Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias latipes

In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms

A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells

BACKGROUND: Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. METHODS: In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. RESULTS: The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC(50 )values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC(50 )values > 50 μM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47D cancer cells just in 10 min, to effectively kill these cells and induce approximately 80% apoptotic cell death but not in normal cells. The intravenous administration of TfR-lytic peptide in the athymic mice model significantly inhibited tumor progression. CONCLUSIONS: TfR-lytic peptide might provide a potent and selective anticancer therapy for patients

Designed hybrid TPR peptide targeting Hsp90 as a novel anticancer agent

<p>Abstract</p> <p>Background</p> <p>Despite an ever-improving understanding of the molecular biology of cancer, the treatment of most cancers has not changed dramatically in the past three decades and drugs that do not discriminate between tumor cells and normal tissues remain the mainstays of anticancer therapy. Since Hsp90 is typically involved in cell proliferation and survival, this is thought to play a key role in cancer, and Hsp90 has attracted considerable interest in recent years as a potential therapeutic target.</p> <p>Methods</p> <p>We focused on the interaction of Hsp90 with its cofactor protein p60/Hop, and engineered a cell-permeable peptidomimetic, termed "hybrid Antp-TPR peptide", modeled on the binding interface between the molecular chaperone Hsp90 and the TPR2A domain of Hop.</p> <p>Results</p> <p>It was demonstrated that this designed hybrid Antp-TPR peptide inhibited the interaction of Hsp90 with the TPR2A domain, inducing cell death of breast, pancreatic, renal, lung, prostate, and gastric cancer cell lines <it>in vitro</it>. In contrast, Antp-TPR peptide did not affect the viability of normal cells. Moreover, analysis <it>in vivo </it>revealed that Antp-TPR peptide displayed a significant antitumor activity in a xenograft model of human pancreatic cancer in mice.</p> <p>Conclusion</p> <p>These results indicate that Antp-TPR peptide would provide a potent and selective anticancer therapy to cancer patients.</p

Discovery of Genes Activated by the Mitochondrial Unfolded Protein Response (mtUPR) and Cognate Promoter Elements

In an accompanying paper, we show that the mitochondrial Unfolded Protein Response or mtUPR is initiated by the activation of transcription of chop through an AP-1 element in the chop promoter. Further, we show that the c/ebpβ gene is similarly activated and CHOP and C/EBPβ subsequently hetero-dimerise to activate transcription of mtUPR responsive genes. Here, we report the discovery of six additional mtUPR responsive genes. We found that these genes encoding mitochondrial proteases YME1L1 and MPPβ, import component Tim17A and enzymes NDUFB2, endonuclease G and thioredoxin 2, all contain a CHOP element in their promoters. In contrast, genes encoding mitochondrial proteins Afg3L2, Paraplegin, Lon and SAM 50, which do not have a CHOP element, were not up-regulated. Conversely, genes with CHOP elements encoding cytosolic proteins were not induced by the accumulation of unfolded proteins in mitochondria. These results indicate that mtUPR responsive genes appear to share a requirement for a CHOP element, but that this is not sufficient for the regulation of the mtUPR. A more detailed analysis of promoters of mtUPR responsive genes revealed at least two additional highly conserved, putative regulatory sites either side of the CHOP element, one a motif of 12 bp which lies 14 bp upstream of the CHOP site and another 9 bp element, 2 bp downstream of the CHOP site. Both of these additional elements are conserved in the promoters of 9 of the ten mtUPR responsive genes we have identified so far, the exception being the Cpn60/10 bidirectional promoter. Mutation of each of these elements substantially reduced the mtUPR responsiveness of the promoters suggesting that these elements coordinately regulate mtUPR

The Chop Gene Contains an Element for the Positive Regulation of the Mitochondrial Unfolded Protein Response

