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Introduction As the main component of plant oil and the primary form of stored energy, triacylglycerol (TAG) is involved in multitudinous physiological and biochemical processes (Mao et al. 2018; Zhang [...], Diacylglycerol acyltransferase (DGAT) catalyzes the only rate-limiting step in the pathway of plant oil (TAG) biosynthesis and is involved in plant development. In this study, five DGAT family members were identified from maize genome database. Phylogenetic analysis classified the ZmDGATs into type-I, II, and III clusters. Conserved functional domain analysis revealed that the proteins encoded by ZmDGAT1 contained conserved MBOAT domains, while two ZmDGAT2-encoding proteins harbored LPLAT domains. qRT-PCR analysis showed that ZmDGAT genes exhibited very high relative expression in developing seeds, especially at the early stage of seed development. Under various abiotic stress conditions, differential responses of ZmDGAT genes were observed. An overall significant induction of ZmDGAT genes under cold stress in leaves and a quick and strong response to osmotic stresses in roots were highlighted. This study provides useful information for understanding the roles of DGATs in oil accumulation and stress responses in higher plants. Key words: diacylglycerol acyltransferase (DGAT), triacylglycerol (TAG), gene expression, abiotic stress, maize (Zea mays L.). La diacylglycerol acyltransferase (DGAT) catalyse la seule etape limitante dans le sentier biosynthetique des huiles (TAG) chez les plantes et elle est egalement impliquee dans le developpement. Dans ce travail, cinq membres de la famille DGAT ont ete identifies au sein des banques de donnees pour le genome du mais. Une analyse phylogenetique a classifie les ZmDGAT en groupes de type I, II et III, respectivement. Une analyse des domaines fonctionnels conserves a revele que les proteines codees par les ZmDGAT1 contenaient les domaines conserves MBOAT, tandis que deux ZmDGAT2 codaient pour des proteines contenant des domaines LPLAT. Une analyse qRT-PCR a montre que les ZmDGAT presentaient une très forte expression relative chez les graines en developpement, particulièrement tôt dans le developpement des graines. Sous diverses conditions de stress abiotique, des reponses differentielles des ZmDGAT ont ete observees. Globalement, une induction significative des ZmDGAT lors de stress de froid chez les feuilles, ainsi qu'une reponse rapide et forte aux stress osmotiques chez les racines a ete observee. Cette etude fournit des informations utiles pour comprendre les rôles desDGATdans la production d'huile et la reponse aux stress chez les plantes superieures. [Traduit par la Redaction] Mots-cles : diacylglycerol acyltransferase (DGAT), triacylglycerol (TAG), expression genique, stress abiotique, Zea mays L.

