Immuno-stimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus

Inonotus obliquus BELYU1102 was selected from 12 different strains of Inonotus as a producer of immuno-stimulating polysaccharide. After a batch fermentation of I. obliquus BELYU1102 was carried out in a 300 l pilot vessel, endo-polysaccharide and exo-polysaccharide were both obtained. The proliferation activity of endo-polysaccharide for splenic cells was much higher than the activity of exo-polysaccharide. The active endo-polysaccharide was produced primarily during the late stationary phase. Enhanced proliferation and polyclonal IgM antibody production were observed in B cells by purified water-soluble endo-polysaccharide. Nitrite production and expression of IL-1beta, IL-6, TNF-alpha, and iNOS in macrophages were also enhanced. However, the endo-polysaccharide did not affect the proliferation of T cells, the IL-2 expression of Th1 cells, or the IL-4 expression of Th2 cells. The endo-polysaccharide showed activities similar to lipopolysaccharide (LPS) for B cells and macrophages, but there was a large difference between the two polysaccharides because cellular activations induced by endo-polysaccharide were not affected by polymyxin B, a specific inhibitor of LPS. The endo-polysaccharide appeared to have other cellular binding sites with TLR-4 and did not show a direct toxicity against tumor cells. However, indirect anti-cancer effects via immuno-stimulation were observed. The mycelial endo-polysaccharide of I. obliquus is a candidate for use as an immune response modifier. Submerged mycelial cultures are advantageous for industrial production of polysaccharides.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase II by Platycodon grandiflorum saponins via suppression of nuclear factor-kappaB activation in RAW 264.7 cells

Saponins are glycosidic compounds present in many edible and inedible plants. They exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammation and immunomodulation. In this study, we investigated the effects of seven platycodin saponins on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that 2"-O-acetyl polygalacin D (S1), platycodin A (S2), platycodin D (S3), and polygalacin D (S6) inhibited LPS-induced NO production in a concentration-dependent manner. Furthermore, these compounds inhibited the expression of LPS-induced iNOS and COX-2 protein and mRNA without an appreciable cytotoxic effect on RAW 264.7 macrophages, and could suppress induction by LPS of pro-inflammatory cytokines such as prostaglandin E2 (PGE2). Treatment with these compounds of RAW 264.7 cells transfected with a reporter construct indicated a reduced level of LPS-induced nuclear factor-kappaB (NF-kappaB) activity and effectively lowered NF-kappaB binding as measured by electrophoretic mobility shift assay (EMSA). The suppression of NF-kappaB activation appears to occur through the prevention of inhibitor kappaB (IkappaB) degradation. In vivo, platycodin saponin mixture (PS) and S3 protected mice from the lethal effects of LPS. The 89% lethality induced by LPS/galactosamine was reduced to 60% and 50% when PS and S3, respectively, were administered simultaneously with LPS. These results suggest that the main inhibitory mechanism of the platycodin saponins may be the reduction of iNOS and COX-2 gene expression through blocking of NF-kappaB activation.     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.     

Expression of the glutathione <Emphasis Type="Italic">S</Emphasis>-transferase gene (NT107) in transgenic <Emphasis Type="Italic">Dianthus superbus</Emphasis>

Using an Agrobacterium-mediated transformation method based on wounding cultured immature seeds with carborundum (600 mesh) in liquid, auxin-regulated tobacco glutathione S -transferase (GST) (NT107) constructs were used to transform Dianthus superbusL. A 663 bp DNA band was found in the transgenic plant genome by PCR analysis using NT107-1 and NT107-2 primers, and a Southern blot analysis showed that the DIG-labelled GST gene was hybridized to the expected amplified genomic DNA fragment from transgenic D. superbus. An overexpression of NT107 led to a twofold increase in GST-specific activity compared to the non-transgenic control plants, and the GST overexpression plants showed an enhanced acclimatization in the soil. To investigate whether an increased expression of GST could affect the resistance of photosynthesis to environmental stress, these plants were subjected to drought and various light intensities from 100 to 3000 mol m^–2s^–1. Copper accumulation and the translocation rate were also analysed in the transgenic lines, and the GST overexpression plants were found to synthesize phytochelatin (PC), which functions by sequestering and detoxifying excess copper ions.These two authors contributed equally to this work     

Comparative characteristics of three human embryonic stem cell lines

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.     

The Phosphorylation status of merlin is important for regulating the Ras-ERK pathway

The neurofibromatosis type2 (NF2) tumor suppressor gene product, merlin, is structurally related to the ezrin-radixin-moesin (ERM) family of proteins that anchor the actin cytoskeleton to specific membrane proteins and participate in cell signaling. However, the basis of the tumor suppressing activity of merlin is not well understood. Previously, we identified a role of merlin as an inhibitor of the Ras-ERK signaling pathway. Recent studies have suggested that phosphorylation of merlin, as of other ERM proteins, may regulate its function. To determine whether phosphorylation of merlin affects its suppression of Ras-ERK signaling, we generated plasmids expressing full-length merlin with substitutions of serine 518, a potential phosphorylation site. A substitution that mimics constitutive phosphorylation (S518D) abrogated the ability of merlin to suppress effects of the Ras-ERK signaling pathway such as Ras-induced SRE transactivation, Elk-mediated SRE transactivation, Ras-induced ERK phosphorylation and Ras-induced focus formation. On the other hand, an S518A mutant, which mimics nonphosphorylated merlin, acted like wild type merlin. These observations show that mimicking merlin phosphorylation impairs not only growth suppression by merlin but also its inhibitory action on the Ras-ERK signaling pathway.     

Structure and tissue distribution of a trinucleotide-repeat-containing gene (cag-3) expressed specifically in the mouse brain

Using in silico approaches and RACE we cloned a full length trinucleotide (CAG) repeat-containing cDNA (cag-3). The cDNA is 2478 bp long and the deduced polypeptide consists of 140 amino acids of which 73 are glutamines. The genomic sequence spans approximately 79 kb on mouse chromosome 7 and the gene is composed of four exons. Standard and real-time PCR analyses of several mouse tissues showed that the gene is exclusively expressed in the brain and is not detected in embryonic stages. Within the brain, it is expressed throughout the forebrain region with predominant expression in the hypothalamus and olfactory bulb and very low levels in the mid- and hindbrain.     

Native polysomes of Saccharomyces cerevisiae in liquid solution observed by atomic force microscopy

The native polysomes of Saccharomyces cerevisiae were visualized in liquid solution by atomic force microscopy without external contrasting, such as shadowing and negative staining. This study showed native polysomes as lined particle with a height of ca. 27 nm, which is agreement with the height of 80S ribosomes in previous study. We found a small subparticle, located in a ring-shape or at the end of a linear structure, and visualized mRNA chains between adjacent ribosomes. Although the structures of polysomes have been studied for decades, it has remained difficult to visualize the native three-dimensional form. By the observation in liquid solution, we temporarily stopped the translation using an antibiotic to presenting the native three-dimensional structure and function of the polysomes. Our results provide not only new findings on native eukaryotic polysomes, but also great potential to visualize the influence of various environmental conditions on polysomes.