Enhancement of Macarpine Production in Eschscholzia Californica Suspension Cultures under Salicylic Acid Elicitation and Precursor Supplementation.

Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4'-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.

A drug-repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6-thioguanine as an effective therapeutic agent for TPMT-low cancer cells.

Pancreatic cancer is one of the most difficult cancers to cure due to the lack of early diagnostic tools and effective therapeutic agents. In this study, we aimed to isolate new bioactive compounds that effectively kill pancreatic ductal adenocarcinoma (PDAC) cells, but not untransformed, human pancreatic ductal epithelial (HPDE) cells. To this end, we established four primary PDAC cell lines and screened 4141 compounds from four bioactive-compound libraries. Initial screening yielded 113 primary hit compounds that caused over a 50% viability reduction in all tested PDAC cells. Subsequent triplicate, dose-dependent analysis revealed three compounds with a tumor cell-specific cytotoxic effect. We found that these three compounds fall into a single category of thiopurine biogenesis. Among them, 6-thioguanine (6-TG) showed an IC50 of 0.39-1.13 µm toward PDAC cells but had no effect on HPDE cells. We propose that this cancer selectivity is due to differences in thiopurine methyltransferase (TPMT) expression between normal and cancer cells. This enzyme is responsible for methylation of thiopurine, which reduces its cytotoxicity. We found that TPMT levels were lower in all four PDAC cell lines than in HPDE or Panc1 cells, and that knockdown of TPMT in HPDE or Panc1 cells sensitized them to 6-TG. Lastly, we used a patient-derived xenograft model to confirm that 6-TG has a significant antitumor effect in combination with gemcitabine. Overall, our study presents 6-TG as a strong candidate for use as a therapeutic agent against PDAC with low levels of TPMT.

Production of Recombinant Caffeine Synthase from Guarana (Paullinia cupana var. sorbilis) in Escherichia coli.

Caffeine synthase (CS) is a methyltransferase responsible for the last two steps of the caffeine biosynthesis pathway in plants. CS is able to convert 7-methylxanthine to theobromine (3,7-dimethylxanthine) and theobromine to caffeine (1,3,7-trimethylxanthine) using S-adenosyl-L-methionine as the methyl donor in both reactions. The production of a recombinant protein is an important tool for the characterization of enzymes, particularly when the enzyme has affinity for different substrates. Guarana has the highest caffeine content among more than a hundred plant species that contain this alkaloid. Different from other plants, in which CS has a higher affinity for paraxanthine (1,7-dimethylxanthine), caffeine synthase from guarana (PcCS) has a higher affinity for theobromine. Here, we describe a method to produce a recombinant caffeine synthase from guarana in Escherichia coli and its purification by affinity chromatography. The recombinant protein retains activity and can be used in enzymatic assays and other biochemical characterization studies.

[Expression and purification of a jasmonic acid carboxyl methyltransferase from Salvia miltiorrhiza in E.coli].

Jasmonic acid carboxyl methyltransferase（JMT）, a key enzyme for jasmonate（JA） biosynthesis, catalyzes the methylation of JA to form Me JA. To characterize the function of JMT, a plasmid pGEX-4T- SmJMT1 harboring JMT1（SmJMT1） gene from Salvia miltiorrhiza was successfully transformed into E. coli BL21（DE3） for protein expression. The recombination SmJMT1 was separated using SDS-PAGE and the size of expressed SmJMT1 protein was consistent with the prediction. The bacterial growth conditions were determined for optimal expression, which include growth temperature, incubation time, IPTG concentrations and culture density. The optimal growth conditions for SmJMT1 were that the bacterial cultures were grown to an A600 of 0.8, and induced with IPTG at a final concentration of 0.4 mmol·L-1, and then incubated for 8 h at 20 ℃. The expression of SmJMT1 in E. coli was confirmed by Western blotting, and mass spectrometry analysis of methyltransferase family. The successful expression and purification of JMT in this study provide the basis for more study of JA biosynthetic pathway and JA-regulated secondary metabolism of medicinal plants.

Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera.

Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania.

Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation.

To produce beneficial phenolic acids for medical and commercial purposes, researchers are interested in improving the normally low levels of salvianolic acid B (Sal B) produced by Salvia miltiorrhiza. Here, we present a strategy of combinational genetic manipulation to enrich the precursors available for Sal B biosynthesis. This approach, involving the lignin pathway, requires simultaneous, ectopic expression of an Arabidopsis Production of Anthocyanin Pigment 1 transcription factor (AtPAP1) plus co-suppression of two endogenous, key enzyme genes: cinnamoyl-CoA reductase (SmCCR) and caffeic acid O-methyltransferase (SmCOMT). Compared with the untransformed control, we achieved a greater accumulation of Sal B (up to 3-fold higher) along with a reduced lignin concentration. This high-Sal B phenotype was stable in roots during vegetative growth and was closely correlated with increased antioxidant capacity for the corresponding plant extracts. Although no outward change in phenotype was apparent, we characterized the molecular phenotype through integrated analysis of transcriptome and metabolome profiling. Our results demonstrated the far-reaching consequences of phenolic pathway perturbations on carbohydrate metabolism, respiration, photo-respiration, and stress responses. This report is the first to describe the production of valuable end products through combinational genetic manipulation in S. miltiorrhiza plants. Our strategy will be effective in efforts to metabolically engineer multi-branch pathway(s), such as the phenylpropanoid pathway, in economically significant medicinal plants.

Chromatin-modifying enzymes as modulators of reprogramming.

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Genetic variation predicting cisplatin cytotoxicity associated with overall survival in lung cancer patients receiving platinum-based chemotherapy.

PURPOSE: Inherited variability in the prognosis of lung cancer patients treated with platinum-based chemotherapy has been widely investigated. However, the overall contribution of genetic variation to platinum response is not well established. To identify novel candidate single nucleotide polymorphisms (SNP)/genes, we carried out a genome-wide association study (GWAS) for cisplatin cytotoxicity by using lymphoblastoid cell lines (LCL), followed by an association study of selected SNPs from the GWAS with overall survival (OS) in lung cancer patients. EXPERIMENTAL DESIGN: A GWAS for cisplatin was conducted with 283 ethnically diverse LCLs. A total of 168 top SNPs were genotyped in 222 small cell lung cancer (SCLC) and 961 non-SCLC (NSCLC) patients treated with platinum-based therapy. Association of the SNPs with OS was determined by using the Cox regression model. Selected candidate genes were functionally validated by siRNA knockdown in human lung cancer cells. RESULTS: Among 157 successfully genotyped SNPs, 9 and 10 SNPs were top SNPs associated with OS for patients with NSCLC and SCLC, respectively, although they were not significant after adjusting for multiple testing. Fifteen genes, including 7 located within 200 kb up or downstream of the 4 top SNPs and 8 genes for which expression was correlated with 3 SNPs in LCLs were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which expression levels were correlated with the rs11169748 and rs2440915 SNPs, significantly decreased cisplatin sensitivity in lung cancer cells. CONCLUSIONS: This series of clinical and complementary laboratory-based functional studies identified several candidate genes/SNPs that might help predict treatment outcomes for platinum-based therapy of lung cancer.

Molecular cloning of an O-methyltransferase from adventitious roots of Carapichea ipecacuanha.

Carapichea ipecacuanha produces various emetine-type alkaloids, known as ipecac alkaloids, which have long been used as expectorants, emetics, and amebicides. In this study, we isolated an O-methyltransferase cDNA from this medicinal plant. The encoded protein (CiOMT1) showed 98% sequence identity to IpeOMT2, which catalyzes the 7'-O-methylation of 7'-O-demethylcephaeline to form cephaeline at the penultimate step of emetine biosynthesis (Nomura and Kutchan, J. Biol. Chem., 285, 7722-7738 (2010)). Recombinant CiOMT1 showed both 7'-O-methylation and 6'-O-methylation activities at the last two steps of emetine biosynthesis. This indicates that small differences in amino acid residues are responsible for distinct regional methylation specificities between IpeOMT2 and CiOMT1, and that CiOMT1 might contribute to two sequential O-methylation steps from 7'-O-demethylcephaeline to emetine.

Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase.

We report high-throughput structure-based virtual screening of putative Flavivirus 2'-O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate (7Me)GpppAC(5). Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC(50) value of 60 microM.

Purification and characterization of a mandibular organ protein from the American lobster, Homarus americanus: a putative farnesoic acid O-methyltransferase.

Methyl farnesoate (MF) appears to have important roles in the development, morphogenesis, and reproduction of crustaceans. To better understand the regulation of MF synthesis, we studied farnesoic acid O-methyltransferase (FAOMeT, the final enzyme in the MF biosynthetic pathway) in the American lobster (Homarus americanus). FAOMeT purified from mandibular organ (MO) homogenates had a MW of approximately 38,000. The sequences of trypsin fragments of purified FAOMeT were used to design PCR primers to amplify a cDNA fragment, which was used to isolate a full-length cDNA containing a single open reading frame (ORF) of 828 bp encoding a protein of 276 amino acids. The deduced amino acid sequence of this putative FAOMeT protein contained two copies of a conserved approximately 135 amino acid domain we term the CF (CPAMD8/FAOMeT) domain; single copies of this domain also occur in the human CPAMD8 protein (a member of the alpha-2 macroglobulin family) and an uncharacterized Drosophila protein. The recombinant protein had no FAOMeT activity. However, its addition to MO homogenates from eyestalk ablated (ESA) lobsters increased enzyme activity by up to 75%, suggesting that FAOMeT may require an additional factor or modification (e.g., phosphorylation) for its activation. The mRNA for the putative FAOMeT was primarily found in the proximal region of the MO, the predominant site of MF synthesis. FAOMeT transcripts were found in muscle tissue from ESA animals, but not in green gland, hepatopancreas, or in muscle tissue from intact animals. FAOMeT mRNA was also detected in embryos and larval stages. This is the first comprehensive report of this protein in the lobster, and is an important step in elucidating the functions of MF in these animals.

Retinoid compounds activate and induce hepatic glycine N-methyltransferase in rats.

Glycine N-methyltransferase (GNMT) functions to regulate S-adenosylmethionine (SAM) levels and the ratio of SAM/S-adenosylhomocysteine (SAH). SAM is a universal methyl group donor and the up-regulation of GNMT may lead to wastage of methyl groups required for transmethylation reactions. Previously, we demonstrated that dietary treatment of rats with 13-cis-retinoic acid (CRA) decreased the hepatic concentration of SAM and the SAH ratio. Here, we examined the ability of CRA, as well as all-trans-retinoic acid (ATRA), to regulate hepatic GNMT as a potential basis for our earlier observations. Rats were fed either a control (10% casein + 0.3% L-methionine) diet or a control diet supplemented with L-methionine (10 g/kg diet). Rats from each group were orally given ATRA, CRA (both at 30 micromol/kg body), or vehicle daily for 7 d. For control rats, administration of both CRA and ATRA elevated the hepatic GNMT activity 49% and 34%, respectively, compared with the control group. Similar results were exhibited by rats fed the methionine-supplemented diet. Moreover, the retinoid-induced elevations in enzyme activity were reflected in the abundance of GNMT protein. To our knowledge, this is the first report of a nutritional compound that induces GNMT activity at the transcriptional and/or translational level.

Acquisition of selenium tolerance by a selenium non-accumulating Astragalus species via selection.

