In plants, the generation and supply of methyl units is important in one-carbon (C-1) metabolism, which is essential to all organisms. I have identified a series of cDNA sequences encoding N5, N10-methylenetetrahydrofolate reductase (MTHFR), cobalamin-independent methionine synthase (Met Syn), S-adenosylmethionine synthetase (isoform I, AdoMet Syn2661 and isoform II, AdoMet Syn605), S-adenosylmethionine decarboxylase (SAMDC), serine hydroxymethyltransferase (SHMT) and N5, N10-methenyltetrahydrofolate cyclohydrolase / N5, N10-methylenetetrahydrofolate dehydrogenase (THFC/THFD) in the pathways of generation and supply of methyl units. These are from a cDNA library of mRNA from a susceptible wheat (Triticum monococcum) (Tm) line 441 epidermis, 24 h after inoculation with powdery mildew fungus (Blumeria graminis f. sp. tritici) (Bgt). Phylogenetic tree cluster analysis and subcellular localization prediction by TargetP revealed that MTHFR, Met Syn, AdoMet Syn605, AdoMet Syn2661, SAMDC, and THFC/THFD may be localized in cytosol; SHMT may be localized in mitochondria. Northern blot analysis indicated that expression of MTHFR, Met Syn, AdoMet Syn2661, AdoMet Syn605 and SAMDC genes was up-regulated by powdery mildew infection, abiotic stresses and treatments with stress signal molecules; expression of SHMT and THFC/THFD was either constitutive or down-regulated. These results suggest a close metabolic link between various stresses and the pathways of generation and supply of methyl units in this wheat.

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

In plants, the generation and supply of methyl units is important in one-carbon (C-1) metabolism, which is essential to all organisms. I have identified a series of cDNA sequences encoding N5, N10-methylenetetrahydrofolate reductase (MTHFR), cobalamin-independent methionine synthase (Met Syn), S-adenosylmethionine synthetase (isoform I, AdoMet Syn2661 and isoform II, AdoMet Syn605), S-adenosylmethionine decarboxylase (SAMDC), serine hydroxymethyltransferase (SHMT) and N5, N10-methenyltetrahydrofolate cyclohydrolase / N5, N10-methylenetetrahydrofolate dehydrogenase (THFC/THFD) in the pathways of generation and supply of methyl units. These are from a cDNA library of mRNA from a susceptible wheat (Triticum monococcum) (Tm) line 441 epidermis, 24 h after inoculation with powdery mildew fungus (Blumeria graminis f. sp. tritici) (Bgt). Phylogenetic tree cluster analysis and subcellular localization prediction by TargetP revealed that MTHFR, Met Syn, AdoMet Syn605, AdoMet Syn2661, SAMDC, and THFC/THFD may be localized in cytosol; SHMT may be localized in mitochondria. Northern blot analysis indicated that expression of MTHFR, Met Syn, AdoMet Syn2661, AdoMet Syn605 and SAMDC genes was up-regulated by powdery mildew infection, abiotic stresses and treatments with stress signal molecules; expression of SHMT and THFC/THFD was either constitutive or down-regulated. These results suggest a close metabolic link between various stresses and the pathways of generation and supply of methyl units in this wheat.

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

No description available.

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Protein restriction during rodent pregnancy is a well-established model of developmental programming. Although the Southampton low-protein diet model of developmental programming has been accepted to produce hypertensive offspring, the mechanism of this programming remains unclear. Currently the effects of protein restriction in the Southampton low-protein diet are confounded by a relative elevation of the amino acid methionine. The aim of this project was thus to clarify the roles of protein reduction and methionine elevation within this model, especially within the context of amino acid and one-carbon metabolism. Pregnant Wistar rats were fed casein-based diets ad libitum varying in casein (18% or 9%) and methionine content (0.5% or 1.0%) from day 0 through 20 of pregnancy. Two diets exactly replicated the Southampton control and low-protein diets (Con: 18% casein, 0.5% methionine; LP: 9% casein, 0.5% methionine), while a third low-protein high-methionine diet (LP-MET: 9% casein, 1.0% methionine) was employed as a positive control for methionine stress. On day 20 of gestation dams were sacrificed and the feto-placental unit was excised; maternal and fetal blood was collected for HPLC analysis of free amino acids. Maternal plasma was also analyzed for homocysteine content using a spectrophotometric-based enzyme assay. Diet did not affect maternal weight gain, food consumption, litter size or fetal weight. In dams and fetuses, methionine was significantly elevated in both low-protein groups. Maternal homocysteine was significantly elevated only in dams fed the low-protein high-methionine diet. Reductions in maternal serine, proline and glycine levels also occurred in dams fed the low-protein high-methionine diet; fetuses of these dams had significantly reduced levels of all three branch chain amino acids (leucine, isoleucine and valine). Both low-protein diets resulted in drastic reductions in circulating threonine levels in dams and fetuses. Thus, ingestion of low-protein diets with a relative (0.5%) or overt excess (1.0%) of methionine appears to disrupt one-carbon metabolism at the level of homocysteine remethylation to methionine. This may place strain on the folate cycle, as may be indicated by the reduced levels of threonine, serine and glycine. Further testing is necessary to clarify the extent to which folate stores are being utilized for homocysteine remethylation. Increased competition for placental amino acid transport may explain the alternations in circulating free fetal amino acids. Further investigations into levels of other one-carbon metabolites in dams and fetuses are necessary to fully characterize the effect of low-protein high-methionine diets, particularly within the context of the Southampton model of developmental programming.

Protein restriction during rodent pregnancy is a well-established model of developmental programming. Although the Southampton low-protein diet model of developmental programming has been accepted to produce hypertensive offspring, the mechanism of this programming remains unclear. Currently the effects of protein restriction in the Southampton low-protein diet are confounded by a relative elevation of the amino acid methionine. The aim of this project was thus to clarify the roles of protein reduction and methionine elevation within this model, especially within the context of amino acid and one-carbon metabolism. Pregnant Wistar rats were fed casein-based diets ad libitum varying in casein (18% or 9%) and methionine content (0.5% or 1.0%) from day 0 through 20 of pregnancy. Two diets exactly replicated the Southampton control and low-protein diets (Con: 18% casein, 0.5% methionine; LP: 9% casein, 0.5% methionine), while a third low-protein high-methionine diet (LP-MET: 9% casein, 1.0% methionine) was employed as a positive control for methionine stress. On day 20 of gestation dams were sacrificed and the feto-placental unit was excised; maternal and fetal blood was collected for HPLC analysis of free amino acids. Maternal plasma was also analyzed for homocysteine content using a spectrophotometric-based enzyme assay. Diet did not affect maternal weight gain, food consumption, litter size or fetal weight. In dams and fetuses, methionine was significantly elevated in both low-protein groups. Maternal homocysteine was significantly elevated only in dams fed the low-protein high-methionine diet. Reductions in maternal serine, proline and glycine levels also occurred in dams fed the low-protein high-methionine diet; fetuses of these dams had significantly reduced levels of all three branch chain amino acids (leucine, isoleucine and valine). Both low-protein diets resulted in drastic reductions in circulating threonine levels in dams and fetuses. Thus, ingestion of low-protein diets with a relative (0.5%) or overt excess (1.0%) of methionine appears to disrupt one-carbon metabolism at the level of homocysteine remethylation to methionine. This may place strain on the folate cycle, as may be indicated by the reduced levels of threonine, serine and glycine. Further testing is necessary to clarify the extent to which folate stores are being utilized for homocysteine remethylation. Increased competition for placental amino acid transport may explain the alternations in circulating free fetal amino acids. Further investigations into levels of other one-carbon metabolites in dams and fetuses are necessary to fully characterize the effect of low-protein high-methionine diets, particularly within the context of the Southampton model of developmental programming.

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007