Methylation Step Research Articles

Functional Oligomerization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxyl Methyltransferase, Ste14p

The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26-kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CAAX post-translational processing pathway. Sequence analysis reveals a putative GXXXG dimerization motif located in transmembrane 1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminal-tagged His(10)-myc(3)-Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species.

Atmospheric pressure-thermal desorption (AP-TD)/electrospray ionization-mass spectrometry for the rapid analysis of Bacillus spores

A technique is described where an atmospheric pressure-thermal desorption (AP-TD) device and electrospray ionization (ESI)-mass spectrometry (MS) are coupled and used for the rapid analysis of Bacillus subtilis spores in complex matrices. The resulting AP-TD/ESI-MS technique combines the generation of volatile compounds and/or pyrolysis products with soft-ionization MS detection. In the AP-TD/ESI-MS approach, an electrospray solvent plume was used as the ionization vehicle of thermally desorbed neutrals at atmospheric pressure prior to mass spectrometric analysis using a quadrupole ion trap mass spectrometer. The approach is quantitative with the volatile standard dimethyl methylphosphonate (DMMP) and with the use of an internal standard (diethyl methylphosphonate, DEMP). A linear response was obtained as tested in the 1-50 ppm range (R(2) = 0.991) with a standard error of the estimate of 0.193 (0.9% RSD, n = 5). Bacterial spores were detected by performing pyrolysis in situ methylation with the reagent tetramethylammonium hydroxide (TMAH) for the detection of the bacterial spore biomarker dipicolinic acid (DPA) as the dimethylated derivative (2Me-DPA). This approach allowed spore detection even in the presence of growth media in crude lyophilized samples. Repetitive analyses could be performed with a duty cycle of less than 5 min total analysis time (including sample loading, heating and data acquisition). This strategy proved successful over other direct ambient MS approaches like DESI-MS and AP-TD/ESI-MS without the in situ derivatization step to detect the dipicolinic acid biomarker from spores. A detection limit for the dimethylated DPA biomarker was estimated at 1 ppm (equivalent to 0.01 mug of DPA deposited in the thermal desorption tube), which corresponded to a calculated detection limit of 10(5) spores deposited or 0.1% by weight spore composition in solid samples (assuming a 1 mg sample size). The AP-TD/ESI source used in conjunction with the in situ methylation step allowed the differentiation of bacterial spores from other 'suspicious white powders' using a single stage for mass analysis and with minimum sample preparation, making this approach suitable for simple field-portable MS instrumentation and pattern recognition data analysis.

The Effect of Confined Space on the Growth of Naphthalenic Species in a Chabazite‐Type Catalyst: A Molecular Modeling Study

AbstractMethylation reactions of naphthalenic species over the acidic microporous zeolite with chabazite topology have been investigated by means of two‐layered ab initio computations. Large cluster results combined with van der Waals contributions provide thermodynamic and kinetic results of successive methylation steps. The growth of fused bicyclic species is important as these can act as hydrocarbon pool species within the methanol‐to‐olefin (MTO) process, but ultimately leads to the deactivation of the catalyst. The influence of the confined space of the zeolite pore on the resulting transition state or product shape selectivity is investigated in detail.

Structure–Activity Analysis of Semisynthetic Nucleosomes: Mechanistic Insights into the Stimulation of Dot1L by Ubiquitylated Histone H2B

Post-translational modification of histones plays an integral role in regulation of genomic expression through modulation of chromatin structure and function. Chemical preparations of histones bearing these modifications allows for comprehensive in vitro mechanistic investigation into their action to deconvolute observations from genome-wide studies in vivo. Previously, we reported the semisynthesis of ubiquitylated histone H2B (uH2B) using two orthogonal expressed protein ligation reactions. Semisynthetic uH2B, when incorporated into nucleosomes, directly stimulates methylation of histone H3 lysine 79 (K79) by the methyltransferase, disruptor of telomeric silencing-like (Dot1L). Although recruitment of Dot1L to the nucleosomal surface by uH2B could be excluded, comprehensive mechanistic analysis was precluded by systematic limitations in the ability to generate uH2B in large scale. Here we report a highly optimized synthesis of ubiquitylated H2B bearing a G76A point mutation u(G76A)H2B, yielding tens of milligrams of ubiquitylated protein. u(G76A)H2B is indistinguishable from the native uH2B by Dot1L, allowing for detailed studies of the resultant trans-histone crosstalk. Kinetic and structure-activity relationship analyses using u(G76A)H2B suggest a noncanonical role for ubiquitin in the enhancement of the chemical step of H3K79 methylation. Furthermore, titration of the level of uH2B within the nucleosome revealed a 1:1 stoichiometry of Dot1L activation.

