Genetic characterization and fine mapping of a novel thermo-sensitive genic male-sterile gene tms6 in rice (Oryza sativa L.)

The application of genetic male sterility in hybrid rice production has great potential to revolutionize hybrid seed production methodology. The two-line breeding system by using thermo-sensitive genic male sterility (TGMS) has been discovered and successfully developed as a breeding strategy in rice. One TGMS gene was investigated by a spontaneous rice mutant line, Sokcho-MS, originated from a Korean japonica variety. It was shown that Sokcho-MS is completely sterile at a temperature higher than 27°C and/or lower than 25°C during the development of spikelets, but fertile at the temperature ranging from 25 to 27°C regardless of the levels of day-length. Genetic analysis and molecular mapping based on SSR, STS and EST markers revealed that a single recessive gene locus involved the control of genic male sterility in Sokcho-MS. By using an F₂ mapping population derived from a cross between Sokcho-MS and a fertile indica variety Neda, the new TGMS gene, designated as tms6, was mapped primarily to the long arm of chromosome 5 of Oryza sativa at the interval between markers E60663 (2.0 cM) and RM440 (5.8 cM). Subsequently, tms6 was fine mapped to the interval between markers RM3351 (0.1 cM) and E60663 (1.9 cM). As tms6 appeared to be independent of other mapped TGMS genes in rice, the genetic basis of Sokcho-MS was further discussed.     

Induction method of tyrosine kinase A-mediated cell death in rat pheochromocytoma

Nerve Growth Factor (NGF) is a neurotrophic factor that prevents apoptosis in neuronal progenitor cells. In rat pheochromocytoma (PC12) cells, tyrosine kinase A receptor (TrkA) mediates neurotrophic or protective effects, while p75 neurotrophin receptor (p75NTR) functions as a death receptor. We have determined whether TrkA mediates any cytotoxic effect. Following serum deprivation, TrkA expression increased 2.2-fold and apoptosis began with expression of Bax proapoptotic protein. Application of NGF halved cell viability but this was reversed by K252a, the TrkA inhibitor. These results confirmed the paradoxical cytotoxic effect of neurotrophic NGF via TrkA in PC12 cells following serum deprivation.     

Selective tumor targeting by enhanced permeability and retention effect. Synthesis and antitumor activity of polyphosphazene-platinum (II) conjugates

Nanosized polyphosphazene-platinum (II) conjugates with a wide range of molecular weight from 24,000 to 115,000 were synthesized to study their tumor selectivity by enhanced permeability and retention (EPR) effect and their antitumor activity. It has been found from biodistribution study that the present polyphosphazene-Pt(II) conjugates exhibit high tumor selectivity by EPR effect with the tumor to tissue ratio (TTR) from 3.6 to 13 depending on the molecular size. These polymer conjugates have shown excellent in vivo antitumor activity against both murine and human cancer cell lines. In particular, xenograft trials of the conjugates have shown outstanding tumor inhibition effect on the stomach cancer cell line, YCC-3, which is one of the least responsive to the anticancer agents currently in clinical use, although the reason is not clearly explainable yet. The high in vivo activity seems to be attributed to the controlled-release of the antitumor active platinum (II) moiety, [GlyGluPt(dach] (dach=trans-(+/-)-1,2-diaminocyclohexane) from the phosphazene backbone by degradation in aqueous solution.     

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.     

Molecular mechanisms of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced inverted U-shaped dose responsiveness in anchorage independent growth and cell proliferation of human breast epithelial cells with stem cell characteristics

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a variety of carcinogenic and noncarcinogenic effects in experimental animals, its role in human carcinogenicity remain controversial. A simian virus 40-immortalized cell line from normal human breast epithelial cells with stem cells and luminal characteristics (M13SV1) was used to study whether TCDD can induce AIG positive colony formation and cause increased cell numbers in a inverted U-shaped dose–response manner. TCDD activated Akt, ERK2, and increased the expression of CYP1A1, PAI-2, IL-lb mRNA, and ERK2 protein levels. TCDD was able to increased phosphorylation and expression of ERK2 in same dose–response manner as AIG positive colony formation. Thus, TCDD induced tumorigenicity in M13SV1, possibly through the phosphorylation of ERK2 and/or Akt. Further, cDNA microarray with 7448 sequence-verified clones was used to profile various gene expression patterns after treatment of TCDD. Three clear patterns could be delineated: genes that were dose-dependently up-regulated, genes expressed in either U-shape and/or inverted U-shape. The fact that these genes are intrinsically related to breast epithelial cell proliferation and survival clearly suggests that they may be involved in the TCDD-induced breast tumorigenesis.     

Early events responsible for aluminum toxicity symptoms in suspension-cultured tobacco cells

We investigated the aluminum (Al)-induced alterations in zeta potential, plasma membrane (PM) potential and intracellular calcium levels to elucidate their interaction with callose production induced by Al toxicity. A noninvasive confocal laser microscopy has been used to analyse the live tobacco (Nicotiana tabacum) cell events by means of fluorescent probes Fluo-3 acetoxymethyl ester (intracellular calcium) and DiBAC4 (PM potential) as well as to monitor callose accumulation. Log-phase cells showed no detectable changes in the PM potential during the first 30 min of Al treatment, but sustained large depolarization from 60 min onwards. Measurement of zeta potential confirmed the depolarization effect of Al, but the kinetics were different. The Al-treated cells showed a moderate increase in intracellular Ca2+ levels and callose production in 1 h, which coincided with the time course of PM depolarization. Compared with the Al treatment, cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, facilitated a higher increase in intracellular Ca2+ levels, but resulted in accumulation of only moderate levels of callose. Calcium channel modulators and Al induced similar levels of callose in the initial 1 h of treatment. Callose production induced by Al toxicity is dependent on both depolarization of the PM and an increase in intracellular Ca2+ levels.