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...