Expression Of MTR Research Articles

Dietary Supplementation with Pomegranate and Onion Affects Lipid and Protein Oxidation in the Breast Meat, Thigh, and Liver, Cellular Stress Protein Responses, and Gene Expression of Liver Enzymes Involved in Protein Synthesis in Broilers.

The present study examined the effects of dietary supplementation with extracts of pomegranate (Punica granatum) and onion (Allium cepa), either encapsulated in cyclodextrin (POMALCD group) or in an aqueous (POMALAQ group) form, on breast meat, thigh meat, and liver composition, oxidative stability, cellular signaling pathways, and the gene expression of certain hepatic genes. The results showed that breast and thigh meat contained significantly (p < 0.05) higher moisture content in the group with the aqueous extract, compared to the control and POMALCD groups. Moreover, the protein content was significantly (p < 0.05) higher in the thigh and liver samples of the treated groups in comparison to the control. The iron-induced challenge deteriorated (p < 0.001) the lipid and protein oxidative status of the control group, whereas both supplemented groups showed considerable tolerance in all tissues. The supplementation of pomegranate and onion extracts mitigated or maintained heat shock protein (HSP) levels and elevated (p < 0.05) the Bcl-2/Bad ratio in thigh and breast meat, whereas mitogen-activated protein kinase (MAPK) activation was modulated at a lower rate. After normalization to β-actin expression, quantitative real-time PCR analysis revealed a significant (p < 0.05) induction in the expression of MTR and MSRB1 genes in the liver of the supplemented groups. No differences were observed for the TAT, SMS, and BHMT genes. In conclusion, dietary mixtures of herbal extracts with pomegranate and onion improved protein and lipid oxidative stability in meat, enhanced the hepatic energy status, and exerted ameliorative effects on stress-related proteins. The encapsulated extract of pomegranate and onion, using cyclodextrin as a carrier, appeared to reduce lipid oxidation to a greater extent than the aqueous extract. In contrast, the aqueous extract exhibited higher total antioxidant capacity (TAC) values and provided better protection against protein carbonyl formation.

The inhibitory effect of vitamin D on myocardial homocysteine levels involves activation of Nrf2-mediated methionine synthase

BackgroundHomocysteine (Hcy) is a synthetic amino acid containing sulfhydryl group, which is an intermediate product of the deep metabolic pathway of methionine and cysteine. The abnormal increase in fasting plasma total Hcy concentration caused by various factors is called hyperhomocysteine (HHcy). HHcy is closely relevant to the occurrence and progression of diverse cardiovascular and cerebrovascular diseases, such as coronary heart disease, hypertension and diabetes, etc. Vitamin D/vitamin D receptor (VDR) pathway is pointed out that prevent cardiovascular disease by reducing serum homocysteine levels. Our research is designed to explore the potential mechanism of vitamin D in the prevention and treatment of HHcy. Methods and resultsThe Hcy and 25(OH)D3 levels in mouse myocardial tissue, serum or myocardial cells were detected using ELISA kits. The expression levels of VDR, Nrf2 and methionine synthase (MTR) were observed using Western blotting, immunohistochemistry and real time polymerase chain reaction (PCR). General information of the mice, including diet, water intake and body weight, was recorded. Vitamin D up-regulated the mRNA and protein expression of Nrf2 and MTR in mouse myocardial tissue and cells. CHIP assay determined that the combination of Nrf2 binding to the S1 site of the MTR promoter in cardiomyocytes using traditional PCR and real time PCR. Dual Luciferase Assay was applied to detect the transcriptional control of Nrf2 on MTR. The up-regulation effect of Nrf2 on MTR was verified by Nrf2 knockout and overexpression in cardiomyocytes. The role of Nrf2 in vitamin D inhibition of Hcy was revealed using Nrf2-knockdown HL-1 cells and Nrf2 heterozygous mice. Western blotting, real time PCR, IHC staining and ELISA showed that Nrf2 deficiency could restrain the increase in MTR expression and the decrease in Hcy level induced by vitamin D. The transcriptional activities of Nrf2/MTR were activated by vitamin D/VDR with a decrease in Hcy. ConclusionVitamin D/VDR upregulates MTR in an Nrf2-dependent manner, thereby reducing the risk of HHcy.

