Determinants of the essential one-carbon metabolism metabolites, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine and folate, in cerebrospinal fluid.

BACKGROUND: Disturbances in the levels of one-carbon (1C) metabolism metabolites have been associated with a wide variety of neuropsychiatric diseases. Cerebrospinal fluid (CSF) levels of homocysteine (Hcy) and the other 1C metabolites, nor their interrelatedness and putative determinants, have been studied extensively in a healthy population. METHODS: Plasma and CSF samples from 100 individuals free from neuropsychiatric diseases were analyzed (55 male, 45 female; age 50±17 years). In blood, we measured plasma Hcy, serum folate and serum vitamin B12. In CSF, we measured total Hcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 5-methyltetrahydrofolate (5-methylTHF). Highly selective analytical methods like liquid chromatography combined with either mass spectrometry or fluorescence detection were used. RESULTS: CSF Hcy was inversely correlated with CSF 5-methylTHF and positively with plasma Hcy, independent of serum folate status. CSF SAH correlated with age, lower CSF 5-methylTHF and higher CSF Hcy. CSF 5-methylTHF showed independent negative correlations with age and positive correlations with serum folate. CSF SAM did not correlate with any of the 1C metabolites. CONCLUSIONS: Aging is characterized by a reduction in CSF 5-methylTHF levels and increased CSF levels of the potentially neurotoxic transmethylation inhibitor SAH. CSF 5-methylTHF, which is itself determined in part by systemic folate status, is a powerful independent determinant of CSF levels of Hcy and SAH.

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements: We thank Ms Margaret Fraser, Ms Samantha Flannigan, and Dr Wing Yee Kwong for their expert assistance. The staff at Grampian NHS Pregnancy Counselling Service were essential for collecting fetuses. We thank Professor Geoffrey Hammond and Dr Marc Simard, University of British Colombia for helpful comments on the manuscript. Supported by grants as follows: Scottish Senior Clinical Fellowship (AJD); Chief Scientist Office (Scottish Executive, CZG/1/109 to PAF, & CZG/4/742 (PAF & PJOS); NHS Grampian Endowments 08/02 (PAF, SB & PJOS); the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 212885 (PAF & SMR); the Medical Research Council grants MR/L010011/1 (PAF & PJOS) and MR/K018310/1 (AJD). None of the funding bodies played any role in the design, collection, analysis, and interpretation of data, in the writing of the manuscript, nor in the decision to submit the manuscript for publication

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

The role of pneumococcal carbon metabolism in colonisation and invasive disease

Streptococcus pneumoniae is a common asymptomatic commensal of the human nasopharynx. However, it is better known as a threatening pathogen that causes serious diseases such as pneumonia, meningitis and sepsis, as well as other less severe but more prevalent infections (e.g. otitis media). With the increase of antibiotic resistance and the limited efficacy of vaccines, pneumococcal infections remain a major problem. Therefore, the discovery of new therapeutic targets and preventive drugs are in high demand.(...)

Fermentative 2-carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans.

Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-Coenzyme A (CoA) during fermentation. The aims of our study were: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate-bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate-bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down-stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.

Foliar photochemical processes and carbon metabolism under favourable and adverse winter conditions in a Mediterranean mixed forest, Catalonia (Spain)