Oil content is an important yield trait in Brassica juncea (L.) Coss. Improvements to yield levels of mustard by conventional breeding methods have reached a plateau. The application of transgenic technology is an area that has not yet been explored for improving the oil content of B. juncea. In this study, the effect of overexpression of AtDGAT1 (a key gene involved in oil biosynthesis) on the seed oil content of B. juncea was investigated. For seed-specific overexpression, the gene was linked to Arabidopsis thaliana oleosin promoter and mobilized into mustard through Agrobacterium-mediated transformation. Transformants were selected on MS medium containing 50 mg/L kanamycin, and a transformation frequency of 10.5% was obtained. A total of 10 transgenic events were generated. Analyses of seed weight, oil content, and other yield traits in [T.sub.1] transgenics showed that seed-specific overexpression of AtDGAT1 significantly improved the oil content and seed weight. The maximum oil content increase observed in the transgenic seeds was 8.3% compared with the wild-type plants. Total fatty acid content was increased from 4% to 14% in six of the seven events. However, the content of oleic and linoleic acid was reduced and, of these two, oleic acid content showed drastic reduction. Key words: copy number, oil content, transgenic. Le contenu en huile constitue une importante caracteristique du rendement de Brassica juncea (L.) Coss. Les efforts mis pour ameliorer les niveaux de rendement de la moutarde par les methodes de selection conventionnelles ont atteint un plateau. L'application de la technologie transgenique dans ce domaine n'a pas ete exploree en ce qui a trait a l'amelioration du contenu en huile de B. juncea. Dans cette etude, l'effet de la surexpression de AtDGAT1 (un gene cle de la biosynthese de l'huile), sur le contenu en huile des graines de B. juncea a ete examine. Afin de cibler specifiquement l'expression dans la graine, le gene a ete lie au promoteur de l'oleosine de Arabidopsis thaliana et insere dans la moutarde au moyen d'une transformation par Agrobacterium. Les transformants ont ete selectionnes sur du milieu MS contenant 50 mg/L de kanamycine et une frequence de transformation de 10,5 % a ete obtenue. Dix phenomenes transgeniques au total ont ete generes. Les analyses du poids des graines, du contenu en huile et d'autres caracteristiques de rendement chez les individus transgeniques [T.sub.1] ont montre que la surexpression de AtDGAT1 specifiquement dans les graines ameliorait le contenu en huile et le poids des graines de maniere significative. Le contenu maximal en huile dans les graines observe chez les individus transgeniques etait accru de 8,3 % comparativement aux individus sauvages. Le contenu en acides gras totaux etait accru de 4 a 14 % dans six des sept transgeneses. Cependant, les contenus en acides oleique et linoleique etaient reduits et de ces deux, le contenu en acide oleique etait fortement reduit. [Traduit par la Redaction] Mots-cles: nombre de copie, contenu en huile, transgenique., Introduction Indian mustard (Brassica juncea (L.) Coss.) is an important oilseed crop of the Indian subcontinent. Increasing the oil content is a major goal of mustard breeders. Oil content is [...]

Author(s): John W. Schoggins [sup.1] [sup.15] , Donna A. MacDuff [sup.2] , Naoko Imanaka [sup.1] , Maria D. Gainey [sup.3] , Bimmi Shrestha [sup.4] , Jennifer L. Eitson [sup.5] , [...], The specificity of interferon effectors across an expanded range of viruses is studied, with results indicating that positive-sense single-stranded RNA viruses are more susceptible to interferon-stimulated gene activity than negative-sense RNA or DNA viruses; in addition, the DNA sensor cGAS is shown to have an unappreciated role in RNA virus inhibition. cGAS crucial to innate immunity This study reports the use of cell culture models to scan an extensive interferon-stimulated gene (ISG) library for activity against a broad spectrum of viruses. The scan reveals that positive-sense single-stranded (ss)RNA viruses are more susceptible to ISG activities than negative-sense ssRNA viruses or a DNA virus. The DNA sensor cyclic GMP-AMP synthase (cGAS) is shown to inhibit several RNA viruses. The authors also generated cGAS knockout mice and showed an in vivo requirement for cGAS in antiviral responses. The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors.sup.1,2,3. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses.sup.4,5,6,7,8,9,10,11,12,13. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Cholesterol homeostasis in the enterocyte is regulated by the interplay of multiple genes that ultimately determines the net amount of cholesterol reaching the circulation from the small intestine. The effect of deleting these genes, particularly acyl CoA:cholesterol acyl transferase 2 (ACAT2), on cholesterol absorption and fecal sterol excretion is well documented. We also know that the intestinal mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1) increases in [Acat2.sup.-/-] mice. However, none of these studies has specifically addressed how ACAT2 deficiency impacts the relative proportions of esterified and unesterified cholesterol (UC) in the enterocyte and whether the concurrent loss of ABCA1 might result in a marked buildup of UC. Therefore, the present studies measured the expression of numerous genes and related metabolic parameters in the intestine and liver of ACAT2-deficient mice fed diets containing either added cholesterol or ezetimibe, a selective sterol absorption inhibitor. Cholesterol feeding raised the concentration of UC in the small intestine, and this was accompanied by a significant reduction in the relative mRNA level for Niemann-Pick C1-like 1 (NPC1L1) and an increase in the mRNA level for both ABCA1 and ABCG5/8. All these changes were reversed by ezetimibe. When mice deficient in both ACAT2 and ABCA1 were fed a high-cholesterol diet, the increase in intestinal UC levels was no greater than it was in mice lacking only ACAT2. This resulted from a combination of compensatory mechanisms including diminished NPC1L1-mediated cholesterol uptake, increased cholesterol efflux via ABCG5/8, and possibly rapid cell turnover. cholesterol homeostasis; cholesterol absorption; cholesteryl ester; fecal neutral sterol excretion; intestinal mucosa doi: 10.1152/ajpgi.00190.2010.

Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420-421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14-20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the iysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis. gene discovery | lipid enzyme | phospholipid remodeling | retinal degeneration | visual dysfunction doi/ 10.1073/pnas.1002897107

The fungal cell wall plays a crucial role in host-pathogen interactions. Its formation is the result of the coordinated activity of several extracellutar enzymes, which assemble the constituents, and remodel and hydrolyse them in the extracellular space. Canclida albicans Phrl and Phr2 proteins belong to family GH72 of the [beta]-(1,)-glucanosyltransferases and play a crucial role in cell wall assembly. PHR1 and PHR2, homologues of Saccharomyces cerevisiae GAS1, are differently regulated by extracellular pH. PHR1 is expressed when ambient pH is 5.5 or higher, whereas PHR2 has the reverse expression pattern. Their deletion causes a pH-conditional defect in morphogenesis and virulence. In this work we explored whether PHR1 deletion affects the ability of C. albicans to adhere to and invade human epithelia. PHR1 null mutants exhibited a marked reduction in adhesion to both abiotic surfaces and epithelial cell monolayers. In addition, the mutant was unable to penetrate and invade reconstituted human epithelia. Transcription profiling of selected hyphal-specific and adhesin-encoding genes indicated that in the PHR1 null mutant, HWP1 and ECE1 transcript levels were similarly reduced in both adhesion and suspension conditions. These results, combined with microscopy analysis of the septum position, suggest that PHR1 is not required for the induction of hyphal development but plays a key role in the maintenance of hyphal growth. Thus, the [beta]-(1,)-glucan processing catalysed by Phrl p is of fundamental importance in the maintenance of the morphological state on which the adhesive and invasive properties of C. albicans greatly depend. DOI 10.1099/mic.0.038000-0

The enzyme carnitine palmitoyltransferase (CPT) I is a major regulator of mitochondrial fatty acid oxidation in vertebrates. Numerous genome duplication events throughout evolution have given rise to three (in mammals) or multiple (in fish) genetically and functionally different isoforms of this enzyme. In particular, these isoforms represent a diversification of kinetic and regulatory properties stemming from mutations at the genomic and proteomic levels. Phylogenetic reconstructions reveal a comprehensive view of the CPT I family in vertebrates and genomic modifications leading to structural changes in proteins and functional differences between tissues and taxa. In a model fish species (rainbow trout), the presence of five CPT I isoforms suggests repeated duplication events in bony fishes and salmonids. Subsequently, an array of nucleotide and amino acid substitutions in the isoforms may contribute to a tissue-specific and a previously observed species-specific difference in the [IC.sub.50] for malonyl-CoA. Moreover, all five isoforms are expressed in trout at the mRNA level in skeletal muscle, heart, liver, kidney, and intestine. In general, transcript levels of the [beta]-isoforms were higher in muscle tissues, while levels of the [alpha]-isoforms were higher in other tissues. Rainbow trout also exhibit developmental plasticity in relative mRNA expression of CPT I isoforms from fry to juvenile to adult stage. Thus the evolution of CPT I has resulted in a very diverse family of isoforms. These differences represent a degree of specificity in the ability of species to regulate function at the protein and tissue levels, which, in turn, may allow for precise control of lipid oxidation in individual tissues during physiological perturbations. evolution; lipid oxidation; malonyl-CoA; transmembrane domain doi: 10.1152/ajpregu.00088.2010.

