Hydrodynamic-based delivery of an interleukin-22-Ig fusion gene ameliorates experimental autoimmune myocarditis in rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.     

Rapid PCR-based method for detection and differentiation of Didymiaceae and Physaraceae (myxomycetes) in environmental samples

Ecological studies of myxomycetes have been limited by the absence of universal cultivation techniques and the lack of life stage independent identification methods. We designed a novel PCR primer pair for the specific amplification of small subunit ribosomal RNA gene of Didymiaceae and Physaraceae. The primers produced amplicons from 192 fruiting body samples belonging to 10 genera. Twenty-four samples yielded longer fragments and sequence analysis revealed the presence of intron(s). As for the exonic regions, while sequence heterogeneities within a single species/varietas/forma were frequently observed, identical sequences were obtained only from identical species/varietas. The effectiveness of this primer pair in the analysis of morphologically unidentifiable samples was confirmed with the applications to samples of environmental plasmodium/sclerotium and soil. Denaturing gradient gel electrophoresis analysis was also tested with the soil samples. The results presented here demonstrate this PCR-based method can facilitate further ecological studies of Physaraceae and Didymiaceae in the environment.     

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells

The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High-density lipoprotein. The platelet-derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL-208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3-K, Akt. The constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3-K-Akt pathways participate in PDGF-suppression of ABCA1 expression.     

Effects of lotus root (the edible rhizome of Nelumbo nucifera) on the deveolopment of non-alcoholic fatty liver disease in obese diabetic db/db mice

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that would ameliorate NAFLD is therefore of interest. Lotus root, the edible rhizome of Nelumbo nucifera, contains a high level of polyphenolic compounds, and several health-promoting properties of lotus root have been reported. The present study examines whether dietary lotus root powder can protect db/db mice from hepatic injury. After 3 weeks of feeding, the hepatomegaly, hepatic triglyceride accumulation, and elevated hepatic injury markers in the serum were markedly alleviated in the Lotus diet-fed db/db mice relative to the control mice. These effects were partly attributable to suppression of the lipogenic enzyme activities and mRNA expression by the Lotus diet. The serum levels of adiponectin, which has been reported to have a protective effect against NAFLD, were significantly higher in the Lotus group than in the Control group of the db/db mice. Moreover, the hepatic expression of such inflammatory genes as tumor necrosis factor-alpha and monocyte chemoattractant protein-1 were markedly suppressed by the Lotus diet. We speculate that the development and progression of NAFLD were prevented by suppressing the expression of lipogenic and inflammatory genes as a result of the higher serum adioponectin level in the Lotus diet-fed db/db mice.     

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.     

Large-scale development of expressed sequence tag-derived simple sequence repeat markers and diversity analysis in Arachis spp.

Large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed in peanut (Arachis hypogaea L.) to obtain more informative genetic markers. A total of 10,102 potential non-redundant EST sequences, including 3,445 contigs and 6,657 singletons, were generated from cDNA libraries of the gynophore, roots, leaves and seedlings. A total of 3,187 primer pairs were designed on flanking regions of SSRs, some of which allowed one and two base mismatches. Among the 3,187 markers generated, 2,540 (80%) were trinucleotide repeats, 302 (9%) were dinucleotide repeats, and 345 (11%) were tetranucleotide repeats. Pre-polymorphic analyses of 24 Arachis accessions were performed using 10% polyacrylamide gels. A total of 1,571 EST-SSR markers showing clear polymorphisms were selected for further polymorphic analysis with a Fluoro-fragment Analyzer. The 16 Arachis accessions examined included cultivated peanut varieties as well as diploid species with the A or B genome. Altogether 1,281 (81.5%) of the 1,571 markers were polymorphic among the 16 accessions, and 366 (23.3%) were polymorphic among the 12 cultivated varieties. Diversity analysis was performed and the genotypes of all 16 Arachis accessions showed similarity coefficients ranging from 0.37 to 0.97. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9604-8) contains supplementary material, which is available to authorized users.     

