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1.  Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells 
Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.
doi:10.1590/1414-431X20132936
PMCID: PMC3854426  PMID: 24036910
Macrophage migration inhibitory factor; HL-1 cells; Hydrogen peroxide; Atrial fibrillation; Protein kinases
2.  Critical evaluation of transcription factor Atf2 as a candidate modulator of alcohol preference in mouse and human populations 
In prior work, congenic strains carrying the DBA/2Igb (D2) region of chromosome 2 (Chr2) for alcohol preference were bred onto a C57BL/6Ibg (B6) background and as predicted were found to reduce voluntary consumption. Subsequently, interval-specific congenic recombinant strains (ISCRS) were generated and also tested. These ISCRS strains reduced the quantitative trait loci (QTL) interval to a comparatively small 3.4 Mb region. Here, we have exploited an integrative approach using both murine and human populations to critically evaluate candidate genes within this region. First, we used bioinformatics tools to search for genes relevant to alcohol preference within the QTL region. Second, we searched for single nucleotide polymorphisms (SNPs) within exons of every gene in this region. Third, we conducted follow-up microarray analyses to identify differentially expressed genes between the B6 and ISCRS strains in mice from each group. Fourth, we analyzed correlations between the expression level of candidate genes and phenotypes of alcohol preference in a large family of BXD recombinant inbred strains derived from B6 and D2. Finally, we evaluated SNP segregation in both BXD mouse strains and in humans who were heavy alcohol drinkers or non-drinkers. Among several potential candidate genes in this region, we identified activating transcription factor 2 (Atf2) as the most plausible gene that would influence alcohol preference. However, the candidacy of Atf2 was only weakly supported when we used a genetic network approach and by focused reanalysis of genome-wide association study data from European-American and African-American populations. Thus, we cannot conclude that Atf2 plays a role in the regulation of the QTL of mouse Chr2.
doi:10.4238/2013.November.26.9
PMCID: PMC4108070  PMID: 24338393
Alcohol preference; Atf2; Candidate; QTL; Gene expression
3.  CD123 targeting oncolytic adenoviruses suppress acute myeloid leukemia cell proliferation in vitro and in vivo 
Li, G | Li, X | Wu, H | Yang, X | Zhang, Y | Chen, L | Wu, X | Cui, L | Wu, L | Luo, J | Liu, X Y
Blood Cancer Journal  2014;4(3):e194-.
We report here a novel strategy to redirect oncolytic adenoviruses to CD123 by carry a soluble coxsackie-adenovirus receptor (sCAR)-IL3 expression cassette in the viral genome to form Ad.IL3, which sustainably infected acute myeloid leukemia (AML) cells through CD123. Ad.IL3 was further engineered to harbor gene encoding manganese superoxide dismutase (MnSOD) or mannose-binding plant lectin Pinellia pedatisecta agglutinin (PPA), forming Ad.IL3-MnSOD and Ad.IL3-PPA. As compared with Ad.IL3 or Ad.sp-E1A control, Ad.IL3-MnSOD and Ad.IL3-PPA significantly suppressed in vitro proliferation of HL60 and KG-1 cells. Elevated apoptosis was detected in HL60 and KG-1 cells treated with either Ad.IL3-MnSOD or Ad.IL3-PPA. The caspase-9–caspase-7 pathway was determined to be activated by Ad.IL3-MnSOD as well as by Ad.IL3-PPA in HL60 cells. In an HL60/Luc xenograft nonobese diabetic/severe-combined immunodeficiency mice model, Ad.IL3-MnSOD and Ad.IL3-PPA suppressed cancer cell growth as compared with Ad.IL3. A significant difference of cancer cell burden was detected between Ad.IL3 and Ad.IL3-PPA groups at day 9 after treatment. Furthermore, Ad.IL3-MnSOD significantly prolonged mouse survival as compared with Ad.sp-E1A. These findings demonstrated that Ad.IL3-gene could serve as a novel agent for AML therapy. Harboring sCAR-ligand expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting serotype 5 adenovirus infection.
doi:10.1038/bcj.2014.15
PMCID: PMC3972701  PMID: 24658372
4.  Pinellia pedatisecta agglutinin interacts with the methylosome and induces cancer cell death 
Lu, Q | Li, N | Luo, J | Yu, M | Huang, Y | Wu, X | Wu, H | Liu, X Y | Li, G
Oncogenesis  2012;1(10):e29-.
