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Abstract Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the [...]

Some members of Burkholderiales are able to grow on methanol but lack the genes (mxaFI) responsible for the well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydrogenase that is widespread in methylotrophic Proteobacteria. Here, we characterized novel, mono-subunit enzymes responsible for methanol oxidation in four strains, Methyloversatilis universalis FAM5, Methylibium petroleiphilum PM1, and unclassified Burkholderiales strains RZ18-153 and FAM1. The enzyme from M. universalis FAM5 was partially purified and subjected to matrix-assisted laser desorption ionization-time of fight peptide mass fingerprinting. The resulting peptide spectrum was used to identify a gene candidate in the genome of M. petroleiphilum PM1 (mdh2) predicted to encode a type I alcohol dehydrogenase related to the characterized methanol dehydrogenase large subunits but at less than 35% amino acid identity. Homologs of mdh2 were amplified from M. universalis FAM5 and strains RZ18-153 and FAM1, and mutants lacking mdh2 were generated in three of the organisms. These mutants lost their ability to grow on methanol and ethanol, demonstrating that mdh2 is responsible for oxidation of both substrates. Our findings have implications for environmental detection of methylotrophy and indicate that this ability is widespread beyond populations possessing mxaF, the gene traditionally used as a genetic marker for environmental detection of methanol-oxidizing capability. Our findings also have implications for understanding the evolution of methanol oxidation, suggesting a convergence toward the enzymatic function for methanol oxidation in MxaF and Mdh2-type proteins.

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for [C.sub.1] transfer between formaldehyde and formate, the tetrahydrofolate ([H.sub.4]F)-linked pathway and the tetrahydromethanopterin ([H.sub.4]MPT)-linked pathway. Both pathways are required for growth on [C.sub.1] substrates; however, mutants defective for the [H.sub.4]MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the [H.sub.4]MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous [H.sub.4]F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NA[D.sup.+] can successfully substitute for the [H.sub.4]MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the [H.sub.4]MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the [H.sub.4]MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.

The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at Iow copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a [[sigma].sup.N] promoter 5' of mmoX. In this study the genes encoding [[sigma].sup.N] (rpoN) and a typical [[sigma].sup.N] -dependent transcriptional activator (mmoR) were cloned and sequenced, mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5' of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that [[sigma].sup.N] plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a [[sigma].sup.N] and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.

Indole-3-carbinol (13C), a compound that occurs naturally in Brassica vegetables such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (Cdk6) expression and stimulation of [p21.sup.Waf1/Cip1] gene expression. Construction and transfection of a series of promoter-reporter plasmids demonstrate that the indole-regulated changes in Cdk6 and [p21.sup.Waf1/Cip1] levels are due to specific effects on their corresponding promoters. Mutagenic analysis reveals that 13C signaling targets a composite transcriptional element in the Cdk6 promoter that requires both Sp1 and Ets transcription factors for transactivation function. Analysis of protein-DNA complexes formed with nuclear proteins isolated from 13C-treated and -untreated cells demonstrates that the Sp1 DNA element in the Cdk6 promoter interacts with an 13C-inhibited protein-protein complex that contains the Sp1 transcription factor. In indoletreated cells, a fraction of [[.sup.3]H]I3C was converted into its natural diindole product [sup.3]H-labeled 3-3'-diindolylmethane ([[3.sup]]DIM] which accumulates in the nucleus; this suggests that DIM may have a role in the transcriptional activities of I3C. Mutagenic analysis of the [p21.sup.Waf1/Cip1 promoter reveals that in transfected breast cancer cells, DIM (as well as I3C) stimulates [p21.sup.Waf1/Cip p21] transcription through an indole-responsive region of the promoter that contains multiple Sp1 consensus sequences. Furthermore, DIM treatment regulates the presence of a nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and [p21.sup.Waf1/Cip1] promoters are newly defined downstream targets of the indole-signaling pathway, and that the observed transcriptional effects are due to a combination of the cellular activities of 13C and DIM. KEY WORDS: * indoles * 13C * DIM * antiproliferative pathway * regulated promoter * cell-cycle gene * Sp1 transcription factor * reproductive cancer cell

Studies increasingly indicate that dietary indole-3-carbinol (I3C) prevents the development of estrogen-enhanced cancers including breast, endometrial and cervical cancers. Epidemiological, laboratory, animal and translational studies support the efficacy of I3C. Whereas estrogen increases the growth and survival of tumors, I3C causes growth arrest and increased apoptosis and ameliorates the effects of estrogen. Our long-range goal is to best use I3C together with other nutrients to achieve maximum benefits for cancer prevention. This study examines the possibility that induction of growth arrest in response to DNA damage (GADD) in genes by diindolylmethane (DIM), which is the acid-catalyzed condensation product of I3C, promotes metabolically stressed cancer cells to undergo apoptosis. We evaluated whether genistein, which is the major isoflavonoid in soy, would alter the ability of I3C/DIM to cause apoptosis and decrease expression driven by the estrogen receptor (ER)-[alpha]. Expression of GADD was evaluated by real-time reverse transcription-polymerase chain reaction. Proliferation and apoptosis were measured by a mitochondrial function assay and by fluorescence-activated cell sorting analysis. The luciferase reporter assay was used to specifically evaluate expression driven by ER-[alpha]. The estrogen-sensitive MCF-7 breast cancer cell line was used for these studies. We show a synergistic effect of I3C and genistein for induction of GADD expression, thus increasing apoptosis, and for decrease of expression driven by ER-[alpha]. Because of the synergistic effect of I3C and genistein, the potential exists for prophylactic or therapeutic efficacy of lower concentrations of each phytochemical when used in combination. KEY WORDS: * indole-3-carbinol * diindolylmethane * genistein * estradiol

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/[cm.sup.2]), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Research has been conducted on Bacillus methanolicus MGA3 steady-state methanol-limited cultures. The role of the growth-inhibitory effects of methanol pulses which perturb steady-state methanol concentrations has been investigated and the results are reported.