Cleavage mechanism and anti-tumor activity of 3,6-epidioxy-1,10-bisaboladiene isolated from edible wild plants

A bisabolane sesquiterpene endoperoxide compound, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from edible wild plants grown in the northern area of Japan, Cacalia delphiniifolia and Cacalia hastata, using a mutant yeast (cdc2-1 rad9Δ). It showed cytotoxicity at IC(50) = 3.4 μM and induced apoptosis against the human promyelocytic leukemia cell line HL60 through a new stable rearrangement product (1) when in the presence of FeSO(4). This conversion mechanism is different from another sesquiterpene endoperoxide lactone compound, dihydroartemisinin (DHA), which is an anti-malarial drug. The cytotoxicity of EDBD decreased in the presence of the ferrous ion chelating drug deferoxamine mesylate (DFOM), and this suggested that the structural change of the drug caused by Fe(2+) may be responsible for its biological activities. EDBD induced apoptosis via phosphorylation of p38 mitogen-activated protein kinase (MAPK) in HL60 cells, and was detected by Western blot. EDBD resulted in an immediate increase in DCF fluorescence intensity in HL60 cells using DCFH-DA (2',7'-dichlorofluorescin diacetate) assay. The in vitro reaction of EDBD with FeSO(4) also increased DCF fluorescence intensity in a dose dependent manner. These results showed that the biological activity of EDBD involves an unstable carbon-centered radical intermediate. Furthermore, there was no similarity between the JFCR39 fingerprints of EDBD and DHA (correlation coefficient on COMPARE Analysis γ = 0.158). EDBD showed anti-tumor effects against a xenograft of Lox-IMVI cells in vivo.     

Tumor cell-specific prodrugs using arsonic acid-presenting iron oxide nanoparticles with high sensitivity

We report the tumor cell-selective prodrugs based on the arsonic acid-presenting iron oxide nanoparticles. We synthesized the well-dispersed nanoparticles having arsonoacetic acid which is composed of the low toxic As(V) form. From the analyses of the reaction products, it is suggested that the reduction by dithiothreitol with arsonoacetic acid and the modified nanoparticles could generate the highly-toxic As(III) species. In the MTT assays, it was found that the cell viabilities of HeLaS3 and especially HepG2 were reduced in the presence of the modified nanoparticles. In contrast, a slight effect on viability was observed with primary mouse hepatocytes. The viabilities showed good agreements with the amounts of intracellular reduced glutathione concentrations. Furthermore, the valid concentrations of the modified nanoparticles for tumor-specific cytotoxicity were similar level in MRI measurements. These results indicate that arsonic acid-presenting nanoparticles should be a good platform for developing highly-sensitive tumor-specific prodrugs.     

Synthesis of quinolinomorphinan derivatives as highly selective δ opioid receptor ligands

We have reported previously the novel δ opioid agonist KNT-127 which showed high affinity and selectivity for the δ receptor. Moreover, the analgesic effect of subcutaneously administered KNT-127 was more potent than that of a prototypical δ agonist (?)-TAN-67 in the acetic acid writhing test. This study of the structure–activity relationship of KNT-127 derivatives focused on the introduction of substituents onto the 5′-, 6′-, 7′- or 8′-position of the quinoline ring and revealed that many derivatives with 5′- or 8′-substituents showed high affinities and selectivities for the δ receptor. Especially, SYK-153 with an 8′-OH group showed the highest affinity and the most balanced and highest selectivity for the δ receptor among the synthesized compounds.     

Coordinated control of self-renewal and differentiation of neural stem cells by Myc and the p19ARF–p53 pathway

The modes of proliferation and differentiation of neural stem cells (NSCs) are coordinately controlled during development, but the underlying mechanisms remain largely unknown. In this study, we show that the protooncoprotein Myc and the tumor suppressor p19^ARF regulate both NSC self-renewal and their neuronal and glial fate in a developmental stage–dependent manner. Early-stage NSCs have low p19^ARF expression and retain a high self-renewal and neurogenic capacity, whereas late-stage NSCs with higher p19^ARF expression possess a lower self-renewal capacity and predominantly generate glia. Overexpression of Myc or inactivation of p19^ARF reverts the properties of late-stage NSCs to those of early-stage cells. Conversely, inactivation of Myc or forced p19^ARF expression attenuates self-renewal and induces precocious gliogenesis through modulation of the responsiveness to gliogenic signals. These actions of p19^ARF in NSCs are mainly mediated by p53. We propose that opposing actions of Myc and the p19^ARF–p53 pathway have important functions in coordinated developmental control of self-renewal and cell fate choices in NSCs.