␤-Carotene 15,15-monooxygenase (BCO), formerly known as ␤-carotene 15,15-dioxygenase, catalyzes the first step in the synthesis of vitamin A from dietary carotenoids. We have biochemically and enzymologically characterized the purified recombinant human BCO enzyme. A highly active BCO enzyme was expressed and purified to homogeneity from baculovirusinfected Spodoptera frugiperda 9 insect cells. The K m and V max of the enzyme for ␤-carotene were 7 M and 10 nmol retinal/mg ؋ min, respectively, values that corresponded to a turnover number (k cat ) of 0.66 min ؊1 and a catalytic efficiency (k cat /K m ) of ϳ10 M ؊1 ⅐min؊1 . The enzyme existed as a tetramer in solution, and substrate specificity analyses suggested that at least one unsubstituted ␤-ionone ring half-site was imperative for efficient cleavage of the carbon 15,15-double bond in carotenoid substrates. High levels of BCO mRNA were observed along the whole intestinal tract, in the liver, and in the kidney, whereas lower levels were present in the prostate, testis, ovary, and skeletal muscle. The current data suggest that the human BCO enzyme may, in addition to its well established role in the digestive system, also play a role in peripheral vitamin A synthesis from plasma-borne provitamin A carotenoids.

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We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

S U M M A R YThe symmetrically cleaving ␤ -carotene 15,15 Ј -monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered ␤ -carotene 9 Ј ,10 Ј -monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis. T he enzymatic action of ␤ -carotene 15,15 Ј -monooxygenases (BCO1) is crucial for the conversion of provitamin A carotenoids to retinol (vitamin A) in the epithelial cells of the small intestine mucosa. The enzyme cleaves the ␤ -ionone ring containing carotenoids centrally, which results in two aldehyde molecules with polyene chains of identical length. Thus cleavage of the most common carotenoid ␤ -carotene results in two molecules of retinaldehyde (retinal), which will be further converted to retinol by a reductive retinal reductase enzyme present in the same cell as BCO1. Previously, we studied the cell type-specific expression of BCO1 and found that the enzyme is also expressed in epithelia of several extraintestinal tissues, which suggested that these tissues have the capacity to directly convert locally stored carotenoids to vitamin A. Thus this may serve as a backup pathway of vitamin A synthesis during times of insufficient dietary intake of preformed vitamin A and provitamin A carotenoids.Recently, Kiefer et al. (2001) cloned and characterized a second carotenoid-cleaving enzyme termed ␤ -carotene 9 Ј ,10 Ј -monooxygenase (BCO2) because it catalyzes asymmetric cleavage of carotenoids, thus yielding one molecule of ␤ -apo-10 Ј -carotenal and one molecule of ␤ -ionone when ␤ -carotene is used as substrate. Interestingly, the BCO2 enzyme appears to accept a wider variety of substrates as compared with BCO1, including the acyclic carotenoid lycopene, which implies that this enzyme may have physiological roles other than providing precursors for vitamin A synthesis.BCO1 and BCO2 belong to a superfamily of nonheme iron-containing oxygenases, many of whose functions are unknown. The physiological role of BCO1 is now well establ...

Ten male pseudohermaphrodites with 17 beta-hydroxysteroid dehydrogenase 3 (17 beta-HSD3) deficiency were evaluated in 1 clinic with an average follow-up of 10.1 years. The diagnoses were made by demonstrating low to normal serum testosterone levels, high androstenedione levels, and high ratios of serum androstenedione to testosterone in the basal state or after treatment with human chorionic gonadotropin. The molecular features of the underlying mutations were identified in all 7 families. Two additional males in the same families are believed to be affected on the basis of history obtained from family members. All of the 46,XY individuals in these families were registered at birth and raised as females (despite the presence of ambiguous genitalia in all or most), and all virilized after the time of expected puberty due to a rise in serum testosterone to or toward the normal male range. The age at diagnosis varied from 4 to 37 years. Ten individuals were studied by the same psychologist, and change of gender role (social sex) from female to male occurred in 3 subjects and in the 2 presumed affected subjects not studied. The individual with the highest serum testosterone level maintained female sexual identity, and in 2 families some of the affected males changed gender role and others did not. Thus, while androgen action plays a role in the process, additional undefined psychological, social, and/or biologic factors must be determinants of gender identity/role behavior. Management of the 7 individuals who chose to maintain female sex roles included castration, clitoroplasty, vaginal enlargement procedures when appropriate, treatment of hirsutism, cricoid cartilage reduction, and estrogen replacement. Three of the 7 are married (2 twice), 1 is involved in a long-term heterosexual relationship, 1 is engaged to be married, and the other 2 are not married and not believed to be sexually active. The 3 subjects who changed gender role behavior to male underwent hypospadias repair, and 1 was given supplemental testosterone therapy. One of these men is divorced, and the other 2 (aged 29 and 35 years) are unmarried. The diagnosis in 8 of these subjects was made after the time of expected puberty; it is unclear whether the functional and social outcomes would have been different if the diagnosis had been made and therapy begun earlier in life.