Selection of cultured cells of the selenium sensitive and non-accumulating Astragalus cicer for tolerance to stepwise increasing concentrations of selenite in the medium lead to a variant able to grow at 75 microM selenite. The Se-tolerant culture synthesized a selenocysteine methyltransferase immunologically related but not identical to that of the accumulating A. bisulcatus species and produced Se-methyl-selenocysteine in vivo. Re-cultivation in selenium-free medium lead to breakdown of tolerance and the disappearance of the methyltransferase from cellular proteins. The results prove that the non-accumulating species A. cicer has the cryptic capacity for synthesis of a selenocysteine methyltransferase and also demonstrate that synthesis of the organoselenium compounds in Se-accumulating plants are contributing to selenium tolerance.

Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene.

We previously showed that rat liver betaine-homocysteine methyltransferase (BHMT) mRNA content and activity increased 4-fold when rats were fed a methionine-deficient diet containing adequate choline, compared with rats fed the same diet with control levels of methionine (Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541-545). A further 2-fold increase was observed in rats fed the methionine-deficient diet with supplemental betaine. The nutrition studies reported here were designed to determine whether other methyl donors would induce rat liver BHMT gene expression when added to a methionine-deficient diet and to define the relationship between the degree of methionine restriction and level of methyl donor intake on BHMT expression. Therefore, rats were fed amino acid-defined diets varying in methionine and methyl donor composition. The effect of diet on BHMT expression was evaluated using Northern, Western, and enzyme activity analyses. Similar to when betaine was added to a methionine-deficient diet, choline or sulfonium analogs of betaine induced BHMT expression. The diet-induced induction of hepatic BHMT activity was mediated by increases in the steady-state level of its mRNA and immunodetectable protein. Using methyl donor-free diets, we found that methionine restriction was required but alone not sufficient for the high induction of BHMT expression. Concomitant with methionine restriction, dietary methyl groups were required for high levels of BHMT induction, and a dose-dependent relationship was observed between methyl donor intake and BHMT induction. Furthermore, the severity of methionine restriction influenced the magnitude of BHMT induction. To study the molecular mechanisms that regulate the expression of BHMT, we have cloned the human BHMT gene. This gene spans about 20 kilobases of DNA and contains 8 exons and 7 introns. Using RNA isolated from human liver and hepatoma cells, a major transcriptional start site has been mapped using the 5' rapid amplification of cDNA ends technique, and this start site is 26 nucleotides downstream from a putative TATA box.

Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis.

A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed. COQ3 encodes a 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p-hydroxybenzoic acid. A full length cDNA encoding a homologue of DHHB-methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant. The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant. The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35,360 Da. The predicted amino acid sequence contained all consensus regions for S-adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB-methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene. Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene. The NH2-terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence. The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein. Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes. This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.

Stress responses in alfalfa (Medicago sativa L). XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase.

Medicarpin, the major phytoalexin in alfalfa, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. The methyl group at the 9 position of medicarpin is generally accepted to arise via the methylation of the 4' position (B-ring) of daidzein. Surprisingly, the isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of daidzein in vitro, a reaction that probably does not occur in vivo. Utilizing internal amino acid sequence information from purified alfalfa IOMT, we have isolated three full-length IOMT cDNA clones. A search of the protein databases revealed sequence similarities to O-methyltransferases from various sources. The highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum. The molecular weight of alfalfa IOMT expressed in Escherichia coli was similar to that of purified IOMT from alfalfa cell cultures (41 kDa by SDS-PAGE). The recombinant enzyme catalyzed the O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also methylate the pterocarpan (+) 6a-hydroxymaackiain. Alfalfa contains multiple IOMT genes, and closely related sequences are present in the genomes of chickpea and cowpea, species that also produce B-ring methylated isoflavonoids in vivo. Northern blot analysis indicated that IOMT transcripts are rapidly induced following elicitation, prior to the increase in IOMT activity and medicarpin accumulation. The possible role of the isoflavone 7-OMT in the synthesis of formononetin in vivo is discussed.

Identification and characterization of an S-adenosyl-L-methionine: delta 24-sterol-C-methyltransferase cDNA from soybean.

