid (TFA). The total RNA was then isolated for screening differentially expressed genes related to PTH/nonPLC/PKC pathway using Affymetrix mouse 12x135K gene expression profile microarray, and the identified genes were confirmed by real-time quantitative PCR. MC3T3-E1 cells treated with [Gly(1), Arg(19)]hPTH(1-28)+RP-cAMP, [Gly(1), Arg(19)]hPTH(1-34)+RP-cAMP, [Gly(1), Arg(19)]hPTH(1-34)+ RP-cAMP +100 nmol/L Go6983, or 0.1% TFA were also examined for GR(1-28)- or GR(1-34)-mediated gene expression changes using real-time quantitative PCR.

RESULTSAlizarin red staining visualized red mineralized nodules in the osteoblasts at 28 days of culture. According to the genechip results, we selected 56 target genes related to PTH/nonPLC/PKC pathway, among which CITED1 showed higher expressions in [Gly(1), Arg(19)]hPTH(1-34)+ RP-cAMP group than in both the control group and [Gly(1), Arg(19)]hPTH(1-28)+RP-cAMP group (P<0.05), and its expression was the highest in PTH(1-34) group (P<0.05). RT-PCR of MC3T3-E1 cells yielded consist results with those in the primary osteoblasts, and the cells treated with Go6983 (a PKC inhibitor) did not show GR(1-28)- or GR(1-34)-mediated differential expression of CITED1.

CONCLUSIONThe activation of PLC-independent protein kinase C signaling pathway of PTH enhances the expression of CITED1 in mouse osteoblasts to mediate the effect of PTH on bone metabolism, and this pathway is not dependent on the activation of PLC or PKA signaling.

Animals ; Cells, Cultured ; Indoles ; Maleimides ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins ; physiology ; Osteoblasts ; physiology ; Parathyroid Hormone ; physiology ; Protein Kinase C ; physiology ; Signal Transduction ; Skull ; Trans-Activators ; physiology ; Type C Phospholipases

Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.
4-Butyrolactone ; analogs & derivatives ; chemistry ; metabolism ; Bacterial Proteins ; metabolism ; Iron ; chemistry ; metabolism ; Pseudomonas aeruginosa ; physiology ; Quorum Sensing ; physiology ; Signal Transduction ; Trans-Activators ; metabolism ; Virulence

This study aims to investigate the genetic characteristics of BZLF1 gene and its promoter Zp of the epidemic strains in children with primary Epstein-Barr virus (EBV)-associated diseases. Total DNA was extracted from the peripheral blood of 134 children with EBV-associated infectious mononucleosis (EBV-IM) and 32 children with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) who were admitted to Beijing Children's Hospital from 2006 to 2011. The EBNA3C, BZLF1, and Zp genes were amplified by PCR assay. Typing of EBV was performed according to the size of the amplification product of EBNA3C gene; the amplification products of BZLF1 and Zp genes were subjected to direct sequencing, and sequence analysis was performed using BioEdit 7. 0. 9. The results were as follows: (1) EBV-1 was present in 140 samples (97.2%, 140/144) and EBV-II in 4 samples (2.8%, 4/144). (2) Three BZLF1 genotypes and their 12 subtypes (including 6 newly found subtypes) were detected in this study; there were no significant differences in the frequencies of BZLF1-A and BZLF1-B between the children with EBV-IM and EBV-HLH (P = 0.083); BZLF1-A1 was the dominant genotype in children with EBV-associated diseases; t BZLF1-A mostly had three 29-bp repeats in the first intron of BZLF1 gene, and BZLF1-B mostly had 30-bp repeats (P = 0.000), with the number of repeats varying from 1 to 13. (3) Four Zp genotypes were detected in this study, including Zp-P, Zp-V3, Zp-V4, and Zp-V1; there were no significant differences in the frequencies of these Zp genotypes between children with EBV-IM and EBV-HLH (P = 0.272, 0.252, 1.0, and 1.0, respectively). (4) The linkage analysis of BZLF1 gene and its promoter Zp showed that BZLF1-A1 was highly associated with Zp-V3 (P = 0.000), while BZLF1-B4 with Zp-P (P = 0.000); EBV-I + BZLF1 A1 was highly associated with Zp-V3 (P = 0.000), while EBV-I+BZLF1-B4 with Zp-P (P = 0.000). The conclusions are as follows: (1) BZLF1-A1 is the dominant genotype in children with EBV-associated diseases; there are mostly 29-bp repeats in the first intron of BZLF1 gene for BZLF1-A genotype and 30-bp repeats for BZLF1-B genotype. (2) Zp-P and Zp-V3 are dominant Zp genotypes of EBV in children, which shared similar detection rates. (3) BZLF1-A1 is highly associated with Zp-V3, while BZLF1-B4 with Zp-P; EBV-I+BZLF1-A1 is highly associated with Zp-V3, while EBV-I+BZLF1-B4 with Zp-P.
Child ; Child, Preschool ; China ; epidemiology ; Epstein-Barr Virus Infections ; epidemiology ; virology ; Female ; Genotype ; Herpesvirus 4, Human ; genetics ; physiology ; Humans ; Infant ; Infant, Newborn ; Introns ; genetics ; Male ; Promoter Regions, Genetic ; genetics ; Repetitive Sequences, Nucleic Acid ; genetics ; Trans-Activators ; genetics

Bel1, a transactivator of prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have shown that Bel1 bears a nuclear localization signal (NLS), but its amino acid sequence remains unclear and the corresponding importins have not been identified. In this report, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment could direct nuclear localization, which accords with the consensus sequence K(K/R)X(K/R) of monopartite NLS. Point mutation experiments revealed that K218, R219, and R221 are essential for the nuclear localization of Bel1. The results of the GST-pulldown showed that the Bel1 fragment with residues 215-223, which bears the NLS, interacts with KPNA1, KPNA6, and KPNA7. This result suggests that KPNA1, KPNA6, and KPNA7 maybe involved in Bel1 nuclear translocation.
Cell Line ; Cell Nucleus ; genetics ; metabolism ; virology ; Humans ; Nuclear Localization Signals ; genetics ; metabolism ; Protein Binding ; Protein Transport ; Retroviridae Infections ; genetics ; metabolism ; virology ; Retroviridae Proteins ; chemistry ; genetics ; metabolism ; Spumavirus ; chemistry ; genetics ; physiology ; Trans-Activators ; chemistry ; genetics ; metabolism ; alpha Karyopherins ; genetics ; metabolism

In order to study function of NAC transcription in development, hormone regulation and the stress response of Salvia miltiorrhiza, the NAC transcription was cloned and analyzed. By retrieving cDNA database of S. miltiorrhiza hairy root one NAC unigene was found, then a full length of cDNA was cloned by designing specific primers and PCR amplifying. Using ORF finder it was found that the cDNA containing a NAC-AB conserved domain in N-terminal, so the cDNA was a NAC transcription factor, named as SmNAC1 (kF006346). Bioinformatics analysis showed that SmNAC1 had an open reading frame (ORF) of 591 bp encoding 196 amino acids. The calculated protein had isoelectric point (pI) of 4.36 with molecular weight about 21.66 kDa. The transcription level of SmNAC1 after dealing with yeast extract (YE) and silver ion (Ag+) in S. miltiorrhiza hairy root was markedly stimulated up regulating. It was 1.4 fold compared with the control after induction 2 h, and maintained 2.0 fold on 4-12 h after induction. SmNAC1 may participate in regulation of stress response of YE + Ag+.
Cloning, Molecular ; Computational Biology ; Phylogeny ; Plant Proteins ; genetics ; physiology ; Plant Roots ; chemistry ; Salvia miltiorrhiza ; chemistry ; genetics ; Trans-Activators ; genetics ; physiology

OBJECTIVETo investigate the roles of the hepatitis B virus (HBV)-encoded X protein (HBx), including the full-length and truncated isoforms, and in conjunction with the host-encoded damaged DNA binding protein 1 (DDB1) in HBV replication.

METHODSRecominant expression plasmids carrying the wild-type HBV genome (pGEM-HBV1.2) or with deletion of the full-length HBx protein (pHBV-deltaX), or carrying the full-length HBx protein (pSI-X) or the HBx1to101 (pSI-X1to101) or HBx43to154 (pSI-X43to154) isoforms were constructed for transfection into HepG2 cells. The pcDNA6.2-GW/EmGFP-miR (DDB1-miRNA) vector was constructed for silencing of the DDB1 gene in co-transfected HepG2 cells. At 72 h after transfections, DDB1 silencing was confirmed by western blot analysis and real-time quantitive reverse transcription PCR, HBV DNA copies number was assessed by real time PCR, and levels of hepatitis B surface antigen (HbsAg) and hepatitis B e antigen (HbeAg) were determined by ELISA. Differences between groups was statistically analyzed by single-factor analysis of variance and the t-test.

RESULTSTransfection with pHBV-deltaX led to reductions in DDB1 mRNA (to 52.74% of that in the wild-type pGEM-HBV1.2 transfected cells), HBV replication (to 55.49%), HBsAg level (48.05%), and HBeAg level (46.22%). Co-transfection with pSI-X or pSI-X43to154, but not with pSI-X1to101, restored the pHBV-deltaX-induced reductions in DDB1 mRNA, HBV replication, HBsAg and HBeAg to wild-type levels. The quantity of DDB1 mRNA was approximately parallel with the quantity of HBV DNA copies in all the HepG2 transfection groups.

CONCLUSIONThe COOH-terminal amino acids of HBx are required for HBV replication in hepatocytes, possibly involving the host-encoded DDB1 protein.

DNA-Binding Proteins ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Host-Pathogen Interactions ; Humans ; Protein Isoforms ; metabolism ; Trans-Activators ; metabolism ; Transfection ; Virus Replication

Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.
Actin Cytoskeleton ; physiology ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Cell Proliferation ; Drosophila Proteins ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism

OBJECTIVETo investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro.

METHODSThe hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS(1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups.

CONCLUSIONhAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.

Amnion ; cytology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; Flow Cytometry ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; RNA, Messenger ; genetics ; Trans-Activators ; metabolism ; beta 2-Microglobulin ; metabolism