Preliminary studies of liver regeneration induced by partial hepatectomy (PHE) identified a substantial depletion of hepatic retinoid stores, by greater than 70%, in regenerating livers of wild-type C57Bl/6J mice. To understand this, we compared responses of wild-type and lecithin:retinol acyltransferase (Lrat)-deficient mice, which totally lack hepatic retinoid stores, to PHE. The Lrat-deficient livers showed delayed regeneration in the first 24 h after PHE. At 12 h after PHE, we observed significantly less mRNA expression for growth factors and cytokines implicated in regulating the priming phase of liver regeneration, specifically for Hgf and Tgfα, but not Tgfβ. Compared with wild-type mice, the changes in mRNA levels for p21 and cyclins E1, B1, and A2 mRNAs and for hepatocellular BrdU incorporation and mitoses were delayed (i.e., shifted to later times) in regenerating Lrat−/− livers. Concentrations of all-trans-retinoic acid were significantly lower in the livers of Lrat−/− mice following PHE, and this was accompanied by diminished expression of known retinoid-responsive genes. At later times after PHE, the rate of liver weight restoration for Lrat−/− mice was parallel to that of wild-type mice, although additional biochemical differences were observed. Thus, hepatic retinoid stores are required for maintaining expression of signaling molecules that regulate cell proliferation and differentiation immediately after hepatic injury, accounting for the delayed restoration of liver mass in Lrat−/− mice.