Research has been conducted on the carbaporphyrins. The reaction of these compounds with silver(I) acetate in methanol-CH (sub)2 Cl (sub)2 has produced silver(III) complexes and the details are reported.

Research has been conducted on the calcium hypochlorite. The reaction of this compound with 2,4,6-trichlorophenol and 2,4,6-trichloro-m-cresol in methanol to produce dimer-type ketals and the ketals' conversion to quinones have been investigated and discussed.

Research has been conducted on the carbaprephenate and 4-epi-carbaprephenate dimethyl esters. The preparation of these esters is described and their reactivity in the presence and absence of different proteins with chorismate mutase activity has been investigated.

Research has been conducted on the palladium complexes immobilized on polyaminoamido dendrimers supported on silica. The use of these complexes as catalysts for the iodobenzene carbonylation in methanol to produce methyl benzoate is described.

Research has been conducted on Candida biodinii, a methylotrophic yeast with formaldehyde dehydrogenase activity exhibited during growth on methanol. The cloning of the structural gene from C. biodinii genomic library has been carried out and the results indicate that this gene is responsible for the yeast growth on methanol.

Research has been conducted on the methanol's photosensitized addition to (R)-(+)-limonene. Results indicate that this addition has led to a highly diastereodifferentiating bimolecular asymmetric photoreaction.

Research has been conducted on the dimethylplatinum(II) complexes. The oxidation of these complexes by dioxygen in methanol has been carried out in two steps and the effect of ligand variation on the complexes' oxidation has been investigated and the details are reported.

Research has been conducted on the alpha-lambda (super)3 -iodanyl ketones. The generation of these ketones from (Z)-(2-acetoxyvinyl)(phenyl)-lambda (super)3 -iodanes has been carried out and the results indicate that the generation method involves ester exchange of (Z)-2-acetoxyvinyl-lambda (super)3 -iodanes with methanol in triethylamine.

Research has been conducted on the methylenecyclopropane, bicyclopropylidene and spirocyclopropanated methylenecyclopropanes. The bromine additions to these compound carried out in methanol have been investigated and the results indicate the enhanced bromination rate with the increased number of spiroannelated three-membered rings.

Research has been conducted on the Diels-Alder reaction. The route to the spiro[2.4]hepta-4,6-diene-4-carboxylic acid esters and spiro[2.4]hepta-4,6-diene-4-methanol has been developed via the use of the hetero-Diels-Alder reaction and the utilization of these dienes for the rac-10-hydroxycamphor synthesis via carbo-Diels-Alder reaction has been carried out.

Research has been conducted on the Ala24/Asp61 (results in) Asp24/Asn61 substituted subunit c structure of Escherichia coli. The determination of this structure via the heteronuclear multidimensional NMR in a solvent mixture of water, methanol and monophasic chloroform has been carried out and described.

Research has been conducted on the ring-methoxylate diarylmethane and diarylmethanol radical cations. The reactivities and properties of these compounds generated in aqueous solution via pulse and gamma-radiolysis have been investigated and the results of the studies are reported.

Research has been conducted on the chiral perhydro-1,3-benzoxazines. Results indicate that the intramolecular Diels-Alder reactions on these benzoxazines derived from (-)-8-amino methanol occur with high stereoselection and in high yields.

Research has been conducted on alpha, alpha'-bis(trifluoromethyl)-9,10-anthracenedimethanol. The enantiopure forms of this compound and the corresponding perdeuterated isotopomers have been prepared and studied via (super)1 H NMR spectroscopy and the results demonstrate discrimination for some racemic mixtures.

Research has been conducted on the nitrocefin. The investigation of its methanolysis in anhydrous methanol has been carried out and the results suggest that this methanolysis has occurred in the active site of Zn (super)2+ (super)- beta-lactamases.

Research has been conducted on the chloroperoxidase which catalyzes cyclopropylmethanols' enantioselective oxidation. Results indicate that the enantioselectivity of cis-cyclopropanes' chloroperoxidase oxidation is higher than with the trans isomers.

Research has been conducted on the enantiomeric methylenecyclopropylcarbinols. The absolute configuration of these compounds has been obtained via the reaction of (R)- and (S)-epichlorohydrin with methylenetriphenylphosphorane and the details are presented.

Research has been conducted on the chlorine isotope effects. The effects have been measured for the base-promoted elimination reaction of 1-(2-chloro-2-propyl)indene in methanol and the results are discussed.

Research has been conducted on the photooxygenation reactions of indolizine derivatives in mixed benzene-methanol solvents. The results of these reactions which proceed via the excitation of their ground state charge transfer complex with the molecular oxygen are reported.

Research has been conducted on the dinuclear (phosphine)gold(I) complexes of the ambidentate 1,3,4-thiadiazoledithiolate ligand. The preparation of these complexes from the corresponding (phosphine)gold(I) chlorides and K (sub)2 (SSS) in methanol is reported.

Research has been conducted on the tetraallylic, tetraallenic, and tetrapropargylic stannanes. The reactions of these stannanes with aldehydes in methanol in producing unsaturated alcohols have been investigated and the results indicate that these reactions proceed with the allylic rearrangement for the tetra(2-butenyl)tin and tetra(1,2-butadienyl)-tin.