The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the competition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by down-regulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells.     

A manganese porphyrin complex is a novel radiation protector

Exposure of cells to ionizing radiation leads to formation of reactive oxygen species, which are associated with radiation-induced cytotoxicity. Therefore, compounds that scavenge reactive oxygen species may confer radioprotective effects. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of manganese(III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on radiation-dependent toxicity. We investigated the protective role of MnTMPyP against ionizing radiation in U937 cells and mice. On exposure to ionizing radiation, there was a distinct difference between control cells and cells pretreated with MnTMPyP with respect to viability, cellular redox status, and oxidative damage to cells. Lipid peroxidation, oxidative DNA damage, and protein oxidation were significantly lower in the cells treated with MnTMPyP when the cells were exposed to ionizing radiation. The [GSSG]/[GSH + GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP+ + NADPH] ratio was lower in control cells compared with MnTMPyP-treated cells. Ionizing radiation-induced mitochondrial damage, as reflected by the altered mitochondrial permeability transition, increase in accumulation of reactive oxygen species, reduction of ATP production, and morphological change, was significantly higher in control cells than in MnTMPyP-treated cells. MnTMPyP administration for 14 days at a daily dosage of 5 mg/kg provided substantial protection against killing and oxidative damage in mice exposed to whole-body irradiation. These data indicate that MnTMPyP may have great application potential as a new class of in vivo, non-sulfur-containing radiation protectors.     

Enhanced protein glutathiolation and oxidative stress in cigarette smokers

There are many functional assays of oxidative damage to DNA, protein, and lipids but few reliable markers of chronic oxidative stress. The glutathiolation of proteins at key Cys residues is considered an important redox-sensitive, posttranslational signaling mechanism in the regulation of critical cellular functions. To determine whether protein bound glutathione (GSSP) is a sensitive indicator of oxidative stress, red blood cell and plasma concentrations were measured and compared between smokers and nonsmokers. In a community-based study conducted in Westchester County, New York, USA, blood samples were obtained from 354 cigarette smokers and 97 never smokers. The mean concentration of blood GSSP (micromol/L) was 32% higher in cigarette smokers and 43% higher when standardized by hemoglobin concentrations (p <.01). Plasma GSSP levels were also 20% higher in smokers than in nonsmokers (p <.001). The relationship was dose-dependent, with blood GSSP levels significantly correlated with cigarettes smoked per day, plasma cotinine, and plasma thiocyanate (r values ranged from .25 to .40). In smokers, there were no significant differences in GSSP and GSH levels by GSTM1 or GSTM3 genotype. Intraindividual variation in blood samples, as determined by taking serial samples over a 2-week period, was low (CV = 12.1%, n = 8). GSSP levels are stable over time but increase in response to the abundant free radicals in cigarette smoke. These findings support the use of GSSP as a sensitive biomarker of oxidative stress.     

Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.     

Effect of transferrin on enhancing bioavailability of iron

Transferrin was isolated and purified from bovine plasma. An intestinal segment in situ experiment showed that 19.2% of injected iron was absorbed when FeCl(3) (80 microg Fe/ml) was injected into a duodenum segment of iron-deficient rats. With addition of 10 and 20 mg of purified transferrin/ml, however, ratios of absorbed iron through duodenum segments were significantly increased to 52.7 and 57.9%, respectively. After transferrin-rich extract was isolated by batch type ion exchange chromatography, a soluble ferric complex of the transferrin extract was prepared by adding ferric salts to transferrin extract followed by dialysis, sterilization, and freeze drying. Results of the animal experiment for comparing bioavailabilities of different irons showed that irons in Fe-transferrin extract was most efficiently absorbed and incorporated into hemoglobin generation in anemic rats.     

Anti-fungal sesquiterpenoid from the root exudate of Solanum abutiloides

The Solanum abutiloides plant is highly resistant to soil-borne pathogens such as Fusarium oxysporum f. sp. melongenae, Verticillium dahliae, and Ralstonia solanacearum. This species is utilized as a mating source of resistant cultivars and is also used as a rootstock. The root exudate of Solanum abutiloides was extracted from a soil system composed of charcoal and vermiculite. Anti-fungal activity was found in the extract, and an active ingredient was isolated. The chemical structure of the active compound was determined to be 3-beta-acetoxysolavetivone, a new sesquiterpenoid. The anti-fungal activity of 3-beta-acetoxysolavetivone examined by the inhibition of spore germination of Fusarium oxysporum was close to that of lubimin, and higher than that of solavetivone.     

Comparative study on the modulation of IgE and cytokine production by Phellinus linteus grown on germinated brown Rice, Phellinus Linteus and germinated brown rice in murine splenocytes

We compared the immunomodulating activities in mice of extracts from Phellinus linteus grown on germinated brown rice (PB), Phellinus linteus (PL) alone, and germinated brown rice (BR) alone. The PL, BR and PB-treated mice were administered with the respective extract (2 mg/head/day) by oral gavage for 4 weeks. All extracts markedly decreased the IgE production and allergic responses in serum and splenocytes. PL and PB increased the proportion of CD4(+) but not CD8(+) T cells in splenocytes. Cytokine production was significantly augmented in all treated mice; the concentration of IFN-gamma was greater in the PL, BR and PB mice than in the control group. The concentration of IL-10 was lower in the BR group than in the other groups. These results may be related to the suppression of IgE production. We conclude that PB modulated the immune responses of IgE production and Th1/Th2 cytokine secretion in murine splenocytes.     

The unique hydrogen bonded water in the reduced form of<Emphasis Type="Italic"> Clostridium pasteurianum</Emphasis> rubredoxin and its possible role in electron transfer

Rubredoxin is a small iron-sulfur (FeS₄) protein involved in oxidation–reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was ~50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the S of Cys9 serves as a port for an electron acceptor. Lastly, the Fe–S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between S of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent.     

BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are perceived by the plasma membrane-localized leucine-rich-repeat-receptor kinase BRI1. Based on sequence similarity, we have identified three members of the BRI1 family, named BRL1, BRL2 and BRL3. BRL1 and BRL3, but not BRL2, encode functional BR receptors that bind brassinolide, the most active BR, with high affinity. In agreement, only BRL1 and BRL3 can rescue bri1 mutants when expressed under the control of the BRI1 promoter. While BRI1 is ubiquitously expressed in growing cells, the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells. Loss-of-function of brl1 causes abnormal phloem:xylem differentiation ratios and enhances the vascular defects of a weak bri1 mutant. bri1 brl1 brl3 triple mutants enhance bri1 dwarfism and also exhibit abnormal vascular differentiation. Thus, Arabidopsis contains a small number of BR receptors that have specific functions in cell growth and vascular differentiation.