s 279S by gest on S etem er 1, 2017 jn.nition.org D ow nladed fom with eicosapentaenoic acid [20:5(n-3)] concentration in red blood cell membranes. No clear correlations were found between adduct levels and other fatty acids. Surprisingly, a positive correlation was seen between plasma vitamin C concentrations and adduct levels. No significant correlations were observed between adduct levels and plasma concentrations of other antioxidants. Socioeconomic status significantly correlated with adduct levels: the higher the status, the higher the adduct levels. The individual 1,N-propanodeoxyguanosine adduct level can be modulated not only by lifestyle and nutrition factors but also by the individual DNA repair capacity. We also studied the influence of repair polymorphisms. With the XPD AspAsn variants, no changes in adduct levels were seen. Adduct levels increased in the XPD LysGln variants from 54.3 mean adducts/10 nucleotides in Lys/Lys (n 1⁄4 126) to 60.6 in the Lys/Gln (n 1⁄4 126) and to 63.2 in Gln/Gln (n 1⁄4 40). In the XPA (24G/A) variants, the adduct levels increased from 56.4 in variant G/G (n1⁄4 112) to 58.3 in G/A (n1⁄4 126) and to 64.4 in A/A (n 1⁄4 40). After adjustment for nutrition factors, a significantly increased odds ratio of 2.5 (CI 1.05, 5.8) was determined for having high adduct levels in XPA A/A carriers, suggesting a higher cancer risk in these individuals. DNA from Food as a Biomarker for Human Diet. Douglas Spangler, Richard Rivlin, and David S. Thaler. Raymond and Beverly Sackler Laboratory of Molecular Genetics and Informatics, New York, NY; Clinical Nutrition Research Unit, Strang Cancer Research Laboratory, Weill Medical College of Cornell University, New York, NY. There is a recognized need to develop new, sensitive, quantitative, and objective biomarkers of human dietary intake (1). Speciesand group (e.g., vegetable)-specific DNA sequences are potential biomarkers. We have developed a methodology of food-specific DNA analysis by Q(quantitative)PCR that can be applied to a wide range of clinical samples including blood, urine, saliva, and stool. Analyzing DNA in human biological fluids has the potential to provide novel biomarkers of dietary intake. Use of these biomarkers in clinical contexts will require and inform the understanding of individual variation in rates of digestion, intestinal transit, absorption, microbial processes, and excretion that are likely to be under genetic, physiological, and microecololgical controls. A clinical study is ongoing at the Rockefeller University Hospital. Our first analyses were consistent with the hypothesis that DNA sequences unique to vegetables and to fish were present in the blood of normal humans who ingest these foods. However, DNA from these blood samples was isolated with commercial kits and columns manufactured by Qiagen. Our subsequent sequencing of the PCR amplicons indicates that at least some of the ‘‘food DNA’’ signals derived from purification reagents, most likely the silicacontaining spin columns. Others have reported finding related contaminants in commercial DNA purification reagents (2,3). Contamination of silica columns may be difficult to avoid because of the way they are currently manufactured. Taken together, we believe that these findings call into question the use of currently available commercial silica-based purification columns for certain contamination-sensitive DNA purifications for research, medical-diagnostic, and forensic applications. [Supported by NCI and by The Sloan Foundation.] 1. Johnson RK. Dietary intake—how do we measure what people are really eating? Obes Res. 2002;10: Suppl 1:63S–8S. 2. van der Zee A, Peeters M, de Jong C, Verbakel H. Qiagen DNA extraction kits for sample preparation for Legionella PCR are not suitable for diagnostic purposes. J Clin Microbiol. 2002;40: 1126–37. 3. Peters RP, Mohammadi T, Vandenbroucke-Grauls CM, Danner SA, van Agtmael MA, Savelkoul PH. Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination? FEMS Immunol Med Microbiol. 2004;42:249–53. Dietary Carbohydrate as a Biomarker of Red Meat and Dairy Intake. Eric I. Park. Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC. High consumption of red meat has been associated with increased risk of colorectal cancer. However, the precise dietary component or the mechanism that accounts for this increased risk is unknown. One abundant component of red meat that is gaining interest is a type of carbohydrate moiety known as N-glycolylneuraminic acid (NGNA). NGNA accumulates on the cell surfaces of colon cancer, breast cancer, and hepatocellular carcinoma cells, whereas the surrounding normal cells have only trace amounts. Even more intriguing is how this accumulation occurs, because normal and cancerous human cells cannot synthesize NGNA. One likely source of NGNA is the consumption of red meat and dairy products, which are abundant in NGNA, and the subsequent metabolism of the NGNA by the same intracellular enzymes and transporters that metabolize endogenous sialic acids. Despite the possibility of NGNA serving as a biomarker of red meat and diary intake and for tagging and identifying cancer cells, little is known about the metabolism of dietary NGNA. Toward this goal, pilot studies were performed to determine the feasibility of using NGNA as a marker of red meat and dairy intake. First, a human cell line experiment showed that the amount of NGNA accumulation on the surface of cells depends on the NGNA concentration in the cell medium. Second, HPLC analyses showed that concentrations of NGNA were the same in raw and cooked meats. Third, the amounts of NGNA in human serum and urine samples were detected by HPLC and NMR methods, respectively. These studies suggest that the NGNA concentration in human samples may be useful as a marker of red meat and dairy consumption and a measurement to validate the real consumption levels of self-reported red meat and dairy intakes.

Energy metabolism and, specifically, the coupling of mitochondria to growth and survival is controlled by the cAMP-PKA pathway in yeast. In higher eukaryotes, cAMP signaling originating at the plasma membrane is distributed to different subcellular districts by cAMP waves received by PKA bound to PKA anchor proteins (AKAPs) tethered to these compartments. This review focuses on the subgroup of AKAPs that anchor PKA to the mitochondrial outer membrane (mtAKAPs). Only PKA anchored to mtAKAPs can efficiently transmit cAMP signals to mitochondria. mtAKAP complexes are remarkably heterogeneous. In addition to PKA regulatory subunits, they may include mRNAs, tyrosine phosphatase(s) and tyrosine kinase(s). Selective regulation of these components by cAMP-PKA integrates various signal transduction pathways and can determine which subcellular compartment receives the signal. Unveiling the interactions among the components of these large complexes will shed light on how cAMP and PKA regulate vital mitochondrial processes.