Glutathione PEGylated (GSH-PEG) liposomes were evaluated for their ability to enhance and prolong blood-to-brain drug delivery of the opioid peptide DAMGO (H-Tyr-d-Ala-Gly-MePhe-Gly-ol). An intravenous loading dose of DAMGO followed by a 2 h constant rate infusion was administered to rats, and after a washout period of 1 h, GSH-PEG liposomal DAMGO was administered using a similar dosing regimen. DAMGO and GSH-PEG liposomal DAMGO were also administered as a 10 min infusion to compare the disposition of the two formulations. Microdialysis made it possible to determine free DAMGO in brain and plasma, while the GSH-PEG liposomal encapsulated DAMGO was measured with regular plasma sampling. The antinociceptive effect of DAMGO was determined with the tail-flick method. All samples were analyzed using liquid chromatography-tandem mass spectrometry. The short infusion of DAMGO resulted in a fast decline of the peptide concentration in plasma with a half-life of 9.2 ± 2.1 min. Encapsulation in GSH-PEG liposomes prolonged the half-life to 6.9 ± 2.3 h. Free DAMGO entered the brain to a limited extent with a steady state ratio between unbound drug concentrations in brain interstitial fluid and in blood (Kp,uu) of 0.09 ± 0.04. GSH-PEG liposomes significantly increased the brain exposure of DAMGO to a Kp,uu of 0.21 ± 0.17 (p < 0.05). By monitoring the released, active substance in both blood and brain interstitial fluid over time, we were able to demonstrate that GSH-PEG liposomes offer a promising platform for enhancing and prolonging the delivery of drugs to the brain.

BackgroundPhysical activity has multiple health benefits; however, the majority of children around the world do not attain the recommended levels of daily physical activity. Research has shown that the game Poké mon GO has increased the amount of physical activity of players and that the game has the potential to reach populations that traditionally have low levels of physical activity. Therefore, there is a need to understand which game components can promote initial and sustained physical activity. By using a qualitative research approach, it is possible to achieve rich descriptions and enhance a deep understanding of the components promoting physical activity among children in a game such as Poké mon GO.ObjectiveThe objective of this study was to explore children’s and parents’ experiences playing Poké mon GO.MethodsEight families comprising 13 children (aged 7-12 years) and 9 parents were selected using purposeful sampling. Data collected using focus groups were analyzed using qualitative latent content analysis.ResultsThe following three themes were revealed: (1) exciting and enjoyable exploration; (2) dangers and disadvantages; and (3) cooperation conquers competition. The first centers around the present and possible future aspects of Poké mon GO that promote physical activity. The second focuses on unwanted aspects and specific threats to safety when playing the game. The third shows that cooperation and togetherness are highly valued by the participants and that competition is fun but less important.ConclusionsComponents from Poké mon GO could enhance the efficacy of physical activity interventions. Cooperation and exploration are aspects of the game that preferably could be transferred into interventions aimed at promoting children’s physical activity.

The enzyme carotenoid 15,15'-monooxygenase (CMO1) catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A in the small intestine. Plant carotenoids are an important dietary source of vitamin A (retinol) and the sole source of vitamin A for vegetarians. Vitamin A is essential for normal embryonic development as well as normal physiological functions in children and adults. Here, we describe one heterozygous T170M missense mutation in the CMO1 gene in a subject with hypercarotenemia and mild hypovitaminosis A. The replacement of a highly conserved threonine with methionine results in a 90% reduction in enzyme activity when analyzed in vitro using purified recombinant enzymes. The Michaelis-Menten constant (K(m)) for the mutated enzyme is normal. Ample amounts of carotenoids are present in plasma of persons consuming a normal Western diet, suggesting that the enzyme is saturated with substrate under normal conditions. Therefore, we propose that haploinsufficiency of the CMO1 enzyme may cause symptoms of hypercarotenemia and hypovitaminosis A in individuals consuming a carotenoid-containing and vitamin A-deficient diet.