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Dietary exposures to the seven food, drug, and cosmetic (FD&C) colour additives that are approved for general use in food in the United States were estimated for the US population (aged 2 years and older), children (aged 2–5 years) and teenage boys (aged 13–18 years) based on analytical levels of the FD&C colour additives in foods. Approximately 600 foods were chosen for analysis, based on a survey of product labels, for the levels of FD&C colour additives. Dietary exposure was estimated using both 2-day food consumption data from the combined 2007–10 National Health and Nutrition Examination Survey (NHANES) and 10–14-day food consumption data from the 2007–10 NPD Group, Inc. National Eating Trends – Nutrient Intake Database (NPD NET-NID). Dietary exposure was estimated at the mean and 90th percentile using three different exposure scenarios: low exposure, average exposure and high exposure, to account for the range in the amount of each FD&C colour additive for a given food. For all populations and all exposure scenarios, the highest cumulative eaters-only exposures in food were determined for FD&C Red No. 40, FD&C Yellow No. 5 and FD&C Yellow No. 6. In addition, the eaters-only exposure was estimated for individual food categories in order to determine which food categories contributed the most to the exposure for each FD&C colour additive. Breakfast Cereal, Juice Drinks, Soft Drinks, and Frozen Dairy Desserts/Sherbet (also referred to as Ice Cream, Frozen Yogurt, Sherbet (including Bars, Sticks, Sandwiches)) were the major contributing food categories to exposure for multiple FD&C colour additives for all three populations.

4-Methylimidazole (4-MEI) is formed in caramel colours produced using ammonium compounds (Class III and Class IV caramel colours). 4-MEI can also form in food through Maillard reactions between reducing sugars and amino acids during cooking, roasting or dry-heating. The USFDA has analysed over 700 food and beverage samples collected from 2013 to 2015 for the presence of 4-MEI. These samples include foods containing added caramel colour and foods that are not labelled as containing added caramel colour, but which may contain 4-MEI resulting from thermal treatment. The 4-MEI levels in all food samples were quantified using LC-MS/MS. These data were used to develop a comprehensive dietary exposure assessment for 4-MEI for the U.S. population aged 2 years or more and several sub-populations, using two non-consecutive days of food consumption data from the combined 2009-2012 National Health and Nutrition Examination Survey and 10-14-day food consumption survey data for 2009-2012 from the NPD Group, Inc. National Eating Trends-Nutrient Intake Database. Dietary exposure estimates were prepared for each category of foods labelled as containing added caramel colour and of foods not labelled as containing added caramel colour, but which may contain 4-MEI from thermal treatment. Exposure to 4-MEI from consumption of foods containing added caramel colour was higher than that from foods that contain 4-MEI from thermal treatment for all population groups. Cola-type carbonated beverages were the highest contributors for most populations from foods containing added caramel colour. Coffee was the highest contributor for most populations from foods in which 4-MEI could be formed from thermal treatment. An overall combined exposure to 4-MEI was also estimated that included all foods identified as containing added caramel colour and foods in which 4-MEI could be formed by thermal treatment.

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1. Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary excretion of phosphatidylserine, and we hypothesized that ATP8B1 is a flippase for phosphatidylserine. However, direct evidence for this function is still lacking. In Saccharomyces cerevisiae, members of the Cdc50p/Lem3p family are essential for proper function of the ATP8B1 homologs. We have studied the role of two human members of this family, CDC50A and CDC50B, in the routing and activity of ATP8B1. When only ATP8B1 was expressed in Chinese hamster ovary cells, the protein localized to the endoplasmic reticulum. Coexpression with CDC50 proteins resulted in relocalization of ATP8B1 from the endoplasmic reticulum to the plasma membrane. Only when ATP8B1 was coexpressed with CDC50 proteins was a 250%-500% increase in the translocation of fluorescently labeled phosphatidylserine observed. Importantly, natural phosphatidylserine exposure in the outer leaflet of the plasma membrane was reduced by 17%-25% in cells coexpressing ATP8B1 and CDC50 proteins in comparison with cells expressing ATP8B1 alone. The coexpression of ATP8B1 and CDC50A in WIF-B9 cells resulted in colocalization of both proteins in the canalicular membrane. Conclusion: Our data indicate that CDC50 proteins are pivotal factors in the trafficking of ATP8B1 to the plasma membrane and thus may be essential determinants of ATP8B1-related disease. In the plasma membrane, ATP8B1 functions as a flippase for phosphatidylserine. Finally, CDC50A may be the potential β-subunit or chaperone for ATP8B1 in hepatocytes. (HEPATOLOGY 2007.)

Pyridine clusters are subjected to intense femtosecond laser pulses at fluences around 10 15 W cm −2 at 620 nm, which results in multiple ionization and concomitant Coulomb explosion. A combined covariance map is considered which can be viewed as the overlay of two maps that describe the statistical interaction of the resulting time-of-flight spectra of the pyridine clusters which lead to species having large values of kinetic energy. The combined map is constructed from ones involving both positive covariance values and anti-covariance values obtained in the same experimental study. Significantly, the individual points representing positive and anti-covariance effects arising from the ionization of the clusters via femtosecond laser pulses are found to be mutually exclusive of each other as exemplified in this pyridine study. Our extension of covariance mapping, which was originally formulated by Frasinski et al. for simple molecular interactions, and is further developed in the present study, elucidates the presence of competitive reactions within cluster systems by comparison of the two maps.