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (-)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 [min.sup.-1] [micro][M.sup.-1]) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6.DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, [DELTA]iptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde. doi: 10.1128/JB.01557-09

The enzyme UDP-GIcNAc:[alpha]3-D-mannoside [beta]1,2-N-acetylglucosaminyltransferase I (GnT1, encoded by Mgat1) controls the synthesis of paucimannose N-glycans in Drosophila. We have previously reported that null mutations in Drosophila Mgat1 are viable but exhibit defects in locomotion, brain abnormalities, and a severely reduced life span. Here, we show that knockdown of Mgat1 in the central nervous system (CNS) of wild-type flies decreases locomotor activity and life span. This phenotype is similar to that observed in Drosophila [Mgat1.sup.1] null mutants, demonstrating that Mgat1 is required in the CNS. We also found that neuronal expression of a wild-type Mgat1 transgene rescued the shortened life span of [Mgat1.sup.1] null mutants and resulted in a dramatic 135% increase in mean life span relative to genetically identical controls. Neuronal expression of a wild-type Mgat1 transgene in wild-type flies resulted in a modest 9% increase in mean life span relative to genetically identical controls. In both [Mgat1.sup.1] null mutants and wild-type flies, neuronal expression of wild-type Mgat1 transgene resulted in a significant increase in GnT1 activity and resistance to oxidative stress. Whereas dietary restriction is not absolutely essential for the increased life span, it plays a role in the process. Interestingly, we observe a direct correlation between GnT1 activity and mean life span up to a maximum of ~136 days, showing that the ability of GnT1 activity to increase life span is limited. Altogether, these observations suggest that Mgat1-dependent N-glycosylation plays an important role in the control of Drosophila life span. locomotion | longevity | N-glycosylation | N-acetylglucosaminyltransferase www.pnas.org/cgi/doi/10.1073/pnas.1004431107

To examine the regulation of hepatic acetogenesis in neonatal swine, carnitine palmitoylWansferase I (CPT I) activity was measured in the presence of varying palmitoyl-CoA (substrate) and malonyl-CoA (inhibitor) concentrations, and [1-[sup.14]C]-palmitate oxidation was simultaneously measured. Accumulation rates of [sup.14]C-labeled acetate, ketone bodies, and citric acid cycle intermediates within the acid-soluble products were determined using radio-HPLC. Measurements were conducted in mitochondria isolated from newborn, 24-h (fed or fasted), and 5-mo-old pigs. Acetate rather than ketone bodies was the predominant radiolabeled product, and its production increased twofold with increasing fatty acid oxidation during the first 24-h suckling period. The rate of acetogenesis was directly proportional to CPT I activity. The high activity of CPT I in 24-h-suckling piglets was not attributable to an increase in CPT I gene expression, but rather to a large decrease in the sensitivity of CPT I to malonyl-CoA inhibition, which offset a developmental decrease in affinity of CPIT I for palmitoyl-CoA. Specifically, the [IC.sub.50] for malonyl-CoA inhibition and [K.sub.m] value for palmitoyl-CoA measured in 24-h-suckling pigs were 1.8- and 2.7-fold higher than measured in newborn pigs. The addition of anaplerotic carbon from malate (10 mM) significantly reduced [sup.14]C accumulation in acetate (P < 0.003); moreover, the reduction was much greater in newborn (80%) than in 24-b-fed (72%) and 5-mo-old pigs (55%). The results demonswate that acetate is the primary product of hepatic mitochondrial [beta]-oxidation in Sus scrofa and that regulation during early development is mediated primarily via kinetic modulation of CPT I. acetate; anaplerosis; carnitine palmitoyltransferase I activity; ketone bodies; mitochondria; Sus scrofa doi: 10.1152/ajpregu.00634.2009.

