Maize rbcS promoter activity depends on sequence elements not found in dicot rbcS promoters.

Although the molecular mechanisms of dicot photosynthetic gene regulation have been pursued actively, comparable studies of monocot regulation have been slow to come forth. We show here that monocot (maize and wheat) but not dicot (pea, tobacco, and Arabidopsis) ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoters are active in maize mesophyll protoplasts. The evolutionarily conserved GT and G boxes of dicot rbcS promoters are not essential for light-responsive expression in monocot leaf cells. Instead, at least six constitutive and light-sensitive regulatory elements are likely important for maize rbcS expression. Synergism between upstream and downstream promoter elements is required. Whereas in dicots, light triggers coupled leaf development and photosynthetic gene expression, in monocots, light regulation of rbcS is uncoupled from leaf development. Light regulation of maize rbcS may be divided into direct and indirect contributions mediated by different regulatory elements. Because wheat and maize rbcS promoters show sequence homologies and similar expression patterns in monocot and dicot leaf cells, it appears likely that monocots share conserved regulatory elements irrespective of whether they utilize the C3 or C4 pathway for carbon fixation.     

Fas ligand-induced c-Jun kinase activation in lymphoid cells requires extensive receptor aggregation but is independent of DAXX, and Fas-mediated cell death does not involve DAXX, RIP, or RAIDD

Jun kinase signaling can be elicited by death receptor activation, but the mechanism and significance of this event are still unclear. It has been reported that cross-linking Abs to Fas trigger c-Jun N-terminal kinase (JNK) signaling via caspase-mediated activation of MEKK1 (JNK kinase kinase), elevation of ceramide levels or by recruitment of death domain associated protein (DAXX) to Fas. The effect of physiological ligand for Fas on JNK signaling was never investigated, although evidence is accumulating that Fas ligand is able to induce cellular responses distinct from those evoked by Ab-mediated cross-linking of Fas. Therefore, we investigated the effect of Fas ligand on JNK signaling. Like its ability to induce cell death, Fas ligand reliably activated JNK only upon extensive aggregation of the receptor. Although this was partially dependent on caspase activation, DAXX was not required. DAXX and other death receptor-associated proteins, which have been reported to bind directly or indirectly to Fas, such as receptor interacting protein (RIP) and RIP-associated ICH-1/CED-3-homologous protein with a death domain (RAIDD), were shown to be dispensable for Fas ligand-induced apoptosis.     

Phorbol ester treatment impairs hormone- but not stable GTP analog-induced inhibition of adenylate cyclase

Treatment of intact human platelets and S49 lymphoma cyc- cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, impairs GTP-dependent and hormone-induced inhibition of adenylate cyclase, an action mediated by the inhibitory coupling protein Ni. In contrast, receptor-independent activation of Ni with subsequent adenylate cyclase inhibition induced by the stable GTP analog, guanosine 5'-[gamma-thio]triphosphate, was affected in neither the potency nor onset of Ni activation by the stable GTP analog, in both membrane systems studied. The data indicate that modification of Ni following phorbol ester treatment does not impair its activation by stable GTP analogs.     

Anti-juvenile hormone activity of azacoprostane depends on inhibition of molting

Administration of the anti-ecdysteroid azasteroid 25-azacoprostane (AZCP) to larvae of the tobacco hornworm, Manduca sexta, often inhibits molting without curtailing growth. As a result, AZCP-treated larvae may attain weights 2–3 times greater than normal during the first four instars. This may explain the paradoxical anti-juvenoid activity of AZCP evident in the precocious metamorphosis of AZCP-treated fourth-instar larvae, which was noted only after those larvae attained unusually large weights over 2 g. The weight interval of 2–3 g has been previously identified as a critical threshold for initiation of metamorphosis by normal final (fifth) instar larvae. The premature attainment of this weight threshold by AZCP-treated fourth-instar larvae probably activates the same premetamorphic sequence of events that normally occurs in the fifth instar at this threshold, including activation of potent endogenous anti-juvenoids. Anti-juvenoid activity limited to the penultimate instar is likely to be a general feature of compounds that block molting without inhibiting growth. © 1997 Wiley-Liss, Inc.     