Methylomic change in TET1 and TET2 promoters is increased in children with acute lymphocytic leukemia

Objective: Methylomic changes have been recognized as essential contributors to the development of acute lymphocytic leukemia (ALL). While ALL has traditionally been considered a genetic disease, the increasing importance of epigenetic alterations in leukemogenesis has become evident. Here, our goal was to investigate the changes of DNA methylation level in ALL. Methods: To assess the DNA methylation pattern in ALL, we quantified gene expression of the methylation-related factors, including DNMTs, methionine synthase (MS), and the demethylation factors, TET gene family using qRT PCR. Results: The expression pattern of DNMTs, including DNMT1, DNMT3a, and DNMT3b, was markedly upregulated in samples derived from patients. On the contrary, the expression of the methyl creator, methionine synthase (MTR gene) was down-regulated in samples derived from children with ALL which revealed low concentration of vitamin B12 in their samples. The reduced expression of MTR in children with ALL reveals the disturbance of homocysteine and DNA methylation. Moreover, we examined the expression patterns of TET1, TET2, and TET3 genes implicated in the demethylation process. We found an evident decreasing level of TET1 and TET2 expression in ALL that may refer to an increasing DNA methylation activity in the promoter of the TET gene family. Conclusion: These data suggest the possible alteration in methyaltion-related factors due to methylomic changes within the promoter of TET1 and TET2 genes.

Effects of dietary supplementation with herbal extract as methionine replacer on growth performance, meat composition, oxidative stability and liver gene expression in broiler chickens

Abstract In the present study, an herbal feed additive was tested for partial-to-complete replacement of synthetic methionine in poultry diets, along with its effects on performance, breast and thigh meat chemical composition, oxidative stability during refrigerated storage and the expression of five target genes in liver. In a 35 days trial, 600 one-day-old male chicks were randomly allocated to 4 groups with 10 replicates. Birds in the control group were fed a regular maize–soybean-based diet that covered DL-methionine needs while the second group (Meth40) was similar to control but contained only DL – Methionine at 40% of control diet. Diet in third group contained DLMethionine at 40% of control and the herbal feed additive Methiorep TMMeth40+Mrep) with extracts of Boerhavia diffusa, Azadirachta indica, Vigna mungo and Trigonella foenum-graecum. Diet of fourth group was formulated to totally replace DL Methionine by Methiorep TM (Mrep). Body weight gain and feed consumption were weekly recorded. At the end of the trial, all birds were slaughtered and 2 chickens per pen were selected for meat and liver sampling. The liver was tested for the expression of five target genes, namely Methionine synthase (MTR), Tyrosine aminotransferase (TAT), Spermidine synthase (SMS), Methionine sulfoxide reductase (MSRB1) and Betaine homocysteine S-methyltransferase (BHMT). The results showed that the Meth40 group had reduced body weight compared to the Meth40+Mrep group while the Control and Mrep groups had comparable weights. Feed intake and feed conversion ratio did not differ among the experimental groups. Carcass, breast and thigh meat yield were higher in the Mrep and the Control compared to Meth40 and Meth40+Mrep groups. Also, meat oxidation was significantly lower in herbal groups compared to the control group. After normalization to β-actin expression, quantitative real-time PCR analysis revealed an induction in the expression of MTR and SMS genes in the liver of both herbal treated groups. No changes were observed for the TAT, MSRB1and BHMT genes in the herbal treated groups compared to the control or the Meth40 group. In conclusion, herbal feed additives with specific plant extracts may be able to improve both growth performance and antioxidant activity of broiler chickens, phenolic content; yet, they may also support in amino acid efficient use of broiler.

Associationof vitamin B12/ferritin deficiencyin cancer patients with methylomic changes at promotors of TETmethylcytosine dioxygenases.

Aim: To investigate potential DNA methylation in methylcytosine dioxygenases and correlation of TET genes with vitamin B12/ferritin levels in cancer patients. Materials & methods: 200 blood samples were obtained from both cancer patients and healthy individuals. Results: The expression of DNMT1, DNMT3a and DNMT3b was increased in patients with low vitamin B12 and ferritin levels, while the expression of MTR, TET1 and TET3 significantly decreased. DNA methylation analysis in patients with deficient vitamin B12/ferritin levels showed methylomic changes within the location 318/CG and 385/CG in the promoter region of TET1 and TET3, respectively. Conclusion: Vitamin B12/ferritin deficiency contributes to DNA methylation progress in cancer patients.

O-302 Lower expression of AHCY is associated with increased miscarriage risk

Abstract Study question Are alterations in the transcript levels of genes from the methionine cycle associated with maternal associated miscarriage risk? Summary answer Lower expression of AHCY is associated with a greater number of prior pregnancy losses What is known already Around 15% of pregnancies end in miscarriage, and the risk of recurrence increases with each pregnancy loss. Aberrant differentiation (decidualization) of endometrial stromal cells into specialised decidual cells to accommodate implantation is a key maternal factor for miscarriage risk. Our previous work identified secretory changes in cysteine and methionine metabolites upon decidualization. The methionine cycle contributes to vital cellular functions, including producing methionine for proliferation, regulating cell differentiation, and S-adenosylmethionine (SAM) production. SAM is required for protein, RNA and DNA methylation, thereby influencing pathways at the metabolic, epigenetic, and proteomic levels. AHCY clears S-adenosylhomocysteine (SAH), reducing its inhibition of methylation. Study design, size, duration Endometrial biopsies (n = 250) were collected during the luteal phase (LH + 6-11). Patients were grouped based on their miscarriage history. Accordingly, expression of genes from the methionine cycle were quantified using RT-qPCR. Participants/materials, setting, methods Endometrial biopsies were obtained, with written informed consent, from women attending the Implantation Clinic at University Hospitals Coventry and Warwickshire NHS Trust, following transvaginal ultrasounds to exclude uterine pathology. Isolated RNA was converted into cDNA. Expression of AHCY, AMD1, BHMT2, CBS, MAT2A, MAT2B, and MTR were normalised to L19. Statistical analysis was performed in Graphpad Prism; with significance accepted at p &amp;lt; 0.05. AHCY was silenced in an endometrial cell line to determine its effect on decidualization. Main results and the role of chance This study reports a distinct reduction in expression of methionine cycle genes (MAT2A, AHCY, AMD1, MTR, BHMT2) in the late-luteal phase of the cycle consistent with a reduction in proliferation. By plotting percentile graphs based on the statistical distribution in gene expression for each day of the luteal phase, comparisons have been made between groups. AHCY expression is significantly reduced in patients with increasing number of prior miscarriages, particularly between 0-2 and 5+ previous miscarriages (p = 0.0334). Neither patient age nor BMI are a factor in this reduced expression. In contrast, there is a stromal specific increase in AHCY upon decidualization in vitro, suggesting it is required in the decidua. Silencing AHCY in an endometrial cell line significantly reduces PRL expression upon decidualization. Reduction in AHCY may lead to a decreased “methylation potential” as SAH cannot be cleared. SAH accumulation inhibits methylation, and limits SAM production, thus compounding its effect. Decreased methylation potential could prevent differentiation of the stromal compartment, resulting in lower levels of PRL, and altering decidual timing. Therefore, an embryo may implant into a tissue primed for disintegration, resulting a miscarriage. In summary, AHCY may contribute to aberrant decidualization augmenting the risk of miscarriage. Limitations, reasons for caution Results are based on endometrial biopsies from an implantation clinic, therefore studies into biopsies from women with normal reproductive histories should also be analysed. Further functional studies are needed to ascertain the mechanism of action of AHCY in miscarriage. Wider implications of the findings This study identified that a decrease in AHCY in whole endometrial tissue is associated with increased risk of miscarriage. Further, silencing AHCY perturbed decidual marker expression. Thus, AHCY may act as a biomarker for atypical decidualization, and the clearance of SAH may be a potential treatment. Trial registration number N/A