Evergreen trees in the Mediterranean region must cope with a wide range of environmental stresses from summer drought to winter cold. The mildness of Mediterranean winters can periodically lead to favourable environmental conditions above the threshold for a positive carbon balance, benefitting evergreen woody species more than deciduous ones. The comparatively lower solar energy input in winter decreases the foliar light saturation point. This leads to a higher susceptibility to photoinhibitory stress especially when chilly (< 12 C) or freezing temperatures (< 0 C) coincide with clear skies and relatively high solar irradiances. Nonetheless, the advantage of evergreen species that are able to photosynthesize all year round where a significant fraction can be attributed to winter months, compensates for the lower carbon uptake during spring and summer in comparison to deciduous species. We investigated the ecophysiological behaviour of three co-occurring mature evergreen tree species (Quercus ilex L., Pinus halepensis Mill., and Arbutus unedo L.). Therefore, we collected twigs from the field during a period of mild winter conditions and after a sudden cold period. After both periods, the state of the photosynthetic machinery was tested in the laboratory by estimating the foliar photosynthetic potential with CO2 response curves in parallel with chlorophyll fluorescence measurements. The studied evergreen tree species benefited strongly from mild winter conditions by exhibiting extraordinarily high photosynthetic potentials. A sudden period of frost, however, negatively affected the photosynthetic apparatus, leading to significant decreases in key physiological parameters such as the maximum carboxylation velocity (Vc,max), the maximum photosynthetic electron transport rate (Jmax), and the optimal fluorometric quantum yield of photosystem II (Fv/Fm). The responses of Vc,max and Jmax were highly species specific, with Q. ilex exhibiting the highest and P. halepensis the lowest reductions. In contrast, the optimal fluorometric quantum yield of photosystem II (Fv/Fm) was significantly lower in A. unedo after the cold period. The leaf position played an important role in Q. ilex showing a stronger winter effect on sunlit leaves in comparison to shaded leaves. Our results generally agreed with the previous classifications of photoinhibition-tolerant (P. halepensis) and photoinhibitionavoiding (Q. ilex) species on the basis of their susceptibility to dynamic photoinhibition, whereas A. unedo was the least tolerant to photoinhibition, which was chronic in this species. Q. ilex and P. halepensis seem to follow contrasting photoprotective strategies. However, they seemed equally successful under the prevailing conditions exhibiting an adaptive advantage over A. unedo. These results show that our understanding of the dynamics of interspecific competition in Mediterranean ecosystems requires consideration of the physiological behaviour during winter which may have important implications for long-term carbon budgets and growth trends.

Carbon metabolism in transgenic roots with altered levels of hexokinase and triosephosphate isomerase and growing under different nitrogen status

Ce projet a pour but d’évaluer la capacité de la voie des pentoses phosphates (VPP) dans les racines transgéniques de pomme de terre (Solanum tuberosum) modifiées pour exprimer différents niveaux de l'hexokinase (HK) et de la triosephosphate isomérase cytosolique (cTPI). Dans les racines, la VPP alimente la voie de l’assimilation de l’azote en equivalents réducteurs et permet donc la biosynthèse des acides aminés. Le glucose-6-phosphate produit par l’HK est consommé par la partie oxydative de la VPP catalysée par la glucose-6-phosphate déshydrogénase (G6PDH) et la 6-phosphogluconate déshydrogénase (6PGDH). Les changements dans l'expression de HK et cTPI peuvent affecter le fonctionnement de la VPP et les mécanismes qui sont liés à l’utilisation des équivalents réducteurs produits par la VPP, comme l'assimilation de l’azote et la synthèse des acides aminés. Afin d’évaluer l’effet des manipulations génétiques de l’HK et de la cTPI sur l’assimilation de l’azote, nous avons cultivé les racines transgéniques sur des milieux contenant des concentrations élevées (7 mM) ou basses (0,7 mM) de nitrate d’ammonium comme source d’azote. Les résultats montrent que la culture sur un milieu riche en azote induit les activités G6PDH et 6PGDH. Les données montrent que la capacité de la VPP est plus grande avec des niveaux élevés en HK ou en cTPI. Nous avons aussi pu démontrer une plus grande activité spécifique de l’HK dans les conditions pauvres en azote. Ces données ont été complémentées par des mesures des pools d’acides aminés dans les racines transgéniques cultivées sur différents niveaux d’azote. Aucune tendance notable des pools d’acides aminés n’a été remarquée dans les racines modifiées pour leur contenu en HK suggèrant que la manipulation de HK n’affecte pas l'assimilation de l’azote. Dans les racines transgéniques modifiées pour la cTPI, les ratios Gln/Glu et Asn/Asp sont plus élevés chez les clones antisens, indiquant une assimilation de l’azote plus élevée. Ces résultats ont démontré l'activation de l'assimilation de l’azote chez les clones antisens cTPI dans les conditions élevées et basses d’azote alors que la manipulation de l’HK n’affecte pas l’assimilation de l’azote.