Circulating retinoids (vitamin A and its derivatives) are found predominantly as retinol bound to retinol-binding protein (RBP), which transports retinol from liver stores to target tissues, or as retinyl ester incorporated in lipoproteins of dietary origin. The transport of retinoids from maternal to fetal circulation is poorly understood, especially under conditions of inadequate dietary vitamin A intake. Here we present RBP-/- mice as a tunable model of embryonic vitamin A deficiency. This model has enabled us to analyze metabolic links between maternal nutrition and retinoid delivery to the fetus. Our data show that retinol-RBP is the primary contributor to fetal development, whereas retinyl ester are largely responsible for accumulation of fetal retinoid stores. Furthermore, these studies indicate the importance of embryonic RBP in distributing vitamin A to certain developing tissues under restrictive diets. We also show differences among developing tissues in their dependency on the embryonic retinol-RBP pathway. Finally, we demonstrate that accumulation of embryonic vitamin A stores does not depend on the expression of RBP in the fetal liver.

A-kinase anchor protein 121 (AKAP121) and its spliced isoform AKAP84 anchor protein kinase A (PKA) to the outer membrane of mitochondria, focusing and enhancing cyclic AMP signal transduction to the organelle. We find that AKAP121/84 also binds PTPD1, a src-associated protein tyrosine phosphatase. A signaling complex containing AKAP121, PKA, PTPD1, and src is assembled in vivo. PTPD1 activates src tyrosine kinase and increases the magnitude and duration of epidermal growth factor (EGF) signaling. EGF receptor phosphorylation and downstream activation of ERK 1/2 and Elk1-dependent gene transcription are enhanced by PTPD1. Expression of a PTPD1 mutant lacking catalytic activity inhibits sire and downregulates ERK 1/2 but does not affect the activity of c-Jun N-terminal kinase 1/2 and p38alpha mitogen-activated protein kinase. AKAP121 binds to and redistributes PTPD1 from the cytoplasm to mitochondria and inhibits EGF signaling. Our findings indicate that PTPD1 is a novel positive regulator of src signaling and a key component of the EGF transduction pathway. By binding and/or targeting the phosphatase on mitochondria, AKAP121 modulates the amplitude and persistence of src-dependent EGF transduction pathway. This represents the first example of physical and functional interaction between AKAPs and a protein tyrosine phosphatase.

Although the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP(-/-)) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP-/-). By comparing hRBP(-/-), RBP-/-, and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical.jlr Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed.

Retinoids are required for normal embryonic development. Both embryonic retinoid deficiency and excess result in congenital malformations. There is little understanding of the physiology underlying retinoid transfer from the maternal circulation to the embryo. We now report studies that explore this process using retinol-binding protein-deficient (RBP-/-) mice and mice that express human RBP on the RBP-/-) background. Our studies establish that dietary retinoid, bound to lipoproteins, can serve as an important source for meeting tissue retinoid requirements during embryogenesis. Indeed, retinyl ester concentrations in the circulations of pregnant RBP-/- mice are significantly elevated over those observed in wild-type mice, suggesting that lipoprotein retinyl esters may compensate for the absence of retinol-RBP during pregnancy. We also demonstrate, contrary to earlier proposals, that maternal RBP does not cross the placenta and cannot enter the fetal circulation. Overall, our data indicate that both retinol-RBP and retinyl esters bound to lipoproteins are able to provide sufficient retinoid to the embryo to allow for normal embryonic development.

The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage lambda by blocking transcription elongation on the lambda chromosome. Multiple interactions between Nun and the transcription elongation complex are involved in this reaction. The Nun NH2-terminal arginine-rich motif binds BOXB sequence in nascent lambda transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template. Nun Trp(108) is required for interaction with DNA and transcription arrest. We analyzed the role of the adjacent Lys(106) and Lys(107) residues in the Nun reaction. Substitution of the lysine residues with arginine (K106R/ K107R) had no effect on transcription arrest in vitro or in vivo. Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude lambda. All mutants bound RNA polymerase and BOXB. In contrast to Nun K106R/ K107R and K106A/ K107A, Nun K106D/K107D did not cross-link DNA template. These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys(106) and Lys(107) and negatively charged DNA phosphate groups. These may assist intercalation of Trp(108) into template.