Effects of valproate, an HDAC inhibitor, on the expression of folate carriers and folate metabolism-related genes in the placenta of rats

Valproate (VPA), an antiepileptic drug, is known to inhibit histone deacetylases (HDACs). Exposure to VPA during pregnancy increases several fetal risks. The maintenance of folate level during pregnancy is essential for adequate fetal development, and the placenta plays a critical role in supplying nutrients to the fetus. The aim of this study was to elucidate the effects of VPA on the gene expression of folate carriers and metabolizing enzymes in the rat placenta at both mid and late gestation periods. Pregnant rats were orally administered VPA on a single day or 4 days (repeated administration). Gene expression of folate carriers (Folr1, Slc19a1, Slc46a1) and metabolizing enzymes (Cth, Mtr, Mtrr, Mthfr, Dhfr) was assessed in the placenta on gestational day (GD) 13 or GD20. In the control rats, the expression of Folr1, Slc46a1, Cth, and Mthfr tended to be upregulated, whereas that of Mtrr and Dhfr was downregulated during gestation; the expression of Slc19a1 and Mtr did not change. Repeated VPA administration reduced the placental expression of Folr1and Mtr on GD20 and increased the expression of Dhfr on GD13 compared with the control. These findings indicate that administration of VPA alters the placental gene expression of folate carriers and metabolism-related enzymes.

PSXIV-3 Expression of genes within the methionine-folate cycle are altered by glucose and one-carbon metabolite supplementation in bovine embryonic fibroblasts

Abstract We hypothesized that supplementation with one-carbon metabolites (OCM; methionine, folate, choline, and vitamin B12) in divergent glucose media would alter expression of genes in the methionine-folate cycle of DNA methylation in bovine embryonic tracheal fibroblasts. Cells were cultured in EMEM medium containing 1 g/L (Low) or 4.5 g/L (High) of glucose with no added OCM (CON), or 2.5, 5, or 10 times folate, choline, and B12 (2.5X, 5X, or 10X, respectively) in the CON media. Methionine was limited to 2X CON. The data were analyzed as a completely randomized design with a 2 × 4 factorial arrangement of treatments. Cells were passaged three times in treated media before RNA extraction. Gene expression analysis was conducted on Methionine Adenosyltransferase 2A and 2B (MAT2A and MAT2B) DNA Methyltransferase 1, 3A, and 3B (DNMT1, DNMT3A, and DNMT3B), S-Adenosylhomocysteine Hydrolase (AHCY), and Methionine Synthase (MTR). There was no glucose × OCM interaction for DNMT1, DNMT3A, DNMT3B, AHCY, or MTR (P &amp;gt; 0.17). Expression of MAT2B was greater (P &amp;lt; 0.01) in 2.5X, 5X, and 10X OCM in High media compared with all other treatments. Expression of DNMT1 was greater in CON, 5X, and 10X OCM in Low media compared with all other treatments. Expression of MTR was greater (P = 0.03) in CON and 10X OCM treatments compared with 2.5X and 5X levels. Finally, MAT2A and AHCY expression was affected by glucose level, with MAT2A being greater (P = 0.02) in Low compared with High, and AHCY being greater (P &amp;lt; 0.01) in High compared with Low media. These data suggest that glucose and OCM supplementation differentially affect the expression of genes in the methionine-folate cycle of DNA methylation. Additionally, these data demonstrate that additional work elucidating the changes in protein abundance of these enzymes, concentrations of methionine-folate cycle intermediates (specifically S-adenosylmethionine:S-adenosylhomocysteine), as well as changes in DNA methylation are needed.