Pinellia pedatisecta agglutinin (PPA) is a specific mannose-binding plant lectin accumulated in the tuber of P. pedatisecta. In the work presented, the cytotoxicity of PPA to cancer cells was investigated through exogenous expression. A PPA gene was transduced into normal and cancer cell lines through plasmid vectors, and the effect of PPA expression was examined. Results showed that PPA translocated into the nucleus, colocalized with DNA and induced cell death. A mannose-binding motif and a V103-W130 region directed the nuclear translocation of PPA. Coprecipitation, mass spectrometry and western blotting analysis further indentified that PPA was associated with the methylosome, which contains methylosome protein 50 and protein arginine methyltransferase 5 (PRMT5). Knockdown of PRMT5 significantly inhibited the PPA-induced cell death, suggesting that PPA used the methylosome as a target. Furthermore, Ad.surp-PPA, an adenovirus vector in which the PPA gene was controlled by a survivin promoter (surp), selectively inhibited the proliferation of cancer cell lines. Taken together, the expression of PPA gene elicited significant cytotoxicity to cancer cells through targeting the methylosome and might be developed into a novel agent in cancer gene therapy.
doi:10.1038/oncsis.2012.30
PMCID: PMC3503292  PMID: 23552401
methylosome; Pinellia pedatisecta agglutinin; MEP50; PRMT5; nuclear translocation
5.  Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine. 
We have used the endogenous reverse transcriptase reaction of viral core particles from duck liver to elucidate the mechanism of inhibition of duck hepatitis B virus (DHBV) replication by the nucleoside analog (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC). As is the case in human immunodeficiency virus replication, 3TC-5'-triphosphate (3TC-TP) acts as a chain terminator for the DNA polymerase activities. The results of several different experiments support this conclusion, which explains the potent activity of 3TC against the hepadnaviruses. In isolated DHBV core particles, 3TC-TP inhibited the reverse transcriptase in a manner that resembled competitive inhibition with respect to dCTP. However, the kinetics of inhibition was not linear on a double-reciprocal plot for the highest concentrations of 3TC-TP and the lowest concentration of dCTP. This anomaly would be expected if binding to the nucleotide site was followed by DNA chain termination. Calculations that used only the linear part of the curve yielded a Ki of 0.78 +/- 0.10 microM 3TC-TP. The inhibition of core particles incubated in vitro with 3TC-TP was not reversed by removal of the free inhibitor. 3TC-TP inactivated the reverse transcriptase activity in a concentration-dependent manner. The Km of the chain termination reaction was calculated at 0.71 +/- 0.05 microM. Similar competitive kinetics and irreversible inhibition were also obtained on the endogenous DNA polymerase from viral particles from serum, suggesting that 3TC-TP also acts as a chain terminator of the DNA-directed DNA polymerase of DHBV replication.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC162757  PMID: 7492080
6.  Population and family studies of three disease-related polymorphic genes in systemic lupus erythematosus. 
Journal of Clinical Investigation  1995;95(4):1766-1772.
The contribution to systemic lupus erythematosus (SLE) of three lupus-associated polymorphisms (involving the C4A2 complement component, Humhv3005 and the T cell antigen receptor alpha chain gene) are investigated in 81 individuals from 14 multiplex SLE families, 41 unrelated lupus patients, and 88 unrelated healthy controls. The results show a strong association between C4A deletion and SLE in these families. While the current study confirms the previously reported association between hv3005 deletion and sporadic SLE, the study fails to support this association in familial SLE patients. Moreover, no correlation is detected between the occurrence of hv3005 deletion and C4A null alleles in lupus patients, suggesting that the effects of these genetic polymorphisms on predisposition to lupus are independent. The previously reported lupus-associated T cell receptor (TCR) alpha chain polymorphism is not detected in any of the individuals studied here. The combined data suggest that C4A null alleles predispose strongly to development of lupus, whereas the influence of hv3005 deletion is relatively weak. The results also suggest that contributions of weak susceptibility genes such as hv3005 to disease predisposition may be obscured by the effects of stronger genetic factors and thus need to be examined in patients lacking these factors.
PMCID: PMC295700  PMID: 7706484
7.  The role of metaphosphate in the activation of the nucleotide by TPS and DCC in the oligonucleotide synthesis. 
Nucleic Acids Research  1986;14(6):2699-2706.
The course of the activation of 3'-acetylthymidine 5'-phosphate by TPS and DCC were followed up by 31P FT nmr. The fact that the "metaphosphate" (delta-5.1) first becomes detectable only at later stage of the activation and does coexist with pyrophosphate and triphosphate suggests that the pyridinium derivative of "metaphosphate" is most probably not directly formed from the hypothetical mixed anhydride or active "pseudourea" right at the beginning of the reaction of pdTac with TPS or DCC, but rather formed at later stage of the activation reaction from the degradation of the pyro- and triphosphates by the activating agent. The mixed anhydride or the active "pseudourea" is most possibly the active key intermediate.
PMCID: PMC339692  PMID: 3754328
8.  The reaction mechanism of N-benzoylimidazole with ribonucleotides. 
Nucleic Acids Research  1987;15(10):4291-4305.
The reaction of uridine 3'-phosphate with benzoylimidazole in the absence and presence of a strong base was followed up by 31P and 1H nmr as well as paper electrophoresis. Possible reaction courses were proposed, the reaction rate constants were calculated and the reaction mechanism was discussed. It is possible to selectively acylate ribonucleotides with benzoylimidazole by appropriate choice of the base used.
PMCID: PMC340848  PMID: 3588294

Results 1-8 (8)