Genetic polymorphisms modulate the folate metabolism of Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) carry three copies of the Cystathionine beta-synthase (C beta S) gene. The increase in the dosage of this gene results in an altered profile of metabolites involved in the folate pathway, including reduced homocysteine (Hcy), methionine, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Furthermore, previous studies in individuals with DS have shown that genetic variants in genes involved in the folate pathway influence the concentrations of this metabolism's products. The purpose of this study is to investigate whether polymorphisms in genes involved in folate metabolism affect the plasma concentrations of Hcy and methylmalonic acid (MMA) along with the concentration of serum folate in individuals with DS. Twelve genetic polymorphisms were investigated in 90 individuals with DS (median age 1.29 years, range 0.07-30.35 years; 49 male and 41 female). Genotyping for the polymorphisms was performed either by polymerase chain reaction (PCR) based techniques or by direct sequencing. Plasma concentrations of Hcy and MMA were measured by liquid chromatography-tandem mass spectrometry as previously described, and serum folate was quantified using a competitive immunoassay. Our results indicate that the MTHFR C677T, MTR A2756G, TC2 C776G and BHMT G742A polymorphisms along with MMA concentration are predictors of Hcy concentration. They also show that age and Hcy concentration are predictors of MMA concentration. These findings could help to understand how genetic variation impacts folate metabolism and what metabolic consequences these variants have in individuals with trisomy 21.

Alternative carbon metabolism: Virulence and regulation in Candida albicans

The interaction between C. albicans and innate immune cells is a key determinant to disease progression. Transcriptional profiling showed that C. albicans responds to macrophage phagocytosis by inducing pathways required for alternative carbon metabolism (beta-oxidation, the glyoxylate cycle, and gluconeogenesis), suggesting these pathways are important for virulence of C. albicans. ^ We have shown that deleting key genes (FOX2, FBP1) in these pathways results in virulence defects in an in vivo mouse model for systemic infection. Like icl1Δ/Δ mutants, fbp1Δ/Δ mutants are severely attenuated and fox2Δ/Δ mutants are mildly but significantly attenuated, indicating that carbon starvation is a relevant stress in vivo. ^ However, fox2Δ/Δ mutants also had unexpected phenotypes on certain carbon sources, unlike the case in Saccharomyces cerevisiae, suggesting these pathways are regulated differently in C. albicans. To test this, we identified the C. albicans regulators of these pathways based on those from S. cerevisiae and Aspergillus nidulans. ^ C. albicans has a partly conserved framework, but lacks two regulators (Oaf1p, Pip2p) controlling peroxisome biogenesis and beta-oxidation genes in yeast. Instead, C. albicans has a homolog, CTF1, of the A. nidulans fatty acid catabolism regulators FarA and FarB. We have shown that CTF1 is needed for growth on oleate (like FarA and FarB), expression of beta-oxidation and glyoxylate cycle genes, and full virulence. No function for CTF1 has previously been identified in C. albicans. Our data demonstrate a role for alternative carbon metabolism in the virulence of C. albicans and suggest that the regulation of these pathways is a mixture of the filamentous fungi and budding yeast systems. ^

Drought-Induced Effects on Nitrate Reductase Activity and mRNA and on the Coordination of Nitrogen and Carbon Metabolism in Maize Leaves1

Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3− reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

Growth and carbon metabolism of the seaweed Fucus vesiculosus in the western Baltic Sea ("Kiel Outdoor Benthocosms")

Warming and acidification of the oceans as a consequence of increasing CO2-concentrations occur at large scales. Numerous studies have shown the impact of single stressors on individual species. However, studies on the combined effect of multiple stressors on a multi-species assemblage, which is ecologically much more realistic and relevant, are still scarce. Therefore, we orthogonally crossed the two factors warming and acidification in mesocosm experiments and studied their single and combined impact on the brown alga Fucus vesiculosus associated with its natural community (epiphytes and mesograzers) in the Baltic Sea in all seasons (from April 2013 to April 2014). We superimposed our treatment factors onto the natural fluctuations of all environmental variables present in the Benthocosms in so-called delta-treatments. Thereby we compared the physiological responses of F. vesiculosus (growth and metabolites) to the single and combined effects of natural Kiel Fjord temperatures and pCO2 conditions with a 5 °C temperature increase and/or pCO2 increase treatment (1100 ppm in the headspace above the mesocosms). Responses were also related to the factor photoperiod which changes over the course of the year. Our results demonstrate complex seasonal pattern. Elevated pCO2 positively affected growth of F. vesiculosus alone and/or interactively with warming. The response direction (additive, synergistic or antagonistic), however, depended on season and daylength. The effects were most obvious when plants were actively growing during spring and early summer. Our study revealed for the first time that it is crucial to always consider the impact of variable environmental conditions throughout all seasons. In summary, our study indicates that in future F. vesiculosus will be more affected by detrimental summer heat-waves than by ocean acidification although the latter consequently enhances growth throughout the year. The mainly negative influence of rising temperatures on the physiology of this keystone macroalga may alter and/or hamper its ecological functions in the shallow coastal ecosystem of the Baltic Sea.