Ultrafast pump−probe spectroscopic studies on the hydrated 7-azaindole monomer are presented. The experiments provide evidence that excited-state proton transfer in the gas-phase hydrated 7-azaindole monomer may be possible, as predicted by theory, and can occur by means of a water proton bridge. As explained in the text, full tautomerization may not be occurring, but rather the transfer of a proton from the 7-azaindole monomer to the solvating water molecules. A calculated geometry for the structure of 7-azaindole clustered with four waters is presented and used to explain the rate increase observed in this species compared to the 7-azaindole monomer clustered with two and three waters.

Presented here are femtosecond pump-probe studies on the water-solvated 7-azaindole dimer, a model DNA base pair. In particular, studies are presented that further elucidate the nature of the reactive and nonreactive dimers and also provide new insights establishing that the excited state double-proton transfer in the dimer occurs in a stepwise rather than a concerted manner. A major question addressed is whether the incorporation of a water molecule with the dimer results in the formation of species that are unable to undergo excited state double-proton transfer, as suggested by a recent study reported in the literature [Nakajima, A., Hirano, M., Hasumi, R., Kaya, K., Watanabe, H., Carter, C. C., Williamson, J. M. & Miller, T. (1997) J. Phys. Chem. 101, 392–398]. In contrast to this earlier work, our present findings reveal that both reactive and nonreactive dimers can coexist in the molecular beam under the same experimental conditions and definitively show that the clustering of water does not induce the formation of the nonreactive dimer. Rather, when present with a species already determined to be a nonreactive dimer, the addition of water can actually facilitate the occurrence of the proton transfer reaction. Furthermore, on attaining a critical hydration number, the data for the nonreactive dimer suggest a solvation-induced conformational structure change leading to proton transfer on the photoexcited half of the 7-azaindole dimer.

Direct evidence is reported of the connectiveness of charged clusters with highly charged species (N4+, N3+, and N2+) produced upon the interaction of molecular ammonia clusters with an intense femtosecond laser beam (∼1015 W/cm2 at 120 fs). The value of covariance analysis as a general technique for studying dynamical processes in clusters is demonstrated through elucidating the details of various Coulomb explosion events. Positive covariance determinations identify concerted processes such as the concomitant explosion of protonated cluster ions of unsymmetrical size, while anticovariance mapping is exploited to distinguish competitive reaction channels such as the production of highly charged nitrogen atoms formed at the expense of the protonated members of the cluster ion ensemble. The present study demonstrates the great potential which covariance analysis offers in identifying the precursors and products of dynamical events in clusters and in the present case provides further support to the ignition ...

CDC50 proteins are beta-subunits for P4 ATPases, which upon heterodimerization form a functional phospholipid translocation complex. Emerging evidence in mouse models and men links mutations in P4 ATPase genes with human disease. This study analyzed the tissue distribution and cellular localization of CDC50A, the most abundant and ubiquitously expressed CDC50 homologue in the mouse. The authors have raised antibodies that detect mouse and human CDC50A and studied CDC50A localization and glycosylation status in mouse liver cells. CDC50A is a terminal-glycosylated glycoprotein and is expressed in hepatocytes and liver sinusoidal endothelial cells, where it resides in detergent-resistant membranes. In pancreas and stomach, CDC50A localized to secretory vesicles, whereas in the kidney, CDC50A localized to the apical region of proximal convoluted tubules of the cortex. In WIF-B9 cells, CDC50A partially costains with the trans-Golgi network. Data suggest that CDC50A is present as a fully glycosylated protein in vivo, which presumes interaction with distinct P4 ATPases. (J Histochem Cytochem 60: 205-218, 2012)

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type I (BRIC1), forming a spectrum of cholestatic disease. Whereas PFIC1 is a progressive, endstage liver disease, BRIC1 patients suffer from episodic periods of cholestasis that resolve spontaneously. At present it is not dear how the type and location of the mutations relate to the clinical manifestations of PFIC1 and BRIC1. ATP8B1 localizes to the canalicular membrane of hepatocytes; where it mediates the inward translocation of phosphatidylserine. ATP8B1 interacts with CDC50A, which is required for endoplasmic reticulum exit and plasma membrane localization. In this study we analyzed a panel of missense mutations causing PFIC1 (G308V, D554N, G1040R) or BRIC1 (D70N, 166 11). In addition, we included two mutations that have been associated with intrahepatic cholestasis of pregnancy (ICP) (D70N, R867C). We examined the effect of these mutations on protein stability and interaction with CDC50A in Chinese hamster ovary cells, and studied the subcellular localization in WIF-B9 cells. Protein stability was reduced for three out of six mutations studied. Two out of three PFIC1 mutant proteins did not interact with CDC50A, whereas BRIC1/ICP mutants displayed reduced interaction. Importantly, none of the PFIC1 mutants were detectable in the canalicular membrane of WIF-B9 cells, whereas all BRIC1/ICP mutants displayed the same cellular staining pattern as wild-type ATP8B1. Our data indicate that PFIC1 mutations lead to the complete absence of canalicular expression, whereas in BRIC1/ICP residual protein is expressed in the canalicular membrane. Conclusion: These data provide an explanation for the difference in severity between the phenotypes of PFIC1 and BRIC1. (HEPATOLOGY 2009;50: 1597-1605.)

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2–vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2–vsvG. Furthermore, hCTR2–vsvG was co-immunoprecipitated with hCTR2–FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2–eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)–luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE–luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 μM CuCl2, consistent with the Km of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE–luciferase reporter activation in a copper-dependent manner at 40 and 100 μM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.