High levels of unconjugated bilirubin (UCB) in newborn children is associated with a reduction in hepatic UDP glucuronosyltransferase (UGT) 1A1 activity that can lead to CNS toxicity, brain damage, and even death. Little is known regarding those events that lead to UCB accumulation in brain tissue, and therefore, we sought to duplicate this condition in mice. The human UGT1 locus, encoding all 9-UGT1A genes including UGT1A1, was expressed in [Ugt1.sup.-/-] mice. Because the most common clinical condition associated with jaundice in adults is Gilbert's syndrome, which is characterized by an allelic polymorphism in the UGT1A1 promoter, hyperbilirubinemia was monitored in humanized UGT1 mice that expressed either the Gilbert's UGT1A1*28 allele [Tg([UGT1.sup.A1*28])[Ugt1.sup.-/-] mice] or the normal UGT1A1*1 allele [Tg ([UGT1.sup.A1*1])[Ugt1.sup.-/-] mice]. Adult Tg([UGT1.sup.A1*28])[Ugt1.sup.-/-] mice expressed elevated levels of total bilirubin (TB) compared with Tg([UGT1.sup.A1*1]) [Ugt1.sup.-/-] mice, confirming that the promoter polymorphism associated with the UGT1A1*28 allele contributes to hyperbilirubinemia in mice. However, TB accumulated to near toxic levels during neonatal development, a finding that is independent of the Gilbert's UGT1A1*28 promoter polymorphism. Whereas serum TB levels eventually returned to adult levels, TB clearance in neonatal mice was not associated with hepatic UGT1A1 expression. In ~10% of the humanized UGT1 mice, peak TB levels culminated in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures were associated with developmental milestones and can be prevented by enhancing regulation of the UGT1A1 gene in neonatal mice. Ugt1 knockout | kernicterus | bilirubin | UGT1A1*28 | Gilbert's Syndrome doi/ 10.1073/pnas.0913290107

A putative prenyltransferase gene sirD has been identified in the gene cluster encoding the biosynthesis of the phytotoxin sirodesmin PL in Leptosphaeria maculans. The gene product was found to comprise 449 aa, with a molecular mass of 51 kDa. In this study, the coding region of sirD was amplified by PCR from cDNA, cloned into pQE70, and overexpressed in Escherichia coil The overproduced protein was purified to apparent homogeneity, and characterized biochemically. The dimeric recombinant SirD was found to catalyse the Q-prenylation of L-Tyr in the presence of dimethylallyl diphosphate; this was demonstrated unequivocally by isolation and structural elucidation of the enzymic product. Therefore, SirD catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL. [K.sub.m] values for L-Tyr and dimethylallyl diphosphate were determined as 0.13 and 0.17 mM, respectively. Interestingly, SirD was found to share significant sequence similarity with indole prenyltransferases, which catalyse prenyl transfer reactions onto different positions of indole rings. In contrast to indole prenyltransferases, which accept indole derivatives, but not Tyr or structures derived thereof, as substrates, SirD also prenylated L-Trp, resulting in the formation of 7-dimethylallyltryptophan. A [K.sub.m] value of 0.23 mM was determined for L-Trp. Turnover numbers of 1.0 and 0.06 [S.sup.-1] were calculated for L-Tyr and L-Trp, respectively. DOI 10.1099/mic.0.033886-0

The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on [C.sub.2] compounds by bypassing the C[O.sub.2]-generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICL- and MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases. DOI 10.1099/mic.0.030858-0

Leptin is an adipose tissue-derived hormone implicated in atherosclerosis and macrophage foam cell formation. The current study was conducted to examine the effect of leptin on cholesteryl ester accumulation in human monocytes/macrophages. Exogenously added leptin at 5 nM during differentiation of monocytes into macrophages for 7 days accelerated acetylated LDL (acetyl-LDL)-induced cholesteryl ester accumulation by 30-50%. Leptin did not affect endocytic uptake of acetyl-LDL; however, it increased ACAT activity 1.8-fold and ACAT-1 protein expression 1.9-fold. Among the four ACAT-1 mRNA transcripts, two shorter transcripts (2.8 and 3.6 kb) were upregulated ~1.7-fold upon leptin treatment. The enhanced expression of ACAT-1 protein by leptin was suppressed by inhibitors of Janus-activated kinase2 (JAK2) and phosphatidylinositol 3-kinase (PI3K). HDL-mediated cholesterol efflux was suppressed by leptin, which was canceled by K-604, an ACAT-1 inhibitor. Expression of long form of leptin receptor was upregulated during monocytic differentiation into macrophages and sustained after differentiation. Thus, the results suggest that leptin accelerates cholesteryl ester accumulation in human monocyte-derived macrophages by increasing ACAT-1 expression via JAK2 and PI3K, thereby suppressing cholesterol efflux. leptin receptor; acyl-coenzyme A:cholesterol acyltransferase-1; acylcoenzyme A:cholesterol acyltransferase inhibitor; atherosclerosis

