Vitamin D-dependent rickets type I (VDDR-I), also known as pseudovitamin D deficiency rickets (PDDR), is an autosomal recessive disorder characterized by low or undetectable levels of 1␣,25(OH) 2 D, secondary hyperparathyroidism, hypocalcemia, hypophosphatemia, and severe rachitic lesions (18 -21). VDDR-I is assumed to result from impaired synthesis of 1␣,25(OH) 2 D, and, indeed, a number of 1␣(OH)ase gene mutations have been reported in this disorder that result in diminished or absent 1␣(OH)ase activity (13,(22)(23)(24)(25)(26).To further investigate the functional role of the 1␣(OH)ase enzyme, we generated mice deficient in 1␣(OH)ase by gene targeting. Materials and MethodsMethods including construction of the 1␣(OH)ase targeting vector; transfection of embryonic stem (ES) cells and generation of 1␣(OH)ase-deficient mice; Southern blot and PCR analysis of ES cell and mouse tail DNA; Northern blot analysis; biochemical and hormonal analyses; histological analysis; computer-assisted image analysis; immunohistochemistry; and f luorescenceactivated cell sorter (FACS) lymphocyte phenotyping are presented in the supplemental data (which is published on the PNAS web site, www.pnas.org). ResultsThe targeting vector shown in Fig. 1A was used to inactivate one allele of the 1␣(OH)ase gene in ES cells. The inactivated allele lacked both the hormone-binding domain and the heme-binding domain of the enzyme. Two independent ES cell clones were used to generate two lines of mice heterozygous for the mutation, which were then interbred to generate 1␣(OH)ase null (Ϫ͞Ϫ) mice (Fig. 1B). Litter sizes were no different from normal, and the mutated allele was transmitted to the progeny with the expected Mendelian frequency. Thus, haploinsufficiency of the 1␣(OH)ase did not affect embryonic survival. By reverse transcription (RT)-PCR, renal expression of the kidney 1␣(OH)ase mRNA in (ϩ͞Ϫ) mice was reduced relative to that in (ϩ͞ϩ) mice, and, in (Ϫ͞Ϫ) mice, it was undetectable (Fig. 1C).Circulating concentrations of 1,25(OH) 2 D were undetectable in the homozygous null mice and were somewhat lower (although not significantly so) in the heterozygotes relative to normals at 7 weeks of age (Table 1). Serum 25(OH)D concentrations were elevated in (Ϫ͞Ϫ) mice relative to the heterozygotes and normals. Both serum calcium and phosphate concentrations were reduced in (Ϫ͞Ϫ) mice relative to the (ϩ͞Ϫ) mice that were normal, and urinary phosphate was increased in the homozygous null mice. Serum parathyroid hormone concentrations were markedly elevated, the alkaline phosphatase concentrations were twice normal, and the body weight was substantially reduced in the homozygous null mice at this time (Table 1). The null mutant mice appeared grossly normal from birth until This paper was submitted directly (Track II) to the PNAS office.Abbreviations: 1␣(OH)ase, 25(OH)D-1␣-hydroxylase; VDDR-I, vitamin D dependent rickets type I; VDR, vitamin D receptor.

Apolipoprotein D (apoD) is a 29-kDa glycoprotein that is primarily associated with high density lipoproteins in human plasma. It is an atypical apolipoprotein and, based on its primary structure, apoD is predicted to be a member of the lipocalin family. Lipocalins adopt a beta-barrel tertiary structure and transport small hydrophobic ligands. Although apoD can bind cholesterol, progesterone, pregnenolone, bilirubin and arachidonic acid, it is unclear if any, or all of these, represent its physiological ligands. The apoD gene is expressed in many tissues, with high levels of expression in spleen, testes and brain. ApoD is present at high concentrations in the cyst fluid of women with gross cystic disease of the breast, a condition associated with increased risk of breast cancer. It also accumulates at sites of regenerating peripheral nerves and in the cerebrospinal fluid of patients with neurodegenerative conditions, such as Alzheimer's disease. ApoD may, therefore, participate in maintenance and repair within the central and peripheral nervous systems. While its role in metabolism has yet to be defined, apoD is likely to be a multi-ligand, multi-functional transporter. It could transport a ligand from one cell to another within an organ, scavenge a ligand within an organ for transport to the blood or could transport a ligand from the circulation to specific cells within a tissue.

Summary Human embryonic stem cells (hESCs) readily differentiate to somatic or germ lineages but have impaired ability to form extra-embryonic lineages such as placenta or yolk sac. Here, we demonstrate that naive hESCs can be converted into cells that exhibit the cellular and molecular phenotypes of human trophoblast stem cells (hTSCs) derived from human placenta or blastocyst. The resulting “transdifferentiated” hTSCs show reactivation of core placental genes, acquisition of a placenta-like methylome, and the ability to differentiate to extravillous trophoblasts and syncytiotrophoblasts. Modest differences are observed between transdifferentiated and placental hTSCs, most notably in the expression of certain imprinted loci. These results suggest that naive hESCs can differentiate to extra-embryonic lineage and demonstrate a new way of modeling human trophoblast specification and placental methylome establishment.

