White button mushrooms (Agaricus bisporous) are a potential breast cancer chemopreventive agent, as they suppress aromatase activity and estrogen biosynthesis. Therefore, we evaluated the activity of mushroom extracts in the estrogen receptor-positive/aromatase-positive MCF-7aro cell line in vitro and in vivo. Mushroom extract decreased testosterone-induced cell proliferation in MCF-7aro cells but had no effect on MCF-10A, a nontumorigenic cell line. Most potent mushroom chemicals are soluble in ethyl acetate. The major active compounds found in the ethyl acetate fraction are unsaturated fatty acids such as linoleic acid, linolenic acid, and conjugated linoleic acid. The interaction of linoleic acid and conjugated linoleic acid with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty acids inhibit aromatase with similar potency and that mutations at the active site regions affect its interaction with these two fatty acids. Whereas these results suggest that these two compounds bind to the active site of aromatase, the inhibition kinetic analysis indicates that they are noncompetitive inhibitors with respect to androstenedione. Because only conjugated linoleic acid was found to inhibit the testosteronedependent proliferation of MCF-7aro cells, the physiologically relevant aromatase inhibitors in mushrooms are most likely conjugated linoleic acid and its derivatives. The in vivo action of mushroom chemicals was shown using nude mice injected with MCF-7aro cells. The studies showed that mushroom extract decreased both tumor cell proliferation and tumor weight with no effect on rate of apoptosis. Therefore, our studies illustrate the anticancer activity in vitro and in vivo of mushroom extract and its major fatty acid constituents.

(TMG). These in vivo studies led to the discovery of an ATP-dependent metabolite-activated HPr kinase and an HPr(serine phosphate) IHPr(Ser-P)] phosphatase that reversibly phosphorylate HPr (6). This kinase was shown to phosphorylate serine46 in HPr, and it and the HPr(Ser-P) phosphatase were shown to be present in partial association with the cytoplasmic membrane in a wide variety of low-G+C Gram-positive bacteria (7).Lactobacillus brevis has been shown to possess a lactose/H+ symport permease that exhibits the glucose-promoted phenomenon of inducer efflux (8). This organism possesses HPr and the kinase/phosphatase system that reversibly phosphorylates serine-46 in this protein, but enzyme I and the various sugar-specific enzyme II complexes of the PTS are apparently lacking (9). The function of HPr and its reversible phosphorylation in L. brevis might be supposed to be regulation of non-PTS carbohydrate transport, but no direct evidence for this postulate has been forthcoming.In this paper we describe the development and use of a vesicular system that allowed us to test the postulate that HPr plays a direct role in the regulation of the L. brevis lactose permease. We show that both inducer exclusion and inducer effiux are dependent on intravesicular HPr and a metabolic intermediate such as fructose 1,6-bisphosphate (Fru-P2) or gluconate 6-phosphate. The results provide compelling evidence regarding the mechanism of non-PTS transport regulation in Gram-positive bacteria. MATERIALS AND METHODSOrganisms, Growth, and Vesicle Preparations. Growth conditions and the bacterial strain used, L. brevis strain ATCC367, were described previously (8). Cells were grown for 18 hr at 300C in the presence of 25 mM galactose, harvested, washed, and used directly for transport experiments or for membrane vesicle preparation following the method described by Kaback for Escherichia coli (10) with the following modifications. In the spheroplast formation step, lysozyme (2 mg/ml) in 10 mM potassium EDTA (pH 7.0) was incubated with cells for 180 min at room tempera-

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl I-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly efiluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylates serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of BaciUlus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.Many but not all low-GC gram-positive bacteria possess the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that catalyzes the concomitant uptake and phosphorylation of its sugar substrates. The PTS-catalyzed process requires the sequential phosphorylation of four proteins or protein domains, enzyme I, HPr, IlAsugar and IIBsusgar, before sugar phosphorylation and concomitant transport can occur (8,20). The PTS proteins function in numerous biochemical and genetic regulatory capacities (17)(18)(19)21). In early studies, it was shown that addition of a rapidly metabolizable sugar such as glucose to streptococci, lactococci, or lactobacilli resulted in inhibition of the uptake of other sugars (inducer exclusion) as well as rapid efflux of preaccumulated sugars or sugar metabolites (inducer expulsion). For example, lactose and its nonmetabolizable analog...

LactobaciUlus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles ofL. brevis cells, we recently showed that these regulatory effects could be attributed to the metaboliteactivated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46

Lactococcus lactis takes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase. Uptake of 2DG into L. lactis vesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP). 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles. lntravesicular but not extravesicular wild-type HPr of Bacillus subtiljs alone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DC-P. lntravesicular H I 5A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites. lntravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites. The 5460 mutant HPr protein promoted regulation, even in the absence of a metabolite. The V,,, but not the Km value for 2DG uptake was affected. Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr. The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L. lactis. They suggest that two primary functions of HPr(Ser) phosphorylation are: (1) to feedback-inhibit the activities of carbohydrate permeases and not merely to create a hierarchy of preferred carbon sources, and (2) to regulate the cytoplasmic concentrations of carbohydrate inducers by exclusion and expulsion mechanisms.