Mutational analysis of the CG recognizing DNA methyltransferase SssI: Insight into enzyme–DNA interactions

To characterize important steps of DNA methylation by M.SssI, a prokaryotic DNA-(cytosine C5)-methyltransferase (C5-MTase) sharing the specificity of eukaryotic C5-MTases (5′-CG-3′), ten amino acids, selected on the basis of sequence alignments and a computational model, were subjected to mutational analysis. Wild-type and mutant M.SssI variants were studied to determine methylation activity, DNA binding affinity, capacity to induce base flipping, and ability to form covalent complex with a DNA substrate containing the mechanism-based inhibitor 2-pyrimidinone. Wild-type M.SssI induced strong fluorescence when bound to substrate DNA containing 2-aminopurine in place of the target cytosine, indicating flipping of the target base. Reduced fluorescence, moderate, or drastic loss of methyltransferase activity and reduced DNA binding suggest the involvement of the conserved S145 (motif IV), R232 (motif VIII, QxRx R), and T313 (variable region, conserved TL), as well as of the non-conserved Q147 in base flipping. Replacement of E186 (motif VI, ENV) and R230 (motif VIII, Qx RxR) with alanine resulted in loss of methyltransferase activity without impairing DNA binding affinity. These data are consistent with the catalytic role of E186 and R230, and provide, for the first time, experimental support for the essential function of the hitherto not investigated invariant arginine of motif VIII in C5-MTases.

Influence of Cobalamin on Arsenic Metabolism in Bangladesh

BackgroundArsenic is a carcinogen to which 35 million people in Bangladesh are chronically exposed. The enzymatic transfer of methyl groups to inorganic As (iAs) generates monomethylarsonic (MMA) and dimethylarsinic acids (DMA) and facilitates urinary As (uAs) elimination. This process is dependent on one-carbon metabolism, a pathway in which folate and cobalamin have essential roles in the recruitment and transfer of methyl groups. Although DMAV is the least toxic metabolite, increasing evidence suggests that MMAIII may be the most cytotoxic and genotoxic As intermediary metabolite.ObjectiveWe examined the associations between plasma cobalamin and uAs metabolites.MethodsWe conducted a cross-sectional study of 778 Bangladeshi adults in which we over-sampled cobalamin-deficient participants. Participants provided blood samples for the measurement of plasma cobalamin and urine specimens for As measurements.ResultsCobalamin was inversely associated with the proportion of total uAs excreted as iAs (%iAs) [unstandardized regression coefficient (b) = –0.10; 95% confidence interval (CI), −0.17 to −0.02; p = 0.01] and positively associated with %MMA (b = 0.12; 95% CI, 0.05 to 0.20; p = 0.001). Both of these associations were stronger among folate-sufficient participants (%iAs: b = −0.17; 95% CI, −0.30 to −0.03; p = 0.02. %MMA: b = 0.20; 95% CI, 0.11 to 0.30; p < 0.0001), and the differences by folate status were statistically significant.ConclusionsIn this group of Bangladeshi adults, cobalamin appeared to facilitate the first As methylation step among folate-sufficient individuals. Given the toxicity of MMAIII, our findings suggest that in contrast to folate, cobalamin may not favorably influence As metabolism.

Phenylpropanoid polyamine conjugate biosynthesis in Arabidopsis thaliana flower buds

Phenylpropanoid polyamine conjugates have been identified in flowers of many plant species. Their presence in Arabidopsis thaliana has only been recently established in flower buds and pollen grains. Annotation and location of a cation-dependent O-methyltransferase AtTSM1 specifically to the tapetum of young flower buds enabled the subsequent identification of several genes with a putative role in phenylpropanoid polyamine conjugate biosynthesis. Based on the analysis of several A. thaliana knockout mutants, a biosynthetic pathway of these conjugates is proposed, which involves two methylation steps catalyzed by different cation-dependent O-methyltransferases, a cytochrome P450 (CYP98A8) catalyzed hydroxylation, and a conjugating acyl transfer performed by a BAHD-like, hydroxycinnamoyl (HC)-transferase. LC/MS based metabolite profiling of the cyp98A8 knockout line identified new feruloyl- and 4-coumaroylspermidine conjugates in the corresponding flowers consistent with a role of this gene in the hydroxylation of these conjugates. A pattern of minor amounts of bis- and tris-acylspermidine conjugates, likely the products of additional HC-transferases were identified in wild type as well as in the mutant lines. Transcript suppression of the genes early in the pathway was observed in knockout or RNAi-lines of the genes encoding late enzymatic steps. The implication of these findings for spermidine conjugate biosynthesis in flower buds of A. thaliana is discussed.

Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment.

Nutrient Protection against Arsenic Toxicity: Folate, Cysteine Support Methylation in Children

Nutritional factors are known to influence arsenic metabolism in adults, and poor nutritional status—as reflected in part by a lack of various B vitamins and antioxidants—is thought to confer greater susceptibility to arsenic toxicity. Now researchers working in Bangladesh have reported that deficits in the B vitamin folate and the amino acid cysteine may adversely influence arsenic metabolism in children [EHP 117:825–831; Hall et al.]. The research team also found that, compared with adults, children may metabolize arsenic more efficiently and excrete it more readily, regardless of folate status. Chronic exposure to arsenic, a known human carcinogen, occurs mainly through contaminated drinking water and currently affects about 140 million people worldwide, including 35 million Bangladeshis. Such exposure has been linked to increased risks of cardiovascular disease and cancers of the skin, bladder, lung, and liver. Childhood exposure also increases the risk of intellectual deficits and respiratory disorders, among other health problems. Arsenic metabolism involves two methylation steps that rely on folate: Inorganic arsenic (InAs) is first converted to monomethylarsonic acid (MMA), which is then converted to the less toxic dimethylarsinic acid (DMA); this process facilitates urinary arsenic elimination. In the current study, the team measured urinary levels of InAs, MMA, and DMA, as well as blood levels of the metabolic by-product homocysteine and an array of micronutrients (including folate, cysteine, and cobalamin) in 165 6-year-old children. Among the participants, 4.1% of females and 3.3% of males were classified as folate deficient. Consistent with previous research findings involving adults, higher levels of folate and cysteine correlated with a lower proportion of unmethylated arsenic metabolites in the urine, indicating that adequate levels of these nutrients may be important for arsenic methylation in children. In addition, compared with Bangladeshi adults, children had lower mean proportions of urinary InAs and MMA as well as higher mean proportions of urinary DMA. The study did turn up a surprise: Plasma homocysteine was inversely correlated with the proportion of MMA in urine, especially in males, but was positively correlated with DMA in urine. Compared with previous findings for adults, the children were also more likely to have high homocysteine levels, despite being less likely to be classified as folate deficient. More research is required to confirm this finding and the underlying mechanisms. The authors hypothesize that the one-carbon metabolism mechanism behind methylation may be upregulated during periods of rapid growth to meet high demands for DNA and protein biosynthesis. This upregulation would also be associated with an increase in homocysteine biosynthesis. At the same time, behaviors common among Bangladeshi adults—such as cigarette smoking and the chewing of betel nuts—could also play a role in altering arsenic methylation patterns. Overall, this study’s findings indicate that improved nutritional status could constitute a key strategy for reducing the risk of arsenic-related disease in Bangladeshi children.

Health effects of arsenic and chromium in drinking water: recent human findings.

Even at high concentrations, arsenic-contaminated water is translucent, tasteless, and odorless. Yet almost every day, studies report a continually increasing plethora of toxic effects that have manifested in exposed populations throughout the world. In this article we focus on recent findings, in particular those associated with major contributions since 2006. Early life exposure, both in utero and in childhood, has been receiving increased attention, and remarkable increases in consequent mortality in young adults have been reported. New studies address the dose-response relationship between drinking-water arsenic concentrations and skin lesions, and new findings have emerged concerning arsenic and cardiovascular disease. We also review the increasing epidemiological evidence that the first step of methylation of inorganic arsenic to monomethylated arsenic (MMA) is actually an activation step rather than the first step in detoxification, as once thought. Hexavalent chromium differs from arsenic in that it discolors water, turning the water yellow at high concentrations. A controversial issue is whether chromium causes cancer when ingested. A recent publication supports the original findings in China of increased cancer mortality in a population where well water turned yellow with chromium.