Abstract 4246: Association of transcriptional levels of folate-mediated one-carbon metabolism related genes in cancer cell lines with drug treatment response

Abstract Folate-mediated one-carbon metabolism (OCM) is essential for growth and survival of cancer cells. It affects biosynthesis of nucleotides and amino acids, regulation of redox status, methylation of nucleic acids and proteins, and genome maintenance. We investigated whether the response of cancer cells to antitumor therapy treatment may be partially influenced by variation in expression of one-carbon metabolism genes. We used publicly available gene expression data and drug response measures for 251 antitumor agents in 635 cancer cell lines with matching information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources. We examined whether pre-treatment expression levels of OCM-related genes were associated with drug response. Among the 34 OCM genes examined, expression of GART, TYMS, SHMT2, MTR, ALDH2, BHMT, MAT2B, MTHFD2, NNMT, and SLC46A1 showed modest correlations with response to treatment with a variety of antitumor agents. Higher expression levels of the SLC46A1 transporter gene were associated with resistance to multiple drugs, whereas elevated expression of GART, TYMS, SHMT2, MTR, BHMT, and MAT2B was associated with chemosensitivity to multiple antitumor agents. NNMT expression was bimodally distributed and showed different directions of association with various agents. Correlation of increased NNMT expression with sensitivity to dasatinib was validated in the NCI-60 cancer cell line panel. Expression of several OCM genes was strongly associated with expression of multiple components of drug target pathways. For example, expression of both TYMS and GART was strongly positively correlated with BRCA1 expression, NNMT expression was associated with expression of multiple drug target genes including EGFR and ABL2, and the expression of TYMS, DHFR, and SHMT1 was positively correlated with AURKB expression. Pretreatment expression levels of many OCM genes including DHFR, TYMS, ATIC, GART, AHCY, MTHFD1, SLC19A1, and multiple other genes were positively correlated with each other, suggesting their co-regulation. In contrast, NNMT expression was negatively correlated with expression of several other OCM genes. Further studies could investigate whether correlations of expression levels of individual OCM genes with drug response are related to specific metabolic roles of these genes or whether such associations may be due to the general increase in cellular growth and proliferation and tumor progression, which may affect sensitivity to cancer treatment. Citation Format: Julia Krushkal, Suleyman Vural, Dong-Joon Min. Association of transcriptional levels of folate-mediated one-carbon metabolism related genes in cancer cell lines with drug treatment response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4246.

Association of transcriptional levels of folate-mediated one-carbon metabolism-related genes in cancer cell lines with drug treatment response

Folate-mediated one-carbon metabolism is essential for growth and survival of cancer cells. We investigated whether the response of cancer cells to antitumor treatment may be partially influenced by variation in expression of one-carbon metabolism genes. We used cancer cell line information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources to examine whether variation in pretreatment expression of one-carbon metabolism-related genes was associated with response to treatment. GART, TYMS, SHMT2, MTR, ALDH2, BHMT, MAT2B, MTHFD2, NNMT, and SLC46A1 showed modest statistically significant correlations with response to a variety of antitumor agents. Higher expression levels of SLC46A1 were associated with resistance to multiple agents, whereas elevated expression of GART, TYMS, SHMT2, MTR, BHMT, and MAT2B was associated with chemosensitivity to multiple drugs. NNMT expression was bimodally distributed and showed different directions of association with various agents. Correlation of increased NNMT expression with sensitivity to dasatinib was validated in the NCI-60 cancer cell line panel. Pretreatment expression levels were correlated among many one-carbon metabolism genes. Expression of several folate genes was strongly associated with expression of multiple components of drug target pathways. Molecular mechanisms underlying associations of one-carbon metabolism gene with drug response require further investigation.

Elastin modulation and modification by homocysteine: a key factor in the pathogenesis of Pseudoexfoliation syndrome?

BackgroundPseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF....

Human methionine synthase A2756G polymorphism increases susceptibility to prostate cancer.

Background/Aims: Previous results on the association between MTR gene A2756G polymorphism and PCa risk are inconclusive.Methods: We used odds ratios (ORs) with corresponding 95% confidence intervals (95% CIs) to evaluate the correlation between MTR A2756G polymorphism and risk of PCa in meta-analysis. Serum expression of MTR was detected by ELISA and in-silico tools were utilized to assess this variant.Results: Our study included 2,921 PCa patients and 3,095 control subjects. The results indicated that the MTR A2756G polymorphism is linked with an increased risk of PCa using three genetic models (G-allele vs. A-allele: OR = 1.16, 95%CI = 1.04 - 1.30; GA vs. AA: OR = 1.17, 95%CI = 1.02 - 1.33; GG+GA vs. AA: OR = 1.18, 95%CI = 1.04 - 1.34). Stratified analysis produced similar results. A significant association was also indicated in advanced PCa from the meta-analysis. Finally, our experiments showed evidence that serum MTR levels in PCa patients with AA genotypes were statistically higher than in those with GG/GA genotypes.Conclusions: Our present study suggests that the MTR A2756G polymorphism may contribute to the risk of developing PCa, particularly in Asian and hospital-based studies. Moreover, serum MTR might be utilized in diagnosis of PCa.