Transcriptional silencing is a crucial process that is mediated through chromatin structure. The histone deacetylase Sir2 silences genomic regions that include telomeres, ribosomal DNA (rDNA) and the cryptic mating-type loci. Here, we report an unsuspected role for the enzyme Gas1 in locus-specific transcriptional silencing. GAS1 encodes a [beta]-1,3-glucanosyltransferase previously characterized for its role in cell wall biogenesis. In gas1 mutants, telomeric silencing is defective and rDNA silencing is enhanced. We show that the catalytic activity of Gas1 is required for normal silencing, and that Gas1's role in silencing is distinct from its role in cell wall biogenesis. Established hallmarks of silent chromatin, such as Sir2 and Sir3 binding, H4K16 deacetylation, and H3K56 deacetylation, appear unaffected in gas1 mutants. Thus, another event required for telomeric silencing must be influenced by GAS1. Because the catalytic activity of Gas1 is required for telomeric silencing, Gas1 localizes to the nuclear periphery, and Gas1 and Sir2 physically interact, we propose a model in which carbohydrate modification of chromatin components provides a new regulatory element that may be critical for chromatin function but which is virtually unexplored in the current landscape of chromatin analysis. acetylation | beta-glucan | chromatin | S. cerevisiae | telomeres

Aminoacyl-tRNA protein transferases catalyze the post-translational addition of amino acids to proteins. The eubacterial leucyl/phenylalanyl-tRNA-protein transferase (L/F transferase) catalyzes the transfer of leucine or phenylalanine from their respective aminoacylated tRNAs to the N-termini of substrate proteins possessing an N-terminal lysine or arginine amino acid. Conventional assays to quantify L/F transferase activity involve measuring radioactive amino acid incorporation into substrate proteins. We have developed a quantitative matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure to measure the enzymatic activity of L/F transferase. The procedure utilizes stable isotope labeled substrate and internal standard peptides. The method is used to determine the kinetic parameters of [k.sub.cat] and [K.sub.m] for the enzymatic transfer of phenylalanine and three unnatural amino acid derivatives from an aminoacyl-tRNA to a peptide substrate.

UDP-glucuronosyltransferase (UGT) 1A1 glucuronidates bilirubin, estrogens, and xenobiotic compounds. The UGT1A1*28 polymorphism results in lower promoter activity due to 7 thymine-adenine (TA) repeats rather than the more common 6 TA repeats. Previously, we showed that serum bilirubin, a marker of UGT1A1 activity, was lower among individuals homozygous for the UGT1A1*28 polymorphism (7/7) when randomized to a high fruit and vegetable (F&V) diet, whereas there was no effect in individuals with the wild-type (6/6) and heterozygous (6/7) genotypes. Our objective here was to determine if we could detect genotype x diet interactions on bilirubin concentrations in an observational study. Healthy nonsmoking men (n = 146) and women (n = 147), recruited from the Seattle area, provided blood samples for genotyping and bilirubin measurements. We used multiple linear regression to assess the relationships among UGT1A1 genotype, bilirubin concentrations, and consumption of specific F&V [cruciferous vegetables, citrus fruits, and soy foods (n = 268)] based on FFQ and F&V from 6 botanical families [Cruciferae, Rosaceae, Rutaceae, Umbelliferae, Solanaceae, and Leguminosae (n = 261)] based on 3-d food records. We observed a significant interaction of UGTIA1 genotype and citrus consumption among women. Women with the 7/7 genotype who consumed [greater than or equal to] 0.5 daily servings of citrus fruit or foods from the Rutaceae botanical family had ~30% lower serum bilirubin than those with the same genotype who consumed less, whereas 6/6 and 6/7 genotypes dial not differ by consumption (P for interaction = 0.006 and 0.03, respectively). These results suggest that citrus consumption may increase UGT1A1 activity among women with the 7/7 genotype.

Slow Wallerian degeneration ([Wld.sup.S]) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of [Wld.sup.S] remains controversial. In this study, we characterize [Wld.sup.S]-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than [Wld.sup.S], and enzyme-dead versions of Nmnat1 and [Wld.sup.S] exhibit severely reduced axon-protective function. Interestingly, a 16-amino acid N-terminal domain of [Wld.sup.S] (termed N16) accounts for the differences in axon-sparing activity between [Wld.sup.S] and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in [Wld.sup.S] requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, [Wld.sup.S]-mediated suppression of Wallerian degeneration results from VCP-N16 interactions and Nmnatl activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from [Wld.sup.S]. Thus, nuclear Nmnat activity does not appear to be essential for [Wld.sup.S]-like axon protection.