We have previously reported on the discovery of a mitochondrial specific unfolded protein response (mtUPR) in mammalian cells, in which the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional activation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ, and ClpP, but not those encoding stress proteins of the endoplasmic reticulum (ER) or the cytosol. Analysis of the chaperonin 60/10 bidirectional promoter showed that the CHOP element was required for the mtUPR and that the transcription of the chop gene is activated by mtUPR. In order to investigate the role of CHOP in the mtUPR, we carried out a deletion analysis of the chop promoter. This revealed that the transcriptional activation of the chop gene by mtUPR is through an AP-1 (activator protein-1) element. This site lies alongside an ERSE element through which chop transcription is activated in response to the ER stress response (erUPR). Thus CHOP can be induced separately in response to 2 different stress response pathways. We also discuss the potential signal pathway between mitochondria and the nucleus for the mtUPR

MtUPR increases phosphorylation of JNK and a MEK specific inhibitor blocks mtUPR.

<p>(A): mtUPR increases phosphorylation of JNK 1&2. Extracts from cells transfected with vector or OTCΔ, and treated with or without 10 µM of MEK specific inhibitor PD98059, were subjected to Western transfers probed with antibody against p-JNK. (B) and (C): mtUPR induction of the <i>yme1l1</i>(B) and <i>mpp</i>β(C) promoter is inhibited by the MEK specific inhibitor, PD98059. COS-7 cells co-transfected with vector or OTCΔ and <i>yme1l1</i>and <i>mpp</i>β promoter-reporter constructs, with or without 10 µM of PD98059 were used for luciferase assay 32 h after transfection. The fold activation of the promoter constructs in cells expressing OTCΔ compared with those expressing vector alone, with or without of PD98059, is shown as relative luciferase (RLU) activity. Data represent the mean±SEM from experiments performed in triplicate.</p

<i>Chop</i> and <i>c/ebp</i>β promoters contain AP-1 sites and are inducible by mtUPR.

<p>(A) Nucleotide sequence alignment of the mammalian <i>chop</i> promoters (−278 to −222) from human, bovine, mouse and rat. Bold letters show the highly conserved bases of the AP-1 site and the asterisks show the highly conserved sequence surrounding the AP-1 site in <i>chop</i> promoters. The position of the putative novel element of N 30 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000835#pone.0000835-Xie1" target="_blank">[18]</a> is shown in the box. (B): Nucleotide sequences of mammalian <i>c/ebp</i>β promoter region around AP-1 site (Human, Mouse, and Rat) is compared with the <i>chop</i> promoter sequence (−278 to −233). The numbers refer to the distance from transcription initiation site of human <i>chop</i> or human, mouse, and Rat <i>c/ebp</i>β. The asterisks indicate the conserved nucleotides around the AP-1 site. (C): C/EBPβ expression in response to mtUPR. Extracts from cells transfected with vector or OTCΔ were subjected to western blotting and probed with antibodies against C/EBPβ and tubulin as control and show that C/EBPβ, like CHOP is induced by expression of OTCΔ.</p

Oral administration of ferulic acid or ethyl ferulate attenuates retinal damage in sodium iodate-induced retinal degeneration mice

Epidemiological studies indicate that the daily intake of antioxidants from a traditional Asian diet reduces the risk of developing age-related macular degeneration. Many of the phytochemicals that are abundant in whole grains exhibit a wide variety of biological activity such as antioxidant, anti-inflammatory, and neuroprotective effects. Ferulic acid (FA) is a phenolic acid found in vegetables and grains that has therapeutic potential for diabetes mellitus, Alzheimer’s disease, and other diseases. We investigated the retinal protective effect of FA in a sodium iodate (NaIO3)-induced model of retinal degeneration. In a human retinal pigment epithelial cell line, FA attenuated H2O2-induced injury and lipopolysaccharide- or 7-ketocholesterol-induced inflammation. In mice, the oral administration of FA or its analog, ethyl ferulate, attenuated the morphological and functional features of NaIO3-induced retinal degeneration according to optical coherence tomography and electroretinography. Our results demonstrate that the oral administration of FA provides protective effects to the retina, suggesting that the intake of FA as a daily supplement or daily healthy diet containing rich vegetables and whole grains may prevent age-related macular degeneration