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Enzyme-linked i2mmunosorbent assays (ELISAs) that measure circulating phylloquinone have become commercially available, but their validity is uncertain. The objective of this study was to compare plasma phylloquinone concentrations measured using two commercially available ELISAs with concentrations measured using a validated high-performance liquid chromatography (HPLC) assay in 108 samples obtained from participants in a depletion (~10 mcg phylloquinone/d)-supplementation (~500 mcg phylloquinone/d) study. The geometric mean of plasma phylloquinone measured with ELISA A was 0.70 nmol/L, 37% lower than that measured with HPLC. The mean of the ELISA B measures was 12.4 nmol/L, >700% higher than the HPLC measures. Plasma phylloquinone measured using HPLC was significantly lower during phylloquinone depletion than supplementation (0.4 ± 0.1 compared with 1.2 ± 0.2 nmol/L; P < 0.001). Neither of the two ELISAs detected any significant difference in plasma phylloquinone concentrations between depletion and supplementation (ELISA A, P = 0.76; ELISA B, P = 0.29). These findings reinforce the need to validate plasma phylloquinone assays as they become available. [ABSTRACT FROM AUTHOR]

Background: There has been limited characterization of biological variables that impact vitamin K metabolism. This gap in knowledge can limit the translation of data obtained from preclinical animal studies to future human studies.Objective: The purpose of this study was to determine the effects of diet, sex, and housing on serum, tissue, and fecal vitamin K concentrations and gene expression in C57BL6 mice during dietary vitamin K manipulation.Methods: C57BL6 4-mo-old male and female mice were randomly assigned to conventional or suspended-wire cages and fed control [1400 ± 80 μg phylloquinone (PK)/kg] or deficient (31 ± 0.45 μg PK/kg) diets for 28 d in a factorial design. PK and menaquinone (MK) 4 plasma and tissue concentrations were measured by HPLC. Long-chain MKs were measured in all matrices by LC-atmospheric pressure chemical ionization-mass spectrometry. Gene expression was quantified by reverse transcriptase-polymerase chain reaction in the liver, brain, kidney, pancreas, and adipose tissue.Results: Male and female mice responded differently to dietary manipulation in a tissue-dependent manner. In mice fed the control diet, females had ∼3-fold more MK4 in the brain and mesenteric adipose tissue than did males and 100% greater PK concentrations in the liver, kidney, and mesenteric adipose tissue than did males. In mice fed the deficient diet, kidney MK4 concentrations were ∼4-fold greater in females than in males, and there were no differences in other tissues. Males and females differed in the expression of vitamin K expoxide reductase complex 1 (Vkorc1) in mesenteric adipose tissue and the pancreas and ubiA domain-containing protein 1 (Ubiad1) in the kidney and brain. There was no effect of housing on serum, tissue, or fecal concentrations of any vitamin K form.Conclusions: Vitamin K concentrations and expression of key metabolic enzymes differ between male and female mice and in response to the dietary PK concentration. Identifying factors that may impact study design and outcomes of interest is critical to optimize study parameters examining vitamin K metabolism in animal models. [ABSTRACT FROM AUTHOR]

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca2+ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca2+ transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca2+ [³H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca2+ activation or Ca2+ and Mg2+ inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca2+ homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling. [ABSTRACT FROM AUTHOR]

Histone H3 lysine 27 (H3K27) methylation and H2A monou-biquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyl- transferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltrans- ferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A frommammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications. [ABSTRACT FROM AUTHOR]