[O.sup.6]-methylguanine ([O.sup.6]meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. [O.sup.6]meG is repaired through its demethylation by the [O.sup.6]-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated [O.sup.6] in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system. N-end rule | proteolysis | Ubr1 | Ufd4 | yeast

Hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyl-transferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. Here we present the crystal structures of Saccharomyces cerevisiae DMATase--t[RNA.sup.Cys] complex in four distinct forms, which provide snapshots of the RNA modification reaction catalyzed by DMATase. The structures reveal that the enzyme recognizes the tRNA substrate through indirect sequence readout. The targeted nucleotide A37 flips out from the anticodon loop of tRNA and flips into a channel in DMATase, where it meets its reaction partner dimethylallyl pyrophosphate, which enters the channel from the opposite end. Structural changes accompanying the transfer reaction taking place in the crystal result in disengagement of DMATase--tRNA interaction near the reaction center. In addition, structural comparison of DMATase in the complex with unliganded bacterial DMATase provides a molecular basis of ordered substrate binding by DMATase. RNA modification | substrate specificity | x-ray crystallography

The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecyiprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.

Peptidoglycan fragments released by Neisseria gonorrhoeae contribute to the inflammation and ciliated cell death associated with gonorrhea and pelvic inflammatory disease. However, little is known about the production and release of these fragments during bacterial growth. Previous studies demonstrated that one lytic transglycosylase, LtgA, was responsible for the production of approximately half of the released peptidoglycan monomers. Systematic mutational analysis of other putative lytic transglycosylase genes identified lytic transglycosylase D (LtgD) as responsible for release of peptidoglycan monomers from gonococci. An ltgA ltgD double mutant was found not to release peptidoglycan monomers and instead released large, soluble peptidoglycan fragments. In pulse-chase experiments, recycled peptidoglycan was not found in cytoplasmic extracts from the ltgA ltgD mutant as it was for the wild-type strain, indicating that generation of anhydro peptidoglycan monomers by lytic transglycosylases facilitates peptidoglycan recycling. The ltgA ltgD double mutant showed no growth abnormalities or cell separation defects, suggesting that these enzymes are involved in pathogenesis but not necessary for normal growth.

Several studies are conducted to explain the oligosaccharyltransferase (OTase)-subunit mutations in the nonsyndromic mental retardation (NSMR). The fine regulation of the OTase activity is shown to be extremely essential for normal cognitive-function development.

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.

Genotoxicity screening sensors that measure DNA damage from metabolism of arylamines were developed and evaluated. The sensors feature ultrathin films containing DNA and N-acetyltransferase (NAT) on pyrolytic graphite (PG) electrodes. NAT in the film catalyzed the conversion of the arylamine 2-aminofluorene (2-AF) to 2-acetylaminofluorene (2-AAF) by acetyl coenzyme A (AcCoA) dependent N-acetylation, as verified by liquid chromatography. DNA damage in the films from exposure to reactive 2-AF metabolites was measured subsequent to the enzyme reaction using catalytic voltammetric oxidation with Ru[(bpy).sub.3.sup.2+]. Square wave voltammetric (SWV) peaks increased with enzyme reaction time, and relative DNA damage rates at pH 5.8 were measured within 2 min. Control incubations of DNA/NAT films without AcCoA gave no significant sensor response. CapLC-MS/MS analysis of 2-AAF/DNA reaction products was consistent with 2-AF-guanine adducts formed in the films. DNA damage occurred more rapidly under weakly acidic conditions (pH 5.5-5.8) than at neutral pH, suggesting that genotoxicity from arylamine metabolism by NAT could be more significant in slightly acidic environments.