Association of a MiR-499 SNP and risk of congenital heart disease in a Chinese population

MicroRNAs (miRNAs) play an important role in heart development. Single nucleotide polymorphisms (SNPs) in miRNAs have been shown to associate with congenital heart disease (CHD). Methionine synthase (MTR), a key enzyme of folate metabolism, is involved in the early embryonic development. In this study, we aimed to test whether MTR is a direct target of miR-499, and to estimate the associations between miR-499 polymorphisms and the risk of CHD in Chinese population. Gene polymorphisms were analyzed in 1615 subjects including 792 healthy controls and 823 CHD patients. The miR-499 SNP were genotyped and the associations between the SNP frequencies and CHD were assessed by computing odds ratios (ORs) and 95% confidence intervals (95% CIs), as well as by applying Chi-square tests. Dual reporter assay was carried out to test whether MTR is a direct target gene of miR-499. The miR-499 rs374644 AG genotype was not associated with the CHD risk (AG vs. AA. OR=1.27, 95%CI=0.85-1.81, p=0.20). The GG genotype was associated with a significantly increased CHD risk (GG vs. AA. OR=5.33, 95%CI=1.80-15.83, p=0.001). The AG/GG variants were associated with a significantly increased CHD risk, compared with the AA genotype (OR=1.56, 95%CI=1.16-2.10, p=0.003). MiR-499 mimics inhibits the expression of MTR. MiR-499 directly targeted on MTR. Thus, our study suggested that miR-499 directly targets on MTR and the polymorphisms of rs3746444 may be associated with CHD risk in Chinese individuals.

Attenuated expression of MTR in both prenatally androgenized mice and women with the hyperandrogenic phenotype of PCOS.

Polycystic ovary syndrome (PCOS) is a common endocrine, metabolic and heterogeneous disorder in women of reproductive age, the exact etiology of which remains unknown. To unravel the molecular mechanisms underlying the hyperandrogenic phenotype of PCOS, prenatally androgenized (PNA) mice were used to mimic this phenotype in women with PCOS. Using microarray analysis, 1188 differentially expressed genes, including 671 upregulated and 517 downregulated genes, were identified in ovaries from PNA mice. Five differentially expressed genes (Aldh1a7, Bhmt, Mtr, Nrcam, Ptprg) were validated, and decreased MTR expression was shown in ovaries of PNA mice. In addition, results from qRT-PCR showed decreased MTR expression in granulosa cells (GCs) from women with the hyperandrogenic phenotype of PCOS. Serum levels of S-adenosyl methionine (SAM), the downstream product of MTR, were also decreased in PNA mice and women with the hyperandrogenic phenotype of PCOS. Our study provides evidence that the hyperandrogenic phenotype of PCOS is linked to abnormal folate one-carbon metabolism.

The importance of MTHFR, MTR, MTRR and CSE expression levels in Caucasian women with preeclampsia

