Characterization of a novel satellite virus and a strain of Himetobi P virus (Dicistroviridae) from the brown planthopper, Nilaparvata lugens

Two distinct spherical virus-like particles were purified from the brown planthopper, Nilaparvata lugens. One was a geographical isolate of Himetobi P virus (Cripavirus, Dicistroviridae). The other was 30 nm in diameter and contained positive-stranded RNA. The RNA was 1647 nucleotides in length and encoded only its own capsid protein, indicating that this particle is a satellite virus. Transmission tests showed that the satellite was transmitted vertically; however, its helper virus was unknown. We named this satellite Nilaparvata lugens commensal X virus (NLCXV).     

Probiotic Lactobacillus casei activates innate immunity via NF-kappaB and p38 MAP kinase signaling pathways

Probiotic bacteria are microorganisms that benefit the host through improvement of the balance of intestinal microflora and possibly by augmentation of host defense systems. We examined the mechanisms for the up-regulation of innate immune responses by a probiotic Lactobacillus casei ATCC27139, in vivo. Using mouse models of systemic Listeria monocytogenes infection and MethA fibrosarcoma tumorigenesis in combination with BALB/c and SCID mice, we found that parenteral administration of L. casei ATCC27139 confers a protective effect against L. monocytogenes infection and anti-tumor activity against MethA fibrosarcoma by activation of innate immunity, while L. casei ATCC27139-J1R strains, which are J1 phage-resistant strains that have been selected from MNNG-treated clones, lacked these activities. Substantial differences between ATCC27139 and ATCC27139-J1R strains were observed in the capacity to induce innate cytokines such as TNF-alpha, IL-12, IL-18, and IFN-gamma, and pathogen-associated molecular pattern receptors, TLR2 and Nod2, by spleen cells. In addition, although phosphorylation of NF-kappaB p65 in spleen was equally enhanced in the ATCC27139- and the ATCC27139-J1R-treated groups, phosphorylation of both p38 MAPK and MAPKAPK-2 was significantly induced only by ATCC27139. Furthermore, inhibitors of NF-kappaB (sulfasalazine) and p38 MAPK (SB203580) significantly reduced cytokine production by the spleen cells of the mice treated with L. casei ATCC27139, suggesting that both NF-kappaB and p38 MAPK signaling pathways play important roles in the augmentation of innate immunity by the probiotic L. casei.     

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

Maturation of circulating dendritic cells and imbalance of T-cell subsets in patients with squamous cell carcinoma of the head and neck

Interactions between dendritic cells (DCs) and T cells play a pivotal role in the regulation and maintenance of immune responses. In cancer patients, various immunological abnormalities have been observed in these immune cells. Here, we investigated proportions and the phenotype of DCs and the cytokine profile of T-cell subsets in the peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), using multicolor flow cytometry. The percentage of myeloid (CD11c+), but not plasmacytoid (CD123+) DCs, was significantly lower (P<0.05) and expression of HLA-DR was significantly decreased in total and myeloid DCs of cancer patients compared to healthy donors. Simultaneous analyses of T-cell subsets in the patients’ circulation showed significantly increased proportions of CD4+ T cells expressing Th1 and Th2 cytokines after ex vivo stimulation without any skewing in the Th1/Th2 ratio. The relative level of HLA-DR expression on myeloid or total DCs positively correlated with the Th1/Th2 ratio (P<0.01), and the proportion of total circulating DCs was inversely correlated with that of regulatory CD4+CD25+ T cells (P<0.01). These results suggest that the decreased proportion of circulating DCs and decreased HLA-DR expression in DCs may have a major impact on systemic immune responses in patients with SCCHN.     

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

Preventive effects of phosphorylated ascorbate on ultraviolet-B induced apoptotic cell death and DNA strand cleavage through enrichment of intracellular vitamin C in skin epidermal keratinocytes

Mortality of mouse keratinocytes Pam212 that were irradiated with ultraviolet-B (UVB) was shown to be repressed by pre-irradiated administration with L-ascorbic acid (Asc) or more markedly with Asc-2-O-phosphate (Asc2P), but not with dehydroascorbic acid (DehAsc) or Asc-2-O-alpha-glucoside (Asc2G), although not repressed by post-irradiated administration. The cytoprotection by Asc2P was restricted against UVB below 5-20 mJ/cm2, and exhibited markedly by administration for a period over 2 h, which may be caused by intracellular Asc that was accumulated via dephosphorylation of Asc2P and was increased, 6-24 h after, to levels above twice as abundant as those of Asc-administration. Pre-irradiated Asc2P-administration slightly repressed a DNA ladder-like electrophoretic pattern for UVB-irradiated keratinocytes, containing the histone-bound DNA fragments as shown by ELISA assay, and appreciably repressed the DNA-3'OH cleavage terminals as shown by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain. Thus, prevention of UVB-induced cell death by Asc2P was shown to occur concurrently with inhibition of DNA cleavages and enrichment of intracellular Asc.     

Polyhydroxylated fullerene C60(OH)44 suppresses intracellular lipid accumulation together with repression of intracellular superoxide anion radicals and subsequent PPARγ2 expression during spontaneous differentiation of OP9 preadipocytes into adipocytes

Reactive oxygen species has been suggested to be one of the key factors associated with the development of obesity. During spontaneous differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular superoxide anion radicals (O (2) (-.) ) level markedly increases and is accompanied by a significant elevation of intracellular lipid accumulation. This differentiation-dependent increase in intracellular O (2) (-.) level positively correlated with the intracellular augmentation of the lipid level. Super-highly hydroxylated fullerene (SHH-F; C(60)(OH)(44)), a novel polyhydroxylated fullerene derivative, quenched intracellular O (2) (-.) , and lipid accumulation to 38.7 and 42.7 % of that in the control, respectively. By thin-layer chromatographic analysis of extracted cellular lipid components, SHH-F clearly decreased the triglycerides ratio in the whole lipid droplet fraction, but scarcely influenced other lipids components. PPARγ2 expression, which plays a key role in regulating adipogenic differentiation, was significantly suppressed by SHH-F at the late stage of differentiation, with unaltered PPARγ1 expression. The intracellular superoxide anion radical augmentation preceded expression of PPARγ2, strongly suggesting that the primary O (2) (-.) generation was closely associated with lipid accumulation and subsequent PPARγ2 induction. These results indicate that SHH-F suppresses intracellular lipid accumulation, particularly in lipid droplets, and decreases O (2) (-.) level and subsequent PPARγ2 upregulation during spontaneous differentiation of OP9 preadipocytes into adipocytes.