The sequences of the capsular biosynthetic (cps) loci of 90 serotypes of Streptococcus pneumoniae have recently been determined. Bioinformatic procedures were used to predict the general functions of 1,973 of the 1,999 gene products and to identify proteins within the same homology group, Pfam family, and CAZy glycosyltransferase family. Correlating eps gene content with the 54 known capsular polysaccharide (CPS) structures provided tentative assignments of the specific functions of the different homology groups of each functional class (regulatory proteins, enzymes for synthesis of CPS constituents, polymerases, flippases, initial sugar transferases, glycosyltransferases [GTs], phosphotransferases, acetyltransferases, and pyruvyltransferases). Assignment of the glycosidic linkages catalyzed by the 342 GTs (92 homology groups) is problematic, but tentative assignments could be made by using this large set of cos loci and CPS structures to correlate the presence of particular GTs with specific glycosidic linkages, by correlating inverting or retaining linkages in CPS repeat units with the inverting or retaining mechanisms of the GTs predicted from their CAZy family membership, and by comparing the CPS structures of serotypes that have very similar cps gene contents. These large-scale comparisons between structure and gene content assigned the linkages catalyzed by 72% of the GTs, and all linkages were assigned in 32 of the serotypes with known repeat unit structures. Clear examples where very similar initial sugar transferases or glycosyltransferases catalyze different linkages in different serotypes were also identified. These assignments should provide a stimulus for biochemical studies to evaluate the reactions that are proposed.

A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coil The soluble [His.sub.6]-fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. [K.sub.m] values were 67 [micro]M for DMAPP, and 137 [micro]M for L-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although [Ca.sup.2+] enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31% at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates L-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.

Results reveal that heterologous expression of Escherichia coli glycosyltransferases in Bacillus subtilis is feasible as shown by the functionality of genes in the heterologous host. Data indicate that the enzyme activity in the B. subtilis is between 20 and 75% of that of E.Coli.

DNA methylation regulates important biological processes and is involved in tumorigenesis and several human diseases, such as Rett and immunodeficiency, centromeric instability and facial anomalies (ICF). The major objective of our research is to investigate the roles of DNA methylation in mammals through genetic analysis of DNA methyltransferase genes in mouse and human. Previously, we found that Dnmt1 knockout embryonic stem (ES) cells are capable of methylating retroviral DNA de novo. In search of enzymes responsible for de novo methylation, we have cloned a novel family of mammalian DNA methyltransferase genes, Dnmt3a and Dnmt3b. Although extensive sequence similarity was found between Dnmt3a and Dnmt3b, little homology was observed between Dnmt1 and Dnmt3a/3b in the catalytic domain as well as in the N-terminal domain. Additionally, biochemical analysis revealed that, unlike Dnmt1, neither Dnmt3a nor Dnmt3b had a strong preference to hemimethylated DNA substrates. Genetic analysis demonstrated that Dnmt3a and Dnmt3b were required for de novo methylation activities in ES cells and during early embryogenesis and were essential for early development. Interestingly, phenotype analyses of single homozygous mice for either Dnmt3a or Dnmt3b suggested that the functions of Dnmt3a and Dnmt3b also were required at the late developmental stage and even at the adult stage. KEY WORDS: * DNA methylation * Dnmt1 * Dnmt3a * Dnmt3b * ES cells * knockout mouse

Research reveals the diversity of the two Helicobacter pylori strains with respect to gene profile and the encoded DNA methyltransferases specificity. Twentyfive open reading frames are involved in the coding of the enzyme as evidenced by restriction analysis, homology searches, gene cloning and genomic mapping of the methylated bases.

In order to determine whether or not inactivation of TRI101, the gene encoding trichothecene 3-O-acetyltransferase in Fusarium sporotrichioides, is lethal and whether or not TRI101 acts in both Fusarium self-protection against trichothecenes and trichothecene biosynthesis, a Fusarium sporotrichioides NRRL 3299 cDNA expression library has been screened in a toxin-sensitive Saccharomyces cerevisiae strain with no functional PDR5 gene. Disruption of TRI101 was not lethal, and deletion mutants accumulated isotrichodermol, a 3-hydroxytrichothecene. TRI101 converts isotrichodermol to isotrichodermin and is necessary for biosynthesis of T-2 toxin. Trichothecenes are antibiotics. Their biosynthesis would likely require special protective adaptation by organisms that produce them.