Background Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. Objectives This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. Methods Liver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2+/− and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2+/− and Shmt2+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. Results Shmt2 +/− mice exhibited a 48%–67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2+/− mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/− mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/− mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. Conclusions This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.
Zinc plays an integral role in numerous cellular processes including regulation of gene expression. This randomized placebo-controlled trial in adult women evaluated the effects of 20 mg Zn for 23 days. The mRNA abundance of zinc transporters (ZnT1/ZIP3/ZIP4/ZIP8) and metallothionein (MT1) from peripheral blood mononuclear cells was determined by real-time quantitative polymerase chain reaction. In paired samples (n = 6–9), the ZIP4 (P = 0.036) and ZIP8 (P = 0.038) mRNA abundance decreased following zinc supplementation. ZnT1, ZIP3, and MT1 mRNA abundance did not change significantly. The mean ± standard deviation plasma zinc concentration (by inductively coupled plasma mass spectrometry) at baseline was 680 ± 110 μg/L for the zinc group (n = 24) and 741 ± 92 μg/L for the placebo group (n = 23). At endpoint, plasma zinc in the zinc group increased to 735 ± 80 μg/L (P < 0.01) while in the placebo group (717 ± 100 μg/L) it did not change significantly from baseline. The change in mRNA abundance highlights the importance of further investigating ZIP4 and ZIP8 mRNA abundance as potential zinc status biomarkers.
Background Quantitative real-time polymerase chain reaction (qPCR) is a reliable and efficient method for quantitation of gene expression. Due to the increased use of qPCR in examining nutrient-gene interactions, it is important to examine, develop, and utilize standardized approaches for data analyses and interpretation. A common method used to normalize expression data involves the use of reference genes (RG) to determine relative mRNA abundance. When calculating the relative abundance, the selection of RG can influence experimental results and has the potential to skew data interpretation. Although common RG may be used for normalization, often little consideration is given to the suitability of RG selection for an experimental condition or between various tissue or cell types. In the current study, we examined the stability of gene expression using BestKeeper, comparative delta quantitation cycle, NormFinder, and RefFinder in a variety of tissues obtained from iron-deficient and pair-fed iron-replete rats to determine the optimal selection among ten candidate RG. Results Our results suggest that several commonly used RG (e.g., Actb and Gapdh) exhibit less stability compared to other candidate RG (e.g., Rpl19 and Rps29) in both iron-deficient and iron-replete pair-fed conditions. For all evaluated RG, Tfrc expression significantly increased in iron-deficient animal livers compared to the iron-replete pair-fed controls; however, the relative induction varied nearly 4-fold between the most suitable (Rpl19) and least suitable (Gapdh) RG. Conclusion These results indicate the selection and use of RG should be empirically determined and RG selection may vary across experimental conditions and biological tissues.
Microglial cells are the resident macrophages of the central nervous system (CNS). These cells are a primary form of active immune defense in the CNS. In neurodegenerative disorders such as Alzheimer's and Parkinson's disease, microglia are chronically activated and promote the release of pro‐inflammatory cytokines which further disrupt normal CNS activity. There is considerable interest in examining the extent to which bioactive food components can mitigate the effects of inflammation by decreasing oxidative stress and/or by decreasing pro‐inflammatory gene expression.In this study, BV‐2 cells were treated with Lipopolysaccharide (LPS) for 4 or 24 hours and total RNA was isolated from the cells for gene expression analyses. A control group was also present. The LPS‐treated BV‐2 cells did not show the robust induction of IL‐1β and TNFα that was expected at either 4 hours or 24 hours. To determine if other cells would respond to LPS, a previously characterized LPS‐responsive cell line (RAW 264.7) was examined. In RAW 264.7 cells, LPS treatment increased the expression of IL‐1β, iNOS, and TNFα. Since current research on microglial cells suggests a much larger response to LPS, additional experimentation should be done to show consistent results.
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