Structural basis of tRNA modification with CO2 fixation and methylation by wybutosine synthesizing enzyme TYW4†

Wybutosine (yW), one of the most complicated modified nucleosides, is found in the anticodon loop of eukaryotic phenylalanine tRNA. This hypermodified nucleoside ensures correct codon recognition by stabilizing codon-anticodon pairings during the decoding process in the ribosome. TYW4 is an S-adenosylmethionine (SAM)-dependent enzyme that catalyzes the final step of yW biosynthesis, methylation and methoxycarbonylation. However, the structural basis for the catalytic mechanism by TYW4, and especially that for the methoxycarbonylation, have remained elusive. Here we report the apo and cofactor-bound crystal structures of yeast TYW4. The structures revealed that the C-terminal domain folds into a β-propeller structure, forming part of the binding pocket for the target nucleoside. A comparison of the apo, SAM-bound, and S-adenosylhomocysteine-bound structures of TYW4 revealed a drastic structural change upon cofactor binding, which may sequester solvent from the catalytic site during the reaction and facilitate product release after the reaction. In conjunction with the functional analysis, our results suggest that TYW4 catalyzes both methylation and methoxycarbonylation at a single catalytic site, and in the latter reaction, the methoxycarbonyl group is formed through the fixation of carbon dioxide.

Substrate-specific pharmacokinetic interaction between endothelin receptor antagonists and phosphodiesterase-5 inhibitors - assembling the clues

Substrate-specific pharmacokinetic interaction between endothelin receptor antagonists and phosphodiesterase-5 inhibitors - assembling the clues

A two‐step one‐pot radiosynthesis of the potent dopamine D2/D3 agonist PET radioligand [11C]MNPA

Abstract(R)‐(−)‐2‐[11C]Methoxy‐N‐n‐propylnorapomorphine ([11C]MNPA ([11C]2)) is an agonist radioligand of interest for imaging D2/D3 receptors in vivo. Here we sought to develop an improved radiosynthesis of this radioligand. Reference 2 was synthesized in nine steps with an overall yield of about 5%, starting from codeine. Trimethylsilyldiazomethane proved to be a practical improvement in comparison to diazomethane in the penultimate methylation step. A protected precursor for radiolabeling ((R)‐(−)‐2‐hydroxy‐10,11‐acetonide‐N‐n‐propylnoraporphine, 4) was prepared from (R)‐(−)‐2‐hydroxy‐N‐n‐propylnorapomorphine (1) in 30% yield. [11C]2 was prepared from 4 via a two‐step one‐pot radiosynthesis. The first step, methylation of 4 with [11C]methyl triflate, occurred in quantitative radiochemical yield. The second step, deprotection of the catechol moiety with HCl and heat, yielded 60–90% of [11C]2 giving an overall incorporation yield from [11C]methyl triflate of 60–90%. In a typical run more than 1 GBq of [11C]2, was produced from carbon‐11 generated from a 10‐min proton irradiation (16 MeV; 35 µA) of nitrogen–hydrogen target gas. The radiochemical purity of [11C]2 was &gt; 99% and specific radioactivity at the time of injection was 901±342 GBq/µmol (n=10). The total synthesis time was 35–38 min from the end of radionuclide production. The identity of [11C]2 was confirmed by comparing its LC‐MS/MS spectrum with those of reference 2 and (R)‐(−)‐10‐methoxy‐2,11‐dihydroxy‐N‐n‐propylnoraporphine. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Dietary phenolics: chemistry, bioavailability and effects on health

There is much epidemiological evidence that diets rich in fruit and vegetables can reduce the incidence of non-communicable diseases such as cardiovascular diseases, diabetes, cancer and stroke. These protective effects are attributed, in part, to phenolic secondary metabolites. This review summarizes the chemistry, biosynthesis and occurrence of the compounds involved, namely the C6-C3-C6 flavonoids-anthocyanins, dihydrochalcones, flavan-3-ols, flavanones, flavones, flavonols and isoflavones. It also includes tannins, phenolic acids, hydroxycinnamates and stilbenes and the transformation of plant phenols associated with food processing (for example, production of black tea, roasted coffee and matured wines), these latter often being the major dietary sources. Events that occur following ingestion are discussed, in particular, the deglycosylation, glucuronidation, sulfation and methylation steps that occur at various points during passage through the wall of the small intestine into the circulatory system and subsequent transport to the liver in the portal vein.We also summarise the fate of compounds that are not absorbed in the small intestine, but which pass into the large intestine where they are degraded by the colonic microflora to phenolic acids, which can be absorbed into the circulatory system and subjected to phase II metabolism prior to excretion. Initially, the protective effect of dietary phenolics was thought to be due to their antioxidant properties which resulted in a lowering of the levels of free radicals within the body.However, there is now emerging evidence that themetabolites of dietary phenolics,which appear in the circulatory systemin nmol/L to low mmol/L concentrations, exertmodulatory effects in cells through selective actions on different components of the intracellular signalling cascades vital for cellular functions such as growth, proliferation and apoptosis. In addition, the intracellular concentrations required to affect cell signalling pathways are considerably lower than those required to impact on antioxidant capacity. The mechanisms underlying these processes are discussed.