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1؊/؊ embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of ؊/؊ embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1؊/؊ embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.clk-1 mutants of Caenorhabditis elegans are being studied for their pleiotropic phenotype, in which the rates of many biological processes are deregulated and slowed down on average (1, 2). clk-1 encodes a highly conserved (3, 4) mitochondrial (5, 6) protein that is required for ubiquinone (UQ) 1 biosynthesis in yeast (7) and worms (8, 9). Recent evidence suggests that CLK-1 is a hydroxylase that converts demethoxyubiquinone (DMQ) into 5-hydroxy-UQ (10). Indeed, a bacterial CLK-1 homologue is capable of replacing the function of UbiFp, the unrelated enzyme that carries out this function in Escherichia coli. Consistent with this finding, clk-1 mutants in yeast and worms accumulate DMQ 9 instead of producing UQ 9 (7, 9) (the subscript refers to the length of the isoprenoid side chain). In E. coli, DMQ 8 is able to sustain respiration in isolated membranes although at a lower rate than Q 8 (11). Similarly, DMQ 9 also appears to be capable of sustaining electron transport in clk-1 mutants at almost wild-type levels (6, 9). Furthermore, synthetic DMQ 2 can function as a co-factor for electron transport from Complex I and, albeit more poorly, from Complex II (9).It is not clear how the absence of UQ relates to the other phenotypes of clk-1 mutants as there is no correlation between the biochemical phenotype and the severity of the overall phenotype. Indeed, the quinone phenotype is identical for all three known clk-1 alleles (e2519, qm30, and qm51); UQ 9 is undetectable in the mitochondria in all three cases, and all three accumulate the same amount of DMQ. Yet, most of the features affected in clk-1 mutants are slowed down much more severely in the putative null alleles qm30 and qm51 than they are in the part...

Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNA fmet . One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNA fmet . To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.Folic acid is essential for the synthesis of purines and thymidylate and is also required for the remethylation of homocysteine to methionine to support the many cellular methylation reactions that use [S]adenosylmethionine. Because of its numerous roles, it is not surprising that the status of folic acid has been linked with a number of conditions that include megaloblastic anemia, cardiovascular disease, and birth defects, as well as with several types of cancer (for a review, see reference 13). The metabolically active form, tetrahydrofolate (THF), functions to transfer one-carbon units that exist in different oxidation states (14). These one-carbon folates are interconverted by enzymes that are present in cytosolic and mitochondrial compartments in mammalian cells (Fig. 1). Recently, there has been a considerable interest in understanding the metabolic advantages for this compartmentalization, support...

Background:Cachexia is a metabolic disorder characterised by muscle wasting, diminished response to anti-cancer treatments and poor quality of life. Our objective was to identify blood-based biomarkers of cachexia in advanced cancer patients. Hence, we characterised the plasma cytokine and blood cell mRNA profiles of patients grouped in three cohorts: patients with cachexia, pre-cachexia (no cachexia but high CRP levels: ⩾5 mg l−1) and no cachexia (no cachexia and CRP: <5 mg l−1).Methods:A total of 122 newly diagnosed cancer patients with seven cancer types were studied prior to their initial therapy. Plasma levels of 22 cytokines were quantified using the bio-plex technology. mRNAs isolated from whole blood and expression profiles were determined by the chip array technology and Ingenuity Pathway Analysis (IPA) software.Results:In comparison with non-cachectic individuals, both pre-cachectic and cachectic patients showed an increase (⩾1.5-folds) in mRNA expression of neutrophil-derived proteases (NDPs) and significantly elevated angiotensin II (Ang II) (P=0.005 and P=0.02, respectively), TGFβ1 (P=0.042 and P<0.0001, respectively) and CRP (both P<0.0001) in the plasma. Moreover, cachectic patients displayed a significant increase in IL-6 (P=0.005), IL-8 (P=0.001) and absolute neutrophil counts (P=0.007).Conclusions:Ang II, TGFβ1, CRP and NDP are blood biomarkers for cancer cachexia. These findings contribute to early diagnosis and prevention of cachexia.

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.

Epigenetic modifications on the chromatin do not occur in isolation. Chromatin associated proteins and their modification products form a highly interconnected network, and disturbing one component may rearrange the entire system. We see this increasingly clearly in epigenetically dysregulated cancers. It is important to understand the rules governing epigenetic interactions. Here, we use the mouse embryonic stem cell (mESC) model to describe in detail the relationships within the H3K27-H3K36-DNA methylation subnetwork. In particular, we focus on the major epigenetic reorganization caused by deletion of the histone 3 lysine 36 methyltransferase NSD1, which in mESCs deposits nearly all of the intergenic H3K36me2. Although disturbing the H3K27 and DNA methylation (DNAme) components also affects this network to a certain extent, the removal of H3K36me2 has the most drastic effect on the epigenetic landscape, resulting in full intergenic spread of H3K27me3 and a substantial decrease in DNAme. By profiling DNMT3A and CHH methylation (mCHH), we show that H3K36me2 loss upon Nsd1-KO leads to a massive redistribution of DNMT3A and mCHH away from intergenic regions and towards active gene bodies, suggesting that DNAme reduction is at least in part caused by redistribution of de novo methylation. Additionally, we show that pervasive acetylation of H3K27 is regulated by the interplay of H3K36 and H3K27 methylation. Our analysis highlights the importance of H3K36me2 as a major determinant of the developmental epigenome and provides a framework for further consolidating our knowledge of epigenetic networks.