ObjectivePreeclampsia (PE) is a major cause of maternal and perinatal mortality and morbidity. The studies suggest that both polymorphisms and changes of expression in genes encoding enzymes involved in the methionine and homocysteine metabolism (MHM), such as methylenetetrahydrofolate, reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR) and cystathionine gamma-lyase (CSE), could play a role in the development of hypertension during pregnancy. The aim of the study was to determine the expression level of MTHFR, MTR, MTRR and CSE genes in the development of PE in Caucasian women. Study designThe control group consisted of 74 healthy pregnant women and 90 patients with diagnosed pre-eclampsia. Total RNA was isolated from placenta and the mRNA level of examined genes was to determine using real-time PCR. ResultsThe expression level of MTHFR gene showed no statistically significant difference in the study group as compared to the control group. An increase of mRNA levels for MTR and CTH was observed by 124.7% (p<0.0001) and 26.6% (p>0.05), respectively. However, a decrease of placental expression was noted for MTRR by 50% in preeclamptic women as compared to control group (p<0.0001). ConclusionsOur findings suggest that the elevated RNA expression of MTR in placenta of preeclamptic patients is probably results of a potential compensation mechanism of the MHM while elevated CSE expression indicates that homocysteine may be eliminated through the alternate transsulfuration pathway.

Localization of Folate Metabolic Enzymes, Methionine Synthase and 5,10-Methylenetetrahydrofolate Reductase in Human Placenta

Background: Normal fetal development requires adequate folate levels during pregnancy. Although folate metabolic enzymes have important roles in the maintenance of normal fetal development, the location of folate metabolic enzymes, methionine synthase (MTR) and 5,10-methylenetetrahydrofolate reductase (MTHFR), has not been previously examined. Methods: We investigated the expression of MTR and MTHFR in human term placenta obtained from normal and pregnancy-induced hypertension (PIH) patients. Results: MTR is expressed in the villous syncytiotrophoblast and MTHFR is expressed in the extravillous trophoblast. There was no difference in the quantity and location of these enzymes between control and PIH patients. Conclusion: These results suggest that MTR in the villous trophoblast participates in the metabolism of homocysteine by using folate, and MTHFR in the extravillous trophoblast is associated with extratrophoblast invasion.

Thyroid Hormones Maintain The CTCL Malignant Phenotype Through Membrane- and Nuclear-Initiated Transcriptional Programs

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of T-cell non-Hodgkin lymphomas (T-NHL) derived from skin-homing mature T cells. Thyroid hormones (THs) are crucial regulators of cellular differentiation, growth and metabolism. We recently found that THs stimulate the proliferation and metabolism of T cells and that this pathway is co-opted by T-NHL cells to maintain their malignant phenotype. Accordingly, a decrease in TH levels affects the proliferation of T-NHL cells. We also found that THs modulate these functions by activating both canonical nuclear (TR) and membrane receptors (mTR, that for most cells is represented by integrin αvβ3). We now hypothesized that the characterization of the relative contribution of these receptors to the malignant phenotype of T-NHLs will allow for a more precise therapeutic intervention. The study presented here was conducted to determine (i) whether THs are pro-survival factors in CTCL and (ii) the relative contribution of TR and mTR to the effect of THs in CTCLs.To test this hypothesis, we first analyzed the expression of TR and mTR in CTLC cells (HuT 78, MJ and HuT102) vs. normal T cells (from peripheral blood) by qRT-PCR and immunoblotting. We found that CTCL expressed 2 to 10 fold more THRA, ITGAV and ITGB3 mRNA and protein than their normal counterpart (p<0.05, T test). Moreover, in a tissue microarray of 52 CTCLs, the integrin avβ3 was overexpressed in 92% of the cases. To differentiate between nuclear vs. membrane-initiated effects, CTCL cells were treated with physiological concentrations of free and cell impermeable agarose-bound T3/T4 (TH-ag). After 24h of treatment, cell proliferation increased 20-38% in HuT-78 cells and 18-32% in MJ cells with both forms of THs (p<0.05 for both comparisons vs. control). Long-term exposure (96h) to free TH and TH-ag accelerated HuT-78 doubling time by 17-25% and 16-22%, respectively (p<0.05 for both comparisons). By time course analysis of mRNA abundance (1, 2, 3, 6, 12 and 24 h post TH), we found that TH-induced phenotypic changes were associated with 1.5 to 3 fold up-regulation of proliferation markers such as PCNA, CCND1, CCND2, CCND3, CCNE2 and CCNB1 in addition to a concomitant decrease of the tumor suppressor genes TP53 and CDKN1A.To determine the specific transcriptional programs regulated by mTR and TR, T-NHLs were treated with free T3/T4 (for total TR activity) and agarose-coupled T3/T4 (for mTR activity) for 24h and analyzed by mRNA-sequencing. Differentially regulated genes were further analyzed using Ingenuity Pathway Analysis. We found the TNFR and interleukin-2 pathways (e.g. TNFRSF19, TNFRSF9, IL2RA) among the top programs regulated by free THs through canonical nuclear TR activation whereas mTR activation upregulated genes involved in angiogenesis induction (e.g. PDGFR, VEGFA, VEFGB), lymphocyte proliferation/differentiation and DNA replication (e.g. IL4, DOK2). We independently validated these genes in HuT-78 and MJ cell lines by qRT-PCR. Taken together, these data suggest that TR and mTR regulate distinct transcriptional programs that are complimentary towards CTCL proliferation.Considering that (i) mTR are over-expressed in CTCL vs. normal T cells (in contrast to canonical nuclear TR that are broadly expressed) and (ii) mTR regulate pro-survival pathways in CTLC, we wished to determine whether mTR inhibition will be sufficient to decrease CTCL proliferation. We therefore tested the effect of mTR abrogation by using siRNA to ITGAV and ITGB3 as well as the effect of pharmacological mTR inhibitors (i.e: RGD peptide and Tetrac) in CTCL proliferation. We found that mTR inhibition with siRNA or compounds affected mTR-induced transcriptional programs. In fact, the TH-ag mediated upregulation of VEGFB, VEGFA, DOK2 and IL4 mRNA levels were totally abrogated in CTCL cells transfected with the siRNA to ITGAV and ITGB3. Moreover, almost 100% of the TH-ag-induced and 80% of the free-TH-induced CTCL proliferative effect was abrogated by ITGAV- and/or ITGB3-targeting siRNA.Taken together these data suggest that THs are important inducers of CTCL proliferation by transcriptionally regulating major survival pathways, and inhibition of mTR could constitute a selective chemotherapy-free treatment for CTCLs. Disclosures:No relevant conflicts of interest to declare.

Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase

Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin-oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a 'dam-progeny' rat model developed in our laboratory is associated with long-lasting disabilities of behavior and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of fetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates MDR-1 gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and apoptosis in brain. Vitamin B12 and folate combined deficiency impairs fatty acid oxidation and energy metabolism in liver and heart through epigenomic mechanisms related to imbalanced acetylation/methylation. Some but not all of these effects reflect the upstream role of vitamin B12 in SAM synthesis.

Levels of Key Enzymes of Methionine-Homocysteine Metabolism in Preeclampsia

Objective. To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM) in the physiopathology of preeclampsia (PE). Methods. Plasma and placenta from pregnant women (32 controls and 16 PE patients) were analyzed after informed consent. Protein was quantified by western blot. RNA was obtained with RNA purification kit and was quantified by reverse transcritase followed by real-time PCR (RT-qPCR). Identification of the C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) and A2756G methionine synthase (MTR) SNP was performed using PCR followed by a high-resolution melting (HRM) analysis. S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). The SNP association analysis was carried out using Fisher's exact test. Statistical analysis was performed using a Mann-Whitney test. Results. RNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls. Protein, SAM, and SAH levels showed no significant difference between preeclamptic patients and controls. No statistical differences between controls and PE patients were observed with the different SNPs studied. Conclusion. The RNA expression of MTHFR and MTR is elevated in placentas of PE patients, highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in the physiopathology of this disease.

Expression of telomerase gene mTR in the testis of SD rats and its significance.

To study the expression of mTR gene in the testis of SD rats with varied ages and its significance, in situ hybridization (ISH) techniques were applied to detect the expression of telomerase gene mTR mRNA in the testis of SD rats. The expression of mTR was found in testes of different-age male SD rats. There was a positive correlation between the expression of mTR and the location of germ cells (spermatogonia, spermatocyte, spermatid). In Setoli cells, leydig cell and spermatozoa, no telomerase mTR was detectable. Type A spermatogonia expressed the highest level of telomerase mTR mRNA. It was suggested that the expression of mTR gene in the testis of SD rats is of lifetime and coincides with the telomerase activity.