SpnH from Saccharopolyspora spinosa encodes a rhamnosyl 4′-O-methyltransferase for biosynthesis of the insecticidal macrolide, spinosyn A

Deoxysugar, 2', 3', 4'-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4'-O-methylation of 2', 3'-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4'-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4'-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4'-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.

S-Adenosyl-L-homocysteine Hydrolase, Key Enzyme of Methylation Metabolism, Regulates Phosphatidylcholine Synthesis and Triacylglycerol Homeostasis in Yeast: IMPLICATIONS FOR HOMOCYSTEINE AS A RISK FACTOR OF ATHEROSCLEROSIS

In eukaryotes, S-adenosyl-L-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-L-homocysteine, a product and potent competitive inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. De novo phosphatidylcholine (PC) synthesis requires three AdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-L-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.

A Small Molecule Inhibitor of Isoprenylcysteine Carboxymethyltransferase Induces Autophagic Cell Death in PC3 Prostate Cancer Cells

A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336-4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G(1) phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G(1) cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy.

Influence of partial rootzone drying on the composition and accumulation of anthocyanins in grape berries (Vitis viniferacv. Cabernet Sauvignon)

Background and Aims: The aim of the study was to investigate anthocyanin composition and accumulation in grape berries in response to the partial rootzone drying (PRD) irrigation technique. Method and Results: The experiment was on Vitis vinifera cv. Cabernet Sauvignon, wherein PRD had a 40% water deficit relative to the control treatment. PRD decreased berry weight compared with the control, but did not influence total anthocyanin concentration. A significant increase in glucosides of delphinidin, cyanidin, petunidin and peonidin was found in response to PRD from the onset of anthocyanin accumulation (veraison), while malvidin-glucosides were unaffected by the irrigation treatment. The PRD treatment did not cause changes in the proportions of acetyl-, 3-p-coumaroyl- and monoglucoside anthocyanins. Wines produced from the treatments showed no difference in total monomeric anthocyanin, but relative increases (15%) in wine colour density, total tannin and polymeric pigment occurred in response to the PRD treatment. The anthocyanin composition of the wines reflected the response shown in the grapes, where the relative contribution of non-malvidin anthocyanins to total anthocyanins was significantly increased in wines from the PRD treatment. Conclusions: The differences in anthocyanin composition observed in response to PRD could not be accounted for by changes in bunch microclimate, and most likely reflect differences in the methylation step of anthocyanin synthesis. Significance of the Study: The observed changes in anthocyanin composition under the PRD irrigation system have not been previously reported in response to water deficit, and may reflect a unique response to within-vine signalling induced by PRD.

Comparison of four extraction/methylation analytical methods to measure fatty acid composition by gas chromatography in meat

Four different extraction–derivatization methods commonly used for fatty acid analysis in meat ( in situ or one-step method, saponification method, classic method and a combination of classic extraction and saponification derivatization) were tested. The in situ method had low recovery and variation. The saponification method showed the best balance between recovery, precision, repeatability and reproducibility. The classic method had high recovery and acceptable variation values, except for the polyunsaturated fatty acids, showing higher variation than the former methods. The combination of extraction and methylation steps had great recovery values, but the precision, repeatability and reproducibility were not acceptable. Therefore the saponification method would be more convenient for polyunsaturated fatty acid analysis, whereas the in situ method would be an alternative for fast analysis. However the classic method would be the method of choice for the determination of the different lipid classes.

Interactions between Arsenic-Induced Toxicity and Nutrition in Early Life ,

Exposure to arsenic through drinking water is a major public health problem affecting most countries, although the situation is particularly severe in low-income nations. The health consequences of chronic arsenic exposure include increased risk for various forms of cancer and numerous noncancer effects, including diabetes, skin diseases, chronic cough, and toxic effects on liver, kidney, cardiovascular system, and peripheral and central nervous systems. In recent years increasing reports of effects on fetal and child development have appeared. There seems to be a wide variation in susceptibility to arsenic toxicity, which is likely to be related to factors such as variation in arsenic metabolism, nutrition, host-related defense mechanisms, and genetic predisposition. The main mechanisms of arsenic-nutrition interactions include arsenic-induced oxidative stress, which requires nutrient-dependent defense systems, and arsenic metabolism (methylation) via 1-carbon metabolism, which requires methyl groups, folic acid, vitamin B-12, and betaine for the remethylation of homocysteine to methionine. An efficient first methylation step in combination with a slow second methylation step seems to be most critical from a toxicological point of view. A third mode of arsenic-nutrition interaction involves epigenetic effects and fetal programming via DNA methylation.