Publisher Summary This chapter discusses the bacteriophage HK022 nun protein. Prophage HK022 excludes superinfecting phage λ by terminating transcription on the lambda chromosome. This exclusion is promoted by the 109 amino acid HK022 Nun protein. Nun carries an arginine-rich RNAbinding motif (ARM) at its N terminus. The chapter illustrates the procedure for purification of Nun which is explained in two parts—namely, induction and purification. The Nun overproducer plasmid pT7NunII is a pET-21d (+) derivative that carries nun between the plasmid NcoI and HindIII sites. The nun gene is under the control of the T7 promoter, which is in turn, is controlled by an adjacent lac operator and a lacI gene. Basal Nun expression is highly down-regulated by the T7 lysozyme encoded by the pLysS plasmid. E. coli carrying the pT7NunII plasmid is maintained in LB–ampicillin. The purification step includes soluble extract, SP-sepharose FF column chromatography, and mono S chromatography. The chapter includes the protocol for assaying of Nun proteins. Most of the Nun protein in the induced culture is soluble, and it is readily purified from a supernatant fraction by two chromatographic steps. In vitro transcription is performed with template derived from a HindIII-digested λ genome that contains the pL–nutL segment.

A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.

Phage HK022 Nun protein excludes phage lambda by binding nascent lambda-nut RNA and inducing termination and transcript release. In contrast, in a purified in vitro system, Nun arrests transcription on lambdaDNA templates without dissociation of the transcription elongation complex (TEC). Our evidence indicates that transcription-repair coupling factor (Mfd) frees Nun-arrested RNA polymerase. The activity of Nun is enhanced in an mfd-null mutant, consistent with prolonged association of Nun with the TEC. Furthermore, expression of lambda nut RNA in the mfd mutant titrates Nun, allowing superinfecting lambda to form plaques. Finally, addition of Mfd releases a Nun-arrested transcription complex in vitro.

The Nun protein of lambdoid phage HK022 excludes λ‐phage superinfection by blocking expression of genes downstream from the λ nut sequences. Heteronuclear NMR studies have been performed on a Nun peptide comprising residues 1–49 bound to the nutR BoxB RNA. The pattern of 13C chemical shifts indicates that the arginine‐rich motif of Nun forms an induced α‐helix, consisting of residues 23–43, when bound to BoxB RNA, consistent with the structure of a shorter (residues 22–44) Nun peptide/BoxB RNA complex [Faber, C., Schärpf, M., Becker, T., Sticht, H. and Rösch (2001) J. Biol. Chem. 276, 32064–32070]. The N‐terminal extension, residues 1–22, does not show chemical shifts or nuclear Overhauser effects characteristic of stable secondary structure. Nonetheless, 15N relaxation measurements indicate that this region is not completely disordered, as expected for a random coil peptide. Restriction of conformation flexibility in the N‐terminal extension of Nun may be important for binding to other target molecules involved in transcription termination.

Protein kinase A (PKA) anchoring proteins (AKAPs) tether PKA to various subcellular locations. AKAP121, which tethers PKAII to the outer mitochondrial membrane, includes a K homology (KH) RNA-binding motif. Purified AKAP121 KH domain binds the 3′ untranslated regions (3′UTRs) of transcripts encoding the Fo-f subunit of mitochondrial ATP synthase and manganese superoxide dismutase (MnSOD). Binding requires a structural motif in the 3′UTR and is stimulated by PKA phosphorylation of the domain or a mutation that mimics this phosphorylation. AKAP121 expressed in HeLa cells promotes the translocation of MnSOD mRNA from cytosol to mitochondria and an increase in mitochondrial MnSOD. Both reactions are stimulated by cAMP. Thus, by focusing translation at the mitochondrial membrane, AKAP121 may facilitate import of mitochondrial proteins in response to cAMP stimulation.

Transcription termination in Escherichia coli is controlled by many factors. The sequence of the DNA template, the structure of the transcript, and the actions of auxiliary proteins all play a role in determining the efficiency of the process. Termination is regulated and can be enhanced or suppressed by host and phage proteins. This complex reaction is rapidly yielding to biochemical and structural analysis of the interacting factors. Below we review and attempt to unify into basic principles the remarkable recent progress in understanding transcription termination and anti-termination.