The Moorella thermoacetica aromatic O-demethylase was characterized as an inducible three-component system with similarity to the methanogenic methanol, methylamine, and methanethiol methyltransferases and to the O-demethylase system from Acetobacterium dehalogenans. MtvB catalyzes methyl transfer from a phenylmethylether to the cobalt center of MtvC, a corrinoid protein. MtvA catalyzes transmethylation from MtvC to tetrahydrofolate, forming methyltetrahydrofolate. Cobalamin can substitute for MtvC.Constituting about 25% of the earth's biomass (18), lignin is a polymer composed of hydroxylated and methoxylated phenylpropanoid units linked by C™C and C™O™C bonds that undergoes depolymerization to generate monomeric methoxylated aromatics (or phenylmethylethers) like vanillate and syringate. The anaerobic metabolism of phenylmethylethers (11) has been known since 1979 (13). Although acetogenic bacteria can be selectively isolated by growth on methoxylated aromatics like syringate (2), they do not metabolize the aromatic ring itself but use the O-methyl group as a one-carbon growth substrate (equation 1). Oxidation of one methyl group to CO 2 provides the six electrons required for conversion of three methyl groups to acetate. Moorella thermoacetica can metabolize at least 20 different methoxylated aromatics (5). A 22-kDa protein and aromatic O-demethylase activity are induced when M. thermoacetica is exposed to such compounds (6, 37).The aromatic O-demethylases of Acetobacterium woodii (3), Acetobacterium dehalogenans (27), and M. thermoacetica (8) require H 4 folate as the methyl acceptor, producing CH 3 -H 4 folate (8,15,27). Oxidation of one equivalent of CH 3 -H 4 folate to CO 2 and H 4 folate generates six electrons (equation 2) which drive the synthesis of three equivalents of acetylcoenzyme A (acetyl-CoA) by the Wood-Ljungdahl pathway of acetyl-CoA synthesis ( Fig. 1 and reaction 3). This paper describes a three-component aromatic O-demethylase from M. thermoacetica. MATERIALS AND METHODSBacterial strains and growth conditions. M. thermoacetica was cultivated as described earlier (1) but included a vitamin mixture (36). Dicamba (2 mM) was added at an optical density at 600 nm (OD 600 ) of 0.6. Cells were harvested at an OD 600 of 2.0 using a CEPA continuous centrifuge. The cells were frozen in liquid nitrogen and stored at Ϫ80°C.Analytical methods. Corrinoid concentrations were measured by conversion to the dicyano derivative (23) using the extinction coefficients at 580 and 367 nm of 10.13 ϫ 10 3 M Ϫ1 cm Ϫ1 and 30.82 ϫ 10 3 M Ϫ1 cm Ϫ1. Metals were determined by plasma emission spectroscopy (14). Protein concentrations were determined by the Rose Bengal method (9). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by the method of Laemmli (20), and the gels were stained as described by Blum et al. (4).Electron paramagnetic resonance (EPR) spectra were recorded on a Bruker ESP 300E spectrometer equipped with an Oxford ITC4 temperature controller, a model 5340 automatic frequency c...

LEFTY is expressed in normal endometrium in cells that decidualize. To understand the importance of this expression, we have studied the effect of LEFTY on decidualization in vitro and in vivo. Exposure of human uterine fibroblast (HuF) cells to recombinant LEFTY blocked the induction of the decidual differentiation-specific marker genes, IGFBP1 (IGF-binding protein 1) and PRL (prolactin) in response to medroxyprogesterone acetate, estradiol, and prostaglandin E2. The inhibitory effect was associated with decreased induction of the transcription factors ETS1 and FOXO1, both of which are essential for decidualization. Overexpression of LEFTY in decidualized HuF cells with an adenovirus that transduced LEFTY caused a marked decrease in IGFBP1 secretion, and withdrawal of medroxyprogesterone acetate from decidualized cells resulted in a decrease in IGFBP1 secretion and an increase in LEFTY expression. Moreover, overexpression of LEFTY in decidualized cells reprogrammed the cells to a less differentiated state and attenuated expression of decidual markers. Uterine decidualization was markedly attenuated and litter size was significantly reduced by retroviral transduction of LEFTY in the uterine horns of pregnant mice or by induction of LEFTY expression by doxycycline treatment in Tet-On conditional LEFTY transgenic pregnant mice. In addition, administration of the contraceptive agent drospirenone to ovariectomized mice induced a marked increase in LEFTY expression and inhibited decidualization. Taken together, these finding indicate that LEFTY acts as a molecular switch that modulates both the induction of decidual differentiation and the maintenance of a decidualized state. Because decidual cells express abundant amounts of LEFTY, the action of LEFTY on decidualization occurs by an autocrine mechanism.

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Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.