The glucocorticoid agonist activities of mifepristone (RU486) and progesterone are dependent on glucocorticoid receptor levels but not on EC50 values

Mifepristone is an antagonist of the glucocorticoid receptor (GR) that also has significant agonist activity in some cell types. We examined the partial agonist activity of mifepristone in COS-7 cells transfected with increasing amounts of a glucocorticoid receptor expression vector pmGR. As pmGR levels increased, the response of the reporter, pMTVCAT to dexamethasone increased, consistent with increasing levels of receptor expression; the response to mifepristone also increased but at a higher rate, resulting in increasing mifepristone agonist and decreasing antagonist activity. In contrast, increasing pMTVCAT levels increased CAT activity induced by both dexamethasone and mifepristone, but did not change the relative agonist activity of mifepristone. We also examined the relationship between agonist activity and receptor level in a series of clones of the E8.2.A3 cell line expressing various levels of GR. Again, the relative agonist activity of mifepristone increased as GR increased. This increase was not due to changes in the dose response curves to these two ligands since their EC50 values were independent of receptor levels. These results indicate that the degree of glucocorticoid agonist activity exhibited by mifepristone is dependent on the concentration of GR in the cell. Similar results were obtained with another partial agonist of the GR, progesterone, whereas the complete antagonist ZK98.299 had no agonist activity under any condition. Taken together, these results suggest that the phenomenon of receptor concentration-dependence is a property of partial GR agonists in general.     

A plant growth-promoting rhizobacteria (PGPR) mixture does not display synergistic effects,likely by biofilm but not growth inhibition

Combination of different PGPR strains with complementary characteristics as a mixture to reduce possible instability under fluctuating environment has been considered practical. However, PGPR mixtures do not always play synergistic roles in growth promotion or biological control as reflected in our previous findings and other publications. In this work, we accidentally discovered that a mixture containing two well compatible PGPR strains, Bacillus pumilus WP8 and Erwinia persicinus RA2, did not synergize in growth promotion or biological control of tomato bacterial wilt under field conditions. Considering the importance of PGPR biofilm formation in growth promotion and biocontrol activities, we hypothesized that this phenomenon may be related to inhibition of biofilm formation. In vitro experiments showed that biofilm-formation ability of WP8 was inhibited by both RA2 cells and filtered supernatants collected from RA2 cultures at 12 h (RA2-12) rather than 48 h (RA2-48), even at high-temperatures (within 100°C). An in vivo experiment derived from crystal violet staining yielded similar results. Using liquid chromatography-mass spectrometry (LC-MS), we compared primary and secondary metabolites of RA2 (namely RA2-12 and RA2-48) and found D-glutamine, abundant in RA2-12, as the putative inhibitory factor. Trace amounts of jasmonic acid together with viscous extracellular polysaccharides in RA2-48 likely promoted the rescue of robust biofilm formation. This work suggests that inhibition of biofilm formation should be considered in PGPR mixture development.     

A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor.

We have developed a new ligand-dependent chimeric recombinase (Cre-GRdex) by fusing the site-specific Cre recombinase to the ligand binding domain (LBD) of a mutant human glucocorticoid receptor (GRdex). The synthetic glucocorticoid receptor (GR) ligands dexamethasone, triamcinolone acetonide and RU38486efficiently induce recombinase activity in F9 murine embryonal carcinoma cells expressing constitutively Cre-GRdex. In contrast, no recombinase activity was detected in the absence of ligand or in the presence of the natural GR ligands corticosterone, cortisol or aldosterone. Moreover, physiological concentrations of these natural GR ligands do not affect Cre-GRdexrecombinase activity induced by dexamethasone. Thus, as previously shown using Cre-oestrogen receptor (ER) fusion proteins, Cre-GRdexmight be useful for achieving loxP site-directed mutagenesis in cultured cells and spatio-temporally controlled somatic cell mutagenesis in transgenic mice.     

Farnesoid X receptor suppresses constitutive androstane receptor activity at the multidrug resistance protein-4 promoter

Multidrug resistance protein-4 (MRP4) is a member of the multidrug resistance associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive up-regulation in response to cholestatic injury or bile acid feeding. In this study we demonstrate that farnesoid X receptor (FXR) regulates MRP4 in vivo and in vitro. In vivo deletion of FXR induces MRP4 gene expression. In vitro treatment of HepG2 cells with FXR ligands, chenodeoxycholic acid (CDCA), cholic acid (CA) and the synthetic ligand GW-4064 suppresses basal mRNA level of the MRP4 gene as well as the co-treatment with CDCA and 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), an activator of constitutive androstane receptor (CAR). We found in the human MRP4 promoter a CAR responsive element (CARE) embedded within an FXR responsive element (FXRE). We cloned this region and found that FXR suppresses CAR activity in luciferase assay. Finally, we demonstrated that FXR competes with CAR for binding to this overlapping binding site. Our results support the view that FXR activation in obstructive cholestasis might worsen liver injury by hijacking a protective mechanism regulated by CAR and provides a new molecular explanation to the pathophysiology of cholestasis.     

Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.