In plants, the dominant sterols are 24-alkyl sterols, which play multiple roles in plant growth and development, i.e. as membrane constituents and as precursors to steroid growth regulators such as brassinosteroids. The initial step in the conversion of the phytosterol intermediate cycloartenol to the 24-alkyl sterols is catalyzed by S-adenosyl-L-methionine: delta 24-sterol-C-methyl-transferase (SMT), a rate-limiting enzyme for phytosterol biosynthesis. A cDNA clone (SMT1) encoding soybean SMT was isolated from an etiolated hypocotyl cDNA library by immunoscreening using an anti-(plasma membrane) serum. The deduced amino acid sequence of the SMT1 cDNA contained three conserved regions found in S-adenosyl-L-methionine-dependent methyltransferases. The overall structure of the polypeptide encoded by the SMT1 cDNA is most similar to the predicted amino acid sequence of the yeast ERG6 gene, the putative SMT structural gene. The polypeptide encoded by the SMT1 cDNA was expressed as a fusion protein in Escherichia coli and shown to possess SMT activity. The growing soybean vegetative tissues had higher levels of SMT transcript than mature vegetative tissues. Young pods and immature seeds had very low levels of the SMT transcript. The SMT transcript was highly expressed in flowers. The expression of SMT transcript was suppressed in soybean cell suspension cultures treated with yeast elicitor. The transcriptional regulation of SMT in phytosterol biosynthesis is discussed.

Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 nod factors.

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

Relationship between fatty acid accretion, membrane composition, and biologic functions.

Dietary fat affects metabolic pathways for phospholipid biosynthesis in tissues in a coordinated fashion. This may be important to aspects of development that concern phosphatidylcholine metabolism or regulatory processes that depend on signals from a changing milieu in the microenvironment of the membrane. Dietary fat influences the phosphatidylethanolamine (PE) composition in many membranes of the brain and retina and may by altered by small changes in the content of 20:4(6) and 22:6(3). Membrane PE fatty acids that contain one, four, or six double bonds and the ratio of 22:5(6) to 22:6(3) in PE that contains four to six double bonds are also affected. An increase in the omega 6 fatty acid content of membranes is associated with increased PE methyltransferase activity and decreased phosphocholine transferase activity, thus indicating a mechanism by which change in an exogenous factor (e.g., dietary fat intake) may alter neural phospholipid biosynthesis. Small changes in the composition of dietary fat intake change the composition of brain membranes during development. It is provocative to ponder whether diet could be used to induce formation of membrane structures that are more resistant to specific insults that cause degeneration of brain structural material, to ensure optimal functional compositions, or to reverse degenerative changes that occur in neural membrane structure and function.

Cloning of a rat cDNA encoding dihydroxypolyprenylbenzoate methyltransferase by functional complementation of a Saccharomyces cerevisiae mutant deficient in ubiquinone biosynthesis.

3,4-Dihydroxy-5-hexaprenylbenzoate methyltransferase (DHHB-MTase) is the product of the COQ3 gene in Saccharomyces cerevisiae and catalyses the fourth step in the biosynthesis of ubiquinone (coenzyme Q) from p-hydroxybenzoic acid. A full-length cDNA encoding a mammalian homologue of DHHB-MTase was isolated from a newly constructed rat testis cDNA library by functional complementation of a coq3 deletion mutant of S. cerevisiae. The complementing clone contained a 1.1-kb poly(A)(+)-tailed insert with a 858-bp open reading frame and presumably encodes 3,4-dihydroxy-5-polyprenylbenzoate-MTase. The deduced rat amino acid (aa) sequence has a 39% identity over 138 aa with the yeast DHHB-MTase and a 37% identity over this same region with an Escherichia coli protein encoded by the ubiG gene, a MTase that catalyses the terminal step of ubiquinone biosynthesis. The rescue of the yeast coq3 mutant by the rat homologue suggests that yeast and rat synthesize ubiquinone via the same early steps in this pathway.