Reduction of Decoy Receptor 3 Enhances TRAIL-Mediated Apoptosis in Pancreatic Cancer

Most human pancreatic cancer cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the mechanisms by which pancreatic cancer cells utilize their extracellular molecules to counteract the proapoptotic signaling mediated by the TNF family are largely unknown. In this study, we demonstrate for the first time that DcR3, a secreted decoy receptor that malignant pancreatic cancer cells express at a high level, acts as an extracellular antiapoptotic molecule by binding to TRAIL and counteracting its death-promoting function. The reduction of DcR3 with siRNA unmasked TRAIL and greatly enhanced TRAIL-induced apoptosis. Gemcitabine, a first-line drug for pancreatic cancer, also reduced the level of DcR3. The addition of DcR3 siRNA further enhanced gemcitabine-induced apoptosis. Notably, our in vivo study demonstrated that the therapeutic effect of gemcitabine could be enhanced via further reduction of DcR3, suggesting that downregulation of DcR3 in tumor cells could tip the balance of pancreatic cells towards apoptosis and potentially serve as a new strategy for pancreatic cancer therapy.     

Effective immunotherapy of cancer by DNA vaccination.

Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived cell-mediated and humoral immune responses to the antigen encoded by the gene vaccine. In the present study, we used gene vaccination with naked plasmid DNA to induce prophylactic immune responses to tumor associated antigens. MAGE-1 (melanoma antigen 1) is an ideal candidate for cancer vaccines because it belongs to a family of genes that are expressed in a number of human tumors of various histological types but not in normal adult tissues except for the testis, and because both humoral and cell-mediated immune responses against MAGE-1 antigen were detected in tumor patients. Intradermal administration of plasmid DNA encoding MAGE-1 (pcMAGE1) induced anti-MAGE-1-specific antibody in BALB/c mice. In contrast, no detectable level of anti-MAGE-1 antibody was induced by intramuscular injection of pcMAGE1. Also, intradermal injection of pcMAGE1 was capable of generating CTLs reactive with MAGE-1-transfected murine tumor cells, M-MSV-MAGE1. Most of the mice (8 out of 10) immunized with pcMAGE1 rejected the challenge of M-MSV-MAGE1 tumor cells, compared with control animals most of which developed tumors. This suggests that intradermal DNA vaccination could provide a novel immunotherapy of cancer.     

The involvement of cAMP signaling pathway in axis specification in Xenopus embryos

The cAMP signaling system has been postulated to be involved in embryogenesis of many animal species, however, little is known about its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT(1a)R) which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos, ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induced secondary axes but were lacking anterior structures. At the molecular level, 5-HT(1a)R stimulation induced expression of the dorsal mesoderm marker genes, and downregulated expression of the ventral markers but had no effect on expression of the pan mesodermal marker gene in ventral marginal zone explants. In addition, ventral expression and stimulation of the receptor partially restored dorsal axis of UV-irradiated axis deficient embryo. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in patterning early embryos.     

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.     

Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design.

In order to develop a serum-free (SF) medium for the production of erythropoietin (EPO) by suspension culture of recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with Fe(NO3)3.9H2O, CuCl2 and ZnSO4.7H2O which are generally contained in SF medium formulations. Insulin, transferrin and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, glutamate, serine, methionine, phosphatidylcholine, hydrocortisone and pluronic F68 were identified as positive determinants for cell growth. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth in suspension culture. An EPO titer in this optimized SF medium was 79% of that in IMDM supplemented with 5% dialyzed fetal bovine serum (dFBS). Furthermore, the in vitro and in vivo biological activities of EPO produced in the SF medium were comparable to those produced in the serum-supplemented medium. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for the production of EPO by suspension culture of rCHO cells.     

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples.

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.     

Genetic mapping of the Isaac-CACTA transposon in maize

We constructed a genetic linkage map with Isaac-TD, SSR, and SNAP markers in a RIL population which had been derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 368 markers, including 241 Isaac-TD, 121 SSR, and 6 SNAP markers, were assigned to 10 linkage groups, encompassing 1687.0 cM, with an average genetic distance of 4.6 cM between markers. SSR markers were utilized as chromosome anchors, in order to assign the Isaac-TD markers to the chromosomes, and the number of markers in each of the linkage groups ranged between 22 and 49. The majority of the Isaac-TD markers were determined to have been distributed throughout the ten maize chromosomes. In linkage analysis of the Isaac-TD markers with genes of agronomic interest, six genes related with maize kernel starch biosynthesis, ae1, bt2, sh1, sh2, su1, and wx1, were analyzed and shown that they were closely linked with either the Isaac-TD or SSR markers on chromosomes of 3, 4, 5, and 9. We observed and mapped segregation-distorted markers on chromosomes 1, 5, 6, 7, 8, and 10, where these markers were clustered. The Isaac-TD or SSR markers which were closely linked with starch synthesis genes may prove useful in marker-assisted breeding programs.     

Dendroaspis natriuretic peptide protects the post-ischemic myocardial injury

The present study used isolated rat hearts to investigate whether (1) Dendroaspis natriuretic peptide (DNP) is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effects of DNP is related to alteration of Bcl-2 family protein levels. The excised hearts of Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg) and coronary flow (CF, ml/min) were continuously monitored. In the presence of 50 nM DNP, all hearts were perfused for a total of 100 min consisting of a 20 min pre-ischemic period followed by a 30 min global ischemia and 50 min reperfusion. Lactate dehydrogenase (LDH) activity in the effluent was measured during reperfusion. Treatment with DNP alone improved the pre-ischemic LVEDP and post-ischemic LVEDP significantly comparing with the untreated control hearts during reperfusion. However, DNP did not affect the LVDP, heart rate (HR, beats/min), and CF. Bcl-2, an anti-apoptotic protein expressed in ischemic myocardium of DNP+ischemia/reperfusion (I/R) group, was higher than that in I/R alone group. Bax, a pro-apoptotic protein expressed in ischemic myocardium of DNP+I/R group, has no significant difference compared with I/R alone group. These results suggest that the protective effects of DNP against I/R injury would be mediated, at least in part, through the increased ratio of Bcl-2 to Bax protein after ischemia-reperfusion.     

Novel protein kinase interacts with the Cucumber mosaic virus 1a methyltransferase domain

The Cucumber mosaic virus (CMV)-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, we used the yeast two-hybrid system using tobacco cDNA library. One of the cDNA clones encoded a protein homologous to the Arabidopsis putative protein kinase and was designated as Tcoi2 (Tobacco CMV 1a interacting protein 2). Tcoi2 specifically interacted with methyltransferase (MT) domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum. These results suggest Tcoi2 as a novel host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein.