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DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-l-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-3H]AdoMet. A 5′-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1-1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter kcat but not Km for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine. [ABSTRACT FROM AUTHOR]

Although genetic mutagenicity of the chemical ethylmethane sulfonate (EMS) is well established, it remains unclear whether and to what extent this compound is epigenetically mutagenic. This issue was studied in L. (rice) by conducting a parallel investigation using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), on two sets of EMS-treated M0 plants: phenotypically normal and altered. Although more methylation than genetic changes were induced by the EMS-treatments in both sets of plants, the greatest differences were observed in the set of plants with altered phenotypes, suggesting a role of induced epigenetic variation in causing the phenotypic novelties. Moreover, the frequencies of the two types of instabilities induced by the EMS treatment, genetic and epigenetic, are correlated, implicating a common or overlapping mutagenic basis. [ABSTRACT FROM AUTHOR]

Glycine-N methyltransferase (GNMT) is a potential tumor suppressor that is commonly inactivated in human hepatoma. We systematicalIy investigated how GNMT regulates methyl group kinetics and global DNA methylation. HepG2 cells (GNMT inactive, GNMT-) and cells transfected with GNMT expressed vector (GNMT+) were cultured in low (10 μmol/L), adequate (100 μmol/L(, or high (500 μmol/L) L-methionine, each with 2.27 μmol/L folate. Transmethylation kinetics were studied using stable isotopic tracers and GC-MS. Methylation status was determined by S-adenosylmethionine (SAM) and S-adenosylhomocyrteine (SAH) levels, SAM:SAH ratio, DNA methyltransferase (DNMT) activity, and methylated cytidine levels in DNA. Compared with GNMT- cells, GNMT+ cells had lower homocysteine and greater cysteine concentrations. GNMT expression increased methionine clearance by inducing homocysteine transsulfuration and remethylation metabolic fluxes when cells were cultured in high or adequate L-methionine. In contrast, homocysteine remethylation flux was lower in GNMT+ cells than in GNMT- cells and homocysteine transsulfuration fluxes did not differ when cells were cultured in low methionine, suggesting that normal GNMT function helps to conserve methyl groups. Furthermore, GNMT expression decreased SAM and increased SAH levels and reduced DNMT activity in high or adequate, but not low, methionine cultures. In low methionine cultures, restoring GNMT in HepG2 cells did not lead to sarcosine synthesis, which would waste methyl groups. Methylated cytidine levels were significantly lower in GNMT- cells than in GNMT+ cells. In conclusion, we have shown that GNMT affects transmethylation kinetics and SAM synthesis and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes. [ABSTRACT FROM AUTHOR]

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase. [ABSTRACT FROM AUTHOR]

Intraspecific hybrids between the Arabidopsis thaliana accessions C24 and Landsberg erecta have strong heterosis. The reciprocal hybrids show a decreased level of 24-nt small RNA (sRNA) relative to the parents with the decrease greatest for those loci where the parents had markedly different 24-nt sRNA levels. The genomic regions with reduced 24-nt sRNA levels were largely associated with genes and their flanking regions indicating a potential effect on gene expression. We identified several examples of genes with altered 24-nt sRNA levels that showed correlated changes in DNA methylation and expression levels. We suggest that such epigenetically generated differences in gene activity may contribute to hybrid vigor and that the epigenetic diversity between ecotypes provides increased allelic (epi-allelic) variability that could contribute to heterosis. [ABSTRACT FROM AUTHOR]

Abstract: Rapid identification of unknown microorganisms of clinical and agricultural importance is not only critical for accurate diagnosis of infections but also essential for appropriate and prompt treatment. We describe here a rapid method for microorganisms typing based on quantitative analysis of fatty acids by iFAT approach (Isotope-coded Fatty Acid Transmethylation). In this work, lyophilized cell lysates were directly mixed with 0.5M NaOH solution in d3-methanol and n-hexane. After 1min of ultrasonication, the top n-hexane layer was combined with a mixture of standard d0-methanol derived fatty acid methylesters with known concentration. Measurement of intensity ratios of d3/d0 labeled fragment ion and molecular ion pairs at the corresponding target fatty acids provides a quantitative basis for hierarchical clustering. In the resultant dendrogram, the Euclidean distance between unknown species and known species quantitatively reveals their differences or shared similarities in fatty acid related pathways. It is of particular interest to apply this method for typing fungal species because fungi has distinguished lipid biosynthetic pathways that have been targeted for lots of drugs or fungicides compared with bacteria and animals. The proposed method has no dependence on the availability of genome or proteome databases. Therefore, it is can be applicable for a broad range of unknown microorganisms or mutant species. [Copyright &y& Elsevier]

The article presents information on a study on over 9,500 human proteins and developed and optimized a system for proteome-wide identification of novel methylation events that was catalyzed by the protein lysine methyltransferase (PKMT) SETD6. The study identified novel candidate substrates for SETD6 by using two independent detection approaches The study also verified that all targets tested in vitro as well as in cells were genuine substrates. The study described a novel proteome-wide methodology for identifying the new PKMT substrates.

Following its tyrosine phosphorylation. STAT3 is methylated on k140 by the histone methyl transferase SET9 and demethylated by LSD1 when it is bound to a subset of the promoters that it activates. Methylation of K140 is a negative regulatory event, because its blockade greatly increases the steady-state amount of activated STAT3 and the expression of many (i.e., SOCS3) but not all (i.e., CD 14) STAT3 target genes. Biological relevance is shown by the observation that overexpression of SOCS3 when k140 cannot be methylatod blocks the ability of cells to activate STAT3 in response to IL-6. 1(140 methylation does not occur with mutants of STAT3 that do not enter nuclei or bind to DNA. Following treatment with lL-6, events at the SOCS3 promoter occur in an ordered sequence, as shown by chromatin immunoprecipitations. Y705-phosphoryl- STAT3 binds first and 5727 is then phosphorylated, followed by the coincident binding of SET9 and dimethylation of K140, and lastly by the binding of LSD1. We conclude that the lysine methylation of promoter-bound STAT3 leads to biologically important down-regulation of the dependent responses and that SET9. which is known to help provide an activating methylation mark to H3K4, is recruited to the newly activated SOCS3 promoter by STAT3. [ABSTRACT FROM AUTHOR]

Abstract: The thermodynamic stability of methylphenanthrene isomers and the kinetics of reactions involved in their natural destruction/formation have been studied via molecular modelling. The results were combined with published methylphenanthrene abundances in extracts of rocks containing Type III kerogen of a wide maturity range, to evaluate the utility of methylphenanthrene maturity parameters. These parameters track the isomer evolution toward equilibrium, leading to enrichment in the stable 2- and 3-methyl isomers relative to the less stable 9-, 1- and 4-methyl isomers. The pathways from kinetically to thermodynamically controlled distributions remain unclear but probably involve isomerisation, transmethylation and demethylation reactions. To understand the importance of each pathway, ab initio quantum chemical calculations (DFT) have been performed, leading to the identification of possible transition states and to the determination of activation energies for reactions in aqueous solutions. Acid catalysis significantly lowers modelled reaction barriers to an extent consistent with the changes observed in nature. The equilibration can start at very low maturation with acid catalysed 1,2-methyl shifts (isomerisations) regarding low energy barriers: 15.1kcal/mol from 4- to 3-, 22.5kcal/mol from 1- to 2- and 30.2kcal/mol from 3- to 2-methylphenanthrene. Alternative isomerisation pathways through tertiary carbon centres are ineffective. For example the isomerisation between the 9- and 1-isomer requires 39.7kcal/mol, which is ca. 7kcal/mol more than for demethylation coupled with methyl transfer to another molecule (transmethylation). A reverse transmethylation, e.g. modelled methylations of phenanthrene with either a terpenoid alcohol with a gem-dimethyl or polymethyl aryl carotenoid moiety can preferentially lead to 9-methylphenanthrene due to a relatively lower energy barrier. The appearance of 9-methylphenanthrene at very early maturation stages suggests effective heterogenic catalysis by the mineral matrix. The reaction is however reversible, which means that 9-methylphenanthrene tends to demethylate with much higher rates than the other isomers but only when suitable methyl acceptors are available. In turn, free radical demethylation which is likely at advanced levels of maturity would not significantly influence relative proportions of the isomers due to very similar barriers for all the isomers. However the reverse reaction would produce all isomers thus equalising all isomer concentrations. The discussed reactions are only a few of many reactions in which phenanthrene and methylphenanthrenes are potentially involved in nature to mention only biotic (biodegradation) and abiotic methylphenanthrene demethylation, and that there is no a single reaction that determines the fate of these molecules. Therefore, the commonly applied maturity index MPI-1 does not entirely reflect thermal maturity but is instead a molecular expression of complex processes and is dependent partly on catalytic effects of the mineral matrix. Analysis of the published data on methylphenanthrene abundance in rocks containing Type III kerogens indicates that there is no inversion of the MPI-1 trend above R r =1.35% as has been suggested previously. The maturity parameter MPR, defined as the 2-/1-MP ratio seems more closely dependant on thermal maturity than MPI-1. [Copyright &y& Elsevier]

Memory formation and storage require long-lasting changes in memory-related neuronal circuits. Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for an epigenetic mechanism in learning and long-term memory maintenance and raise apparent conundrums and questions. For example, it is unclear how DNA methylation might be reversed during the formation of a memory, how changes in DNA methylation alter neuronal function to promote memory formation, and how DNA methylation patterns differ between neuronal structures to enable both consolidation and storage of memories. Here we evaluate the existing evidence supporting a role for DNA methylation in memory, discuss how DNA methylation may affect genetic and neuronal function to contribute to behavior, propose several future directions for the emerging subfield of neuroepigenetics, and begin to address some of the broader implications of this work. [ABSTRACT FROM AUTHOR]

Background: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nϵ-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nϵ-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal Findings: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation. [ABSTRACT FROM AUTHOR]

Experimental and clinical data support a growth inhibitory role for HER4 in breast cancer. Clinically HER4 expression is extinguished during breast tumorigenesis supporting a tumor suppressor function for HER4, however, a molecular mechanism to explain the selective loss of HER4 expression has remained elusive. Epigenetic mechanisms, for example, aberrant gene promoter hypermethylation, have been shown to ablate tumor suppressor gene expression in breast carcinomas. We identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite-treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Treatment of the HER4-negative BT20 cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC)-enhanced HER4 expression, confirming a role for DNA methylation in suppressed HER4 expression. DAC treatment to reactive HER4 expression in combination with the HER4 ligand heregulin-β1 (HRG) resulted in apoptosis of BT20 cells providing a novel therapeutic strategy for triple-negative tumors. The BT20 cells were rescued from apoptosis when preincubated with HER4 small interfering RNA, thereby confirming a role for HER4 in DAC/HRG-induced apoptosis. We verified HER4 promoter methylation in primary breast carcinomas and detected a significant increase in HER4 promoter methylation in HER4-negative breast tumors (P<0.001). Furthermore, increased levels of HER4 promoter methylation were significantly associated with worse patient prognosis (P=0.0234). Taken together, our data support a tumor suppressor function for HER4, which is epigenetically suppressed in breast tumors through promoter hypermethylation. [ABSTRACT FROM AUTHOR]

Abstract: Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16–22g) were treated with vehicle (Veh; water 15ml/kg ip injections), 250mg/kg APAP (15ml/kg, ip), SAMe (1.25mmol/kg) or SAMe administered 1h after APAP injection (SAMe and SAMe+APAP). Hepatic tissue was collected 2, 4, and 6h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4h by APAP overdose, but not at 2 or 6h. APAP depressed mitochondrial SAMe levels at 4 and 6h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4h following APAP administration. SAMe administration following APAP (SAMe+APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity. [Copyright &y& Elsevier]

Abstract: Narrow-bore GC columns have led to the resolution of complex matrices in a few minutes. To set up a method able to cope with the velocity of ultra fast gas-chromatographic separation, a comparison among four of the most popular lipid transmethylation was applied to pork fat. Different catalysts for transmethylation were tested (NaOCH3, BF3, H2SO4, and KOH), and were compared for yield and rate of transmethylation, cost, and safety. In all cases, KOH gave the best performance of all the considered parameters. For this reason, it is particularly suitable when a large number of samples are being analysed. Although it is very effective in the transesterification of neutral lipids, it does not tolerate water and is unsuitable for free fatty acid derivatization. [Copyright &y& Elsevier]

This study was an investigation of 79 patients referred to the Wessex Regional Genetics Laboratory with suspected Russell–Silver Syndrome or unexplained short stature/intra uterine growth restriction, warranting genetic investigation. Methylation status was analysed at target sequences within eleven imprinted loci (PLAGL1, IGF2R, PEG10, MEST1, GRB10, KCNQ1OT1, H19, IGF2P0, DLK1, PEG3, NESPAS). Thirty seven percent (37%) (29 of 79) of samples were shown to have a methylation abnormality. The commonest finding was a loss of methylation at H19 (23 of 29), as previously reported in Russell–Silver Syndrome. In addition, four of these patients had methylation anomalies at other loci, of whom two showed hypomethylation of multiple imprinted loci, and two showed a complete gain of methylation at IGF2R. This latter finding was also present in five other patients who did not have demonstrable changes at H19. In total, 7 of 79 patients showed a gain of methylation at IGF2R and this was significantly different from a normal control population of 267 individuals (P=0.002). This study in patients with growth restriction shows the importance of widening the epigenetic investigation to include multiple imprinted loci and highlights potential involvement of the IGF2R locus. [ABSTRACT FROM AUTHOR]

AHCY deiciency is a new human methylation disorder, discovered recently in Croatia, and a natural model for investigating processes related to the methylome. Methylation plays an important role in regulating biological processes and is crucial for gene expression, imprinting, signalling, protein synthesis and lipid metabolism. Thus, methylation has broad impact and provides a suitable base for interdisciplinary research. Linking genomics, proteomics, cellomics, lipidomics and metabolomics and other omics approaches may create a new research avenue - 'AHCYdomics' - a new methylation research platform based on AHCY deiciency. Using such research platform will allow to eficiently explore the full potential of the human methylation disorder AHCY deficiency, and to design methods and approaches that will lead to a better understanding of the human methylome. [ABSTRACT FROM AUTHOR]

Restriction–modification (R-M) systems are exclusive to unicellular organisms and ubiquitous in the bacterial world. Bacteria use R-M systems as a defense against invasion by foreign DNA. Analysis of the genome sequences of Helicobacter pylori strains 26 695 and J99 identified an extraordinary number of genes with homology to R-M genes in other bacterial species. All H. pylori strains possess their own unique complement of active R-M systems. All of the methylases that have been studied so far were present in all major human population groupings, suggesting that their horizontal acquisition pre-dated the separation of these populations. The two most strongly conserved methylase genes of H. pylori, hpy IM and hpy IIIM, are both preceded by alternative genes that compete for presence at their loci, and furthermore these genes may be associated with H. pylori pathogenicity. Further study should investigate the roles of H. pylori R-M systems. [ABSTRACT FROM AUTHOR]

The article presents an examination into the history of mid-20th century Canadian psychology, focusing on the innovations of researchers Abram Hoffer and Humphry F. Osmond regarding their 1952 adrenocrome hypothesis of biogenesis for schizophrenia. Details are given overviewing the study presented by Hoffer and Osmond, critically examining the ideological framework of the theory, and offering an integrative analysis of the system into contemporary psychopharmacology by adapting it to the transmethylation theory.

This study was designed to analyze the effect of global and gene-specific DNA methylation patterns on the outcome of patients with acute myeloid leukemia (AML). Methylation of CDKN2B (p15), E-cadherin (CDH) and hypermethylated in cancer 1 (HIC1) promoters and global DNA methylation by luminometric methylation assay (LUMA) was analyzed in 107 AML patients and cytogenetic and molecular mutational analysis was performed. In addition, genome-wide promoter-associated methylation was assessed using the Illumina HumanMethylation27 array in a proportion of the patients. Promoter methylation was discovered in 66, 66 and 51% of the patients for p15, CDH and HIC1, respectively. In multivariate analysis, low global DNA methylation was associated with higher complete remission rate (hazard ratio (HR) 5.9, P=0.005) and p15 methylation was associated with better overall (HR 0.4, P=0.001) and disease-free survival (HR 0.4, P=0.016). CDH and HIC1 methylation were not associated with clinical outcome. Mutational status and karyotype were not significantly associated with gene-specific methylation or global methylation. Increased genome-wide promoter-associated methylation was associated with better overall and disease-free survival as well as with LUMA hypomethylation. We conclude that global and gene-specific methylation patterns are independently associated with the clinical outcome in AML patients. [ABSTRACT FROM AUTHOR]

Abstract: T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine (SAM) levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22–81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age ∼50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly. [Copyright &y& Elsevier]

Abstract: To evaluate the qualitative and quantitative information obtained through three commonly used methods for cutin analysis in plants and soils (saponification, CuO oxidation and transmethylation) and understand the chemical mechanisms involved during the depolymerisation, we compared the three methods using a sample of maize leaves, where cutin might be more intact and quantitatively more important than in soil. Among the aliphatic monomers identified with the different methods, special attention was paid to the hydroxy acids and diacids with a chain length of 16 and 18 carbons, which are considered to be specific for cutin. CuO oxidation afforded seven cutin structural units in low concentration (0.41mgg−1 dry leaves) possibly because of chemical alteration of the monomers under the drastic conditions. Transmethylation afforded only four structural units (0.37mgg−1 dry leaves), possibly because of steric exclusion of the reagent from the more cross-linked parts of the macromolecules. Saponification, affording the highest concentration (1.7mgg−1 dry leaves), including eight different compounds, was also successfully applied to a loamy maize-cultivated soil with low carbon content (12.9mgOCg−1 soil). Except for 9,10-dihydroxyoctadecanoic acid, released from maize leaves, all the structural units specific for maize cutin could be identified and reached a concentration of 19.6μgg−1 soil. The concentrations of the monomers were estimated in soil using this method, with the coefficient of variation ranging from 2% to 9%. [Copyright &y& Elsevier]

The article presents a study which examines the kinetics of methionine and the rate of transsulfuration and transmethylation in healthy women with advancing gestation. Measured where the whole-body rate of appearance of methionine and phenylalanine among health pregnant women during the first, second, and third trimesters of pregnancy. Results indicate that uncomplicated pregnancy in humans is linked with a higher rate of transsulfuration and transmethylation of methionine in gestation.

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS-AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F1 individuals. Genetically identical offspring of asexual F1 plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long-established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions. [ABSTRACT FROM AUTHOR]

Abstract: Hyperhomocysteinaemia was reported in patients with Parkinson''s disease (PD) treated with l-Dopa. The increase in plasma concentration of this sulfur compound arises from the massive methylation of the drug operated by the enzyme catechol-O-methyltransferase (COMT), which acts as a powerful sink of methyl groups. The contemporary occurrence of C677T polymorphism in homozygosity, leading to a temperature-labile variant of the MTHFR enzyme, induces an even more marked increase in tHcy. Here we show that l-Dopa administration in hyperhomocysteinemic PD patients is able to lower intracellular concentration of S-Adenosylmethionine (AdoMet) in erythrocytes (RBC), while the occurrence of hyperhomocysteinaemia causes a significant increase in S-Adenosylhomocysteine (AdoHcy) level. In patients with PD treated with l-Dopa and hyperhomocysteinemic, the remarkable decrease in AdoMet and the concurrent increase in AdoHcy concentration both contribute to significantly lower the transmethylation potential ([AdoMet]/[AdoHcy]), a useful index of the effectiveness of methyl group transfer by methyltransferases. This decrease could indeed contribute to partly attenuate, through a self-limiting kinetic mechanism, the tendency of developing drug resistance, partly mediated in these patients by COMT upregulation. Our results also support the conclusion that COMT inhibitors (entacapone or tolcapone), when administered in PD patients treated with l-Dopa, may potentiate the endogenous AdoHcy-dependent COMT inhibition mechanism already operative in a variable fashion. [Copyright &y& Elsevier]

DNA methylation marks, a key modification of imprinting, are erased in primordial germ cells and sex specifically re-established during gametogenesis. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. We analysed by pyrosequencing the DNA methylation status of 47 CpGs located in differentially methylated regions (DMRs), the DMR0 and DMR2 of the IGF2 gene and in the 3rd and 6th CTCF-binding sites of the H19 DMR in human sperm from men with normal semen and patients with teratozoospermia (T) and/or oligo-astheno-teratozoospermia (OAT). All normal semen samples presented the expected high global methylation level for all CpGs analysed. In the teratozoospermia group, 11 of 19 patients presented a loss of methylation at variable CpG positions either in the IGF2 DMR2 or in both the IGF2 DMR2 and the 6th CTCF of the H19 DMR. In the OAT group, 16 of 22 patients presented a severe loss of methylation of the 6th CTCF, closely correlated with sperm concentration. The methylation state of DMR0 and of the 3rd CTCF was never affected by the pathological status of sperm samples. This study demonstrates that epigenetic perturbations of the 6th CTCF site of the H19 DMR might be a relevant biomarker for quantitative defects of spermatogenesis in humans. Moreover, we defined a methylation threshold sustaining the classification of patients in two groups, unmethylated and methylated. Using this new classification of patients, the observed intrinsic imprinting defects of spermatozoa appear not to impair significantly the outcome of assisted reproductive technologies. [ABSTRACT FROM AUTHOR]

SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. [ABSTRACT FROM AUTHOR]

The methylation reaction of alkyl N-acylthiocarbamates in DMF with dimethyl sulfate in the presence of potassium carbonate takes place with high selectivity affording the S-methylated derivative as the principal reaction product. No isomeric reaction products derived from the N- or the O-methylation were isolable. The new carbonimidothioates 3a-g synthesised were fully characterized by spectroscopic methods. The experimental findings are supported by X-ray and theoretical studies at the B3LYP/6-311G(d.p) level. [ABSTRACT FROM AUTHOR]

Epigenetic modifications play a key role in the patho-physiology of many tumors and the current use of agents targeting epigenetic changes has become a topic of intense interest in cancer research. DNA methyltransferase (DNMT) inhibitors represent a promising class of epigenetic modulators. Research performed yielded promising anti-tumorigenic activity for these agents in vitro and in vivo against a variety of hematologic and solid tumors. These epigenetic modulators cause cell cycle and growth arrest, differentiation and apoptosis. Rationale for combining these agents with cytotoxic therapy or radiation is straightforward since the use of DNMT inhibitor offers greatly improved access for cytotoxic agents or radiation for targeting DNA-protein complex. The positive results obtained with these combined approaches in preclinical cancer models demonstrate the potential impact DNMT inhibitors may have in treatments of different cancer types. Therefore, as the emerging interest in use of DNMT inhibitors as a potential chemo- or radiation sensitizers is constantly increasing, further clinical investigations are inevitable in order to finalize and confirm the consistency of current observations. The present article will provide a brief review of the biological significance and rationale for the clinical potential of DNMT inhibitors in combination with other chemotherapeutics or ionizing radiation. The molecular basis and mechanisms of action for these combined treatments will be discussed herein. [ABSTRACT FROM AUTHOR]

Background: The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells. Results: We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity in vitro. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme. Conclusion: The identification of amodiaquine as an inhibitor of PfPMT in vitro and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs. [ABSTRACT FROM AUTHOR]

There is an increased prevalence of imprinting disorders, such as Beckwith–Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations. [ABSTRACT FROM AUTHOR]

Abstract: Tributyltin (TBT) and triphenyltin (TPT) coexist in freshwater and marine environments. However, the effects of TBT, TPT, and a mixture of the two on DNA methylation in marine fish livers and the mechanism involved remain to be elucidated. Previous study have proved that abnormal methylation patterns are induced by the balance of transmethylation reaction including the tissue level of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) or the activity of DNA (cytosine-5) methyltransferase 1 (DNMT1). Therefore, in the present study, we assessed their ability to cause hepatic DNA hypomethylation in Sebastiscus marmoratus liver and the related mechanism. The results showed that TBT, TPT, and a mixture of the two significantly induced DNA hypomethylation in the fish livers in a dose-dependent manner. Using Pearson correlation coefficient analysis, we identified strong linear correlations between S-adenosylhomocysteine, S-adenosylmethionine, or the SAM to SAH ratio and the hepatic genome-wide 5-methylcytosine content of the DNA, but no correlation between the latter and the DNMT1 expression level. It is therefore proposed that the organotins hypomethylation induced in the marine fish livers was due to altering the balance of the substrate and the product in transmethylation reactions. [Copyright &y& Elsevier]

A simple synthetic route to cinnamyl-sesamols has been developed starting from sesamol via Claisen rearrangement followed by olefin metathesis reaction. [ABSTRACT FROM AUTHOR]

A resin sample was found in the archaeological site of Lixus (Morocco), belonging to the second century BC. The resinous material was found inside an amphora containing iron remains used in the plug as sealing material to hermetically close the pottery. The resinous sample was studied by several analytical techniques, as thermally assisted hydrolysis and methylation-gas chromatography-mass spectrometry (THM-GC-MS), Fourier transformed infrared spectroscopy (FTIR), and light microscopy. The material was identified as a Pinaceae resin. Therefore, a modern pine resin was also analyzed to consider the influence of aging in the archaeological sample. The ancient material was found not too oxidized, owing to the conservation conditions inside the amphora, protected from air and moisture. [ABSTRACT FROM AUTHOR]

Abstract: Lipoic acid is a disulfhydryl-containing compound used in clinical medicine and in experimental models as an antioxidant. We developed a stable isotope dilution capillary gas chromatography/mass spectrometry assay for lipoic acid. We assayed a panel of the metabolites of transmethylation and transsulfuration 30 min after injecting 100 mg/kg lipoic acid in a rat model. Lipoic acid values rose 1000-fold in serum and 10-fold in liver. A methylated metabolite of lipoic acid was also detected but not quantitated. Lipoic acid injection caused a massive increase in serum S-adenosylhomocysteine and marked depletion of liver S-adenosylmethionine. Serum total cysteine was depleted but liver cysteine and glutathione were maintained. Serum total homocysteine doubled, with increases also in cystathionine, N,N-dimethylglycine, and α-aminobutyric acid. In contrast, after injection of 2-mercaptoethane sulfonic acid, serum total cysteine and homocysteine were markedly depleted and there were no effects on serum S-adenosylmethionine or S-adenosylhomocysteine. We conclude that large doses of lipoic acid displace sulfhydryls from binding sites, resulting in depletion of serum cysteine, but also pose a methylation burden with severe depletion of liver S-adenosylmethionine and massive release of S-adenosylhomocysteine. These changes may have previously unrecognized deleterious effects that should be investigated in both human disease and experimental models. [Copyright &y& Elsevier]

Abstract: Changes in the level of lipid composition in a single zebrafish egg have been involved in biological responses to chemical exposures. In this paper, an one-step transmethylation of lipids and simultaneous extraction of resultant fatty acid methyl esters followed by gas chromatography–mass spectrometric analysis was developed for the identification of bonded long chain fatty acids in a single zebrafish egg. The efficiency of transmethylation under different experimental conditions has been investigated. Dried egg homogenates were directly mixed with either 0.5M NaOH or 1% H2SO4 or 4% HCl solution of methanol and then an n-hexane solution was added on the top. Ultrasonication of this immiscible liquid–liquid–solid system produces high velocity impacts between solid particles and liquid phases and thus promotes mass transfer among phases. It was demonstrated that ultrasound irradiation has strong effect on the alkaline-catalyzed transmethylation of lipids but cannot significantly change the acid-catalyzed transmethylation of lipids. With the aid of ultrasonication, transmethylation can be combined with simultaneous extraction of the resultant fatty acid methyl esters into n-hexane phase. This approach simplifies the sample preparation procedure, shortens the reaction time but improves the efficiency of the transmethylation of lipids and reduces sample losses, especially for small size samples. It has been applied to determine bonded fatty acids in a single zebrafish egg. In total, 28 fatty acids from a single zebrafish egg have been identified reproducibly. [Copyright &y& Elsevier]

Aim: Hyperhomocysteinaemia, diagnosed by serum levels, is regarded as an independent risk indicator for cardiovascular events and is associated with various diseases. The pathomechanisms seem to be partly due to concentrations of homocysteine metabolites and their effect on the cellular transmethylation processes. Methods: We compared two common models for experimental hyperhomocysteinaemia – high methionine diet and homocystine-enriched diet – regarding their effects on tissue concentrations of homocysteine metabolites. Results: Both diets induced hyperhomocysteinaemia without affecting renal function or vitamine status. However, the tissue contents of homocysteine and its precursors S-adenosyl-homocysteine (SAH) and S-adenosyl-methionine exhibited major differences between both models. Transmethylation potential was elevated 1.7-fold in liver of rats fed the methionine diet, whereas it was unaltered after homocystine-enriched diet. Kidneys of rats fed the methionine diet did not show any alterations in tissue content of homocysteine and its precursors, whereas in the homocystine group homocysteine and the transmethylation inhibitor SAH were elevated from 23.1 ± 10.4 to 78.0 ± 26.0 nmol g−1 and from 106 ± 4 to 170 ± 22 nmol g−1 respectively. Homocysteine tissue content was elevated in the homocystine, but not in the methionine group. Conclusions: Alterations to homocysteine metabolism are distinct in both models. These findings may explain divergent results, which have been published for these models of hyperhomocysteinaemia and which have resulted in controversial discussions in the past. [ABSTRACT FROM AUTHOR]

Senescence is the process of cellular aging dependent on the normal physiological functions of non-immortalized cells. With increasing data being uncovered in this field, the complex molecular web regulating senescence is gradually being unraveled. Recent studies have suggested two main phases of senescence, the triggering of senescence and the maintenance of senescence. Each has been supported by data implying precise roles for DNA methyltransferases, reactive oxygen species and other factors. We will first summarize the data supporting these claims and then highlight the specific role that we hypothesize that p130/Rbl2 plays in the modulation of the senescence process. [ABSTRACT FROM AUTHOR]

Epigenetic alterations, including gain or loss of DNA methylation, are a hallmark of nearly every malignancy. Changes in DNA methylation can impact expression of cancer-related genes including apoptosis regulators and tumor suppressors. Because such epigenetic changes are reversible, they are being aggressively investigated as potential therapeutic targets. Here we use the Eμ-TCL1 transgenic mouse model of chronic lymphocytic leukemia (CLL) to determine the timing and patterns of aberrant DNA methylation, and to investigate the mechanisms that lead to aberrant DNA methylation. We show that CLL cells from Eμ-TCL1 mice at various stages recapitulate epigenetic alterations seen in human CLL. Aberrant methylation of promoter sequences is observed as early as 3 months of age in these animals, well before disease onset. Abnormally methylated promoter regions include binding sites for the transcription factor FOXD3. We show that loss of Foxd3 expression due to an NF-κB p50/p50:HDAC1 repressor complex occurs in TCL1-positive B cells before methylation. Therefore, specific transcriptional repression is an early event leading to epigenetic silencing of target genes in murine and human CLL. These results provide strong rationale for the development of strategies to target NF-κB components in CLL and potentially other B-cell malignancies. [ABSTRACT FROM AUTHOR]

A novel method to improve the detection of protein ions using a linear ion trap mass spectrometer is presented. A scan function combining charge separation with segmented transmission of multiply charged ions was developed to enhance the sensitivity and resolution of the linear ion trap for the nanoLC-MS analysis of intact proteins. The analytical benefits of the present method are particularly apparent in protein analyses, where the increased proportion of multiply charged ions can exacerbate space-charge effects and compromise the dynamic range of the linear ion trap instrument The enhanced ion storage and charge separation capabilities of our targeted and enhanced multiply charged scan mode provided a 4-fold increase in signal-to-noise and 5-fold increase in resolution, thus enabling the detection of closely related protein isoforms. The application of this method is demonstrated for low femtomole detection of protein standards and nuclear extracts enriched in histone proteins The enhanced resolution of this scan mode also enabled us to monitor subtle changes in the methylation of a subpopulation of histone H3 that occurs in chicken DT40 cells lacking specific methyltransferase activity. The extent of the fold change and PTM site localization was performed using predictive software tools and targeted multiple reaction monitoring analysis of histone peptides. Monomethylation of los 79 in histone H3 (H3K79me1) was down regulated by 240-fold in methyltransferase deficient cells. [ABSTRACT FROM AUTHOR]

Background/Aims: Transmethylation reactions and antioxidant metabolism are linked by transsulfuration, where homocysteine (Hcy) is converted to cysteine and reduced glutathione (GSH). Low protein intake can modulate the balance of this metabolic reaction. The aim of the present investigation was to study the effect of a low-protein diet on Hcy metabolism by monitoring levels of the amino acids involved in these pathways, and relating these levels to GSH levels and lipid peroxidation in rats. Methods: Sixteen rats were divided into 2 groups: control (C; standard AIN-93 diet, 20% protein) and low-protein diet (LPD; 8% protein diet). Rats in both groups were placed on the diets for 28 days. Results: A significant reduction (p < 0.05) in plasma Hcy concentration was found in LPD rats (0.16 ± 0.04 μmol/mg protein) versus C rats (0.25 ± 0.03μmol/mg protein). Methionine levels were not significantly different between the 2 groups (C: 1.24 ± 0.22 μmol/mg protein; LPD: 1.03 ± 0.27 μmol/mg protein). A significant reduction (p < 0.05) in hepatic GSH concentrations (C: 44 ± 10 μmol/mg protein; LPD: 17.4 ± 4.3 μmol/mg protein) was accompanied by an increase in lipid peroxidation (C: 0.13 ± 0.01 μmol/mg protein; LPD: 0.17 ± 0.02 μmol/mg protein; r = –0.62, p < 0.01). Conclusion: Hcy levels were reduced under a low-protein diet, resulting in modulated methyl balance and reduced GSH formation leading to increased susceptibility of hepatic cells to oxidative events. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Centric regions of eukaryotic genomes are packaged into heterochromatin, which possesses the ability to spread along the chromosome and silence gene expression. The process of spreading has been challenging to study at the molecular level due to repetitious sequences within centric regions. A heterochromatin protein 1 (HP1) tethering system was developed that generates "ectopic heterochromatin" at sites within euchromatic regions of the Drosophila melanogaster genome. Using this system, we show that HP1 dimerization and the PxVxL interaction platform formed by dimerization of the HP1 chromo shadow domain are necessary for spreading to a downstream reporter gene located 3.7kb away. Surprisingly, either the HP1 chromo domain or the chromo shadow domain alone is sufficient for spreading and silencing at a downstream reporter gene located 1.9 kb away. Spreading is dependent on at least two H3K9 methyltransfera.ses, with SU(VAR)3-9 playing a greater role at the 3.7-kb reporter and dSETDB1 predominately acting at the 1.9 kb reporter. These data support a model whereby HP1 takes part in multiple mechanisms of silencing and spreading. [ABSTRACT FROM AUTHOR]

Abstract: S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity (“methylation index”). We present a rapid and sensitive LC–MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column (30mm×2.1mm, 3μm particle size) filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC–MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98–105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4°C for 5min and at 25°C for 2min, respectively. Storage of liver tissues at −80°C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues. [Copyright &y& Elsevier]

Human catechol O-methyltransferase (COMT) contains three common polymorphisms (A22S, AS2T, and VIOSM), two of which (A22S and V108M) render the protein susceptible to deactivation by temperature or oxidation. We have performed multiple molecular dynamics simulations of the wild-type, A22S, A52T, and V108M COMT proteins to explore the structural consequences of these mutations In total, we have amassed more than 1.4μs of simulation time, representing the largest set of simulations detailing the feets of polymorphisms on a protein system to date The A52T mutation had no significant effect on COMT structure in accord with experiment, thereby serving as a good negative control for the simulation set Residues 22 (α2) and 108 (α5) interact with each other throughout the simulations and are located in a polymorphic hotspot ∼20 A from the active site. Introduction of either the larger Ser (22) or Met (108) tightens this interaction, pulling α2 and α5 toward each other and away from the protein core. The V108M polymorphism rearranges active-site residues in α5, β3, and β6, increasing the S-adenosylmethionine site solvent exposure. The A22S mutation reorients α2, moving critical carechol-binding residues away from the substrate-binding pocket The A22S and V108M polymorphisms evolved independently in Northern European and Asian population. While the decreased activities of both A22S and V108M COMT are associated with an increases risk for schizophrenia, the V108M-induced destabilization is also linked with improved cognitive function. These results suggest that polymorphisms within this hotspot may have evolved to regulate COMT activity and that heterozygosity for either mutation may be advantageous. [ABSTRACT FROM AUTHOR]

Fatty acids covalently bonded with other molecules have been implicated in many important biological processes. We describe here a rapid approach tanned isotope-coded fatty acid transmethylation (iFAT) that integrates extraction, transmethylation, and isotopic labeling into a single step with the aid of ultrasonic irradiation for comparative analysis of fatly acids by mass spectrometry. In this approach, samples without any prefractionation were mixed with a methanol solution of 0.5 M NaOH and an n-hexane solution. The intense wave shocks and cavitations generated by ultrasonic irradiation not only speed the alkaline-catalyzed transmethylation reaction but also facilitate the simultaneous mass transfer of fatty acid methyl esters into the top n-hexane extraction phase that was injected into a GC/MS system. By using commercially available d3-methanol, we were able to compare the intensity of labeled and unlabeled methyl esters and their corresponding fragment ions. The detection limit can be down to the picogram level. Major advantages of the iFAT strategy are summarized in the following: (1) Efficient heterogeneous reactions. Solid samples such as dried cell lysates or detergent-resistant fractions can be readily transformed and analyzed with the aid of ulirasound irradiation. (2) Accurate quantification of fatty acids. Evaluation of the completeness or losses of transformation reactions across lipid classes has been hampered due to a lack of suitable methods. Isotope labeling can be used as an internal standard for accurate comparison of the fatty acid composition in different cell states. (3) Reduced interferences from complex biological context. The iFAT strategy not only differentially labels fatty acids in different samples, but also volatilizes those molecules, and thus, they are isolated from the bulk background and analyzed by GC/MS. This proposed approach has been applied to quanlitatively determine the fatty acid composition in plant oil and in budding yeast cell lysates and detergent-resistant fractions. It should provide a widely applicable means for quantitative comparison of the fatly acid composition in cells and tissues. [ABSTRACT FROM AUTHOR]

Bauchart-Thevret C, Stoll B, Chacko S, Burrin DG. Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs. Am J Physiol Endocrinol Metab 296: E1239 -E1250, 2009. First published March 17, 2009; doi: 10.1152/ajpendo.9102.2008.-We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pigs were enterally fed a control or an SAA-free diet for 7 days, and then whole body methionine and cysteine kinetics were measured using an intravenous infusion of [1-[sup13]C;methyl-[sup2]H[sub3]]methionine and [`5Nlcysteine. Body weight gain and plasma methionine, cysteine, homocysteine, and taurine and total erythrocyte glutathione concentrations were markedly decreased (-46% to 85%) in SAA-free compared with control pigs. Whole body methionine and cysteine fluxes were reduced, yet methionine utilization for protein synthesis and methionine remethylation were relatively preserved at the expense of methionine transsulfuration, in response to SAA deficiency. Intestinal tissue concentrations of methionine and cysteine were markedly reduced and hepatic levels were maintained in SAA-free compared with control pigs. SAA deficiency increased the activity of methionine metabolic enzymes, i.e., methionine adenosyltransferase, methionine synthase, and cystathionine β-synthase, and S-adenosylmethionine concentration in the jejunum, whereas methionine synthase activity increased and S-adenosylmethionine level decreased in the liver. Small intestine weight and protein and DNA mass were lower, whereas liver weight and DNA mass were unchanged, in SAA-free compared with control pigs. Dietary SAA deficiency induced small intestinal villus atrophy, lower goblet cell numbers, and Ki-67-positive proliferative crypt cells in association with lower tissue glutathione, especially in the jejunum. We conclude that SAA deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs. [ABSTRACT FROM AUTHOR]

Root-hair development is a process involving the interplay between genetic, hormonal, and environmental factors. An Arabidopsis mutant, referred to as sahh1, was initially recovered from a screen for delayed germination. Molecular characterization of the sahh1 mutant revealed that it contained a T-DNA insertion 82 bp 5′ to the coding sequence of S-adenosylhomocysteine hydrolase 1 (SAHH1, At4g13940), resulting in the reduction of SAHH1 expression. The resulting reduction in expression of SAHH1 produced plants with short, hairless roots, delayed germination, and slow growth. All of these phenotypes were restored to normal by complementing the sahh1 mutant with a full length cDNA. In plants, SAHH1 converts S-adenosylhomocysteine to homocysteine in the activated methyl cycle, and is a precursor for methionine and S-adenosylmethionine. Using the root hairless phenotype of the sahh1 mutant as a visual assay, the effects of SAHH1 deficiency on the synthesis of homocysteine, S-adenosylmethionine, 1-cyclopropane-1-carboxylic acid, and spermidine were investigated. [ABSTRACT FROM AUTHOR]