BBA - General Subjects (v.1672, #2)

We have reported that the conversion ratio of tryptophan to niacin increased with increasing dietary concentration of di(2-ethylhexyl)phthalate (DEHP); the conversion ratio was about 2.0% in the control rat, which increased by about 30% in the rat fed with 3.0% DEHP diet. In this study, we investigated whether this abnormal increase in the conversion ratio by DEHP occurred through the alteration of the enzyme activities involved in the metabolism of tryptophan to niacin. Rats were fed with a diet containing 0%, 0.1%, 0.5%, or 1.0% DEHP for 21 days. The nine kinds of enzyme activities involved in the biosynthesis and catabolism in the liver and kidney were measured. Based on previous findings that the formation of quinolinic acid and its' metabolites significantly increased with DEHP administration, we proposed that the activity of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase would be inhibited by DEHP intake. However, we found that the activities in the liver and kidney did not decrease in the rat fed with DEHP-containing diet. We discuss the discrepancy between the metabolite results and the enzyme activities.
Keywords: Phthalate ester; Di(2-ethylhexyl)phthalate; Enzyme activity; Tryptophan metabolism; Niacin; Rat;

Proteolytic fragments of ovalbumin display antimicrobial activity by Antonio Pellegrini; Andreas J Hülsmeier; Peter Hunziker; Ursula Thomas (76-85).
Ovalbumin, one of the major proteins present in avian egg white, was proteolytically digested by trypsin and chymotrypsin and the peptide fragments were investigated for their antimicrobial activity. The antimicrobial peptides were isolated and characterized. From the tryptic digestion, the following five antimicrobial peptide fragments were obtained: SALAM (residues 36–40), SALAMVY (residues 36–42) YPILPEYLQ (residues 111–119), ELINSW (residues 143–148) and NVLQPSS (residues159–165). Digestion of ovalbumin by chymotrypsin yielded the antimicrobial peptides AEERYPILPEYL (residues 127–138), GIIRN (residues 155–159) and TSSNVMEER (residues 268–276). The peptides were synthesized and found to exert antimicrobial activity. They were strongly active against Bacillus subtilis and to a lesser extent against the other bacterial strains examined. A weak fungicidal activity against Candida albicans was also shown by some peptides. Ovalbumin itself was not bactericidal against all the bacteria strains examined. Our results suggest that the food protein ovalbumin may supply the organism with antimicrobial peptides, supporting the immunodefences of the organism.
Keywords: Ovalbumin; Bactericidal peptide; Antimicrobial peptide; Antibiotic; Nutrition; Food intake;

Functional co-expression of xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP-glucuronosyltransferase 1A6, in yeast microsomes by Shin-ichi Ikushiro; Masahiro Sahara; Yoshikazu Emi; Yoshiyasu Yabusaki; Takashi Iyanagi (86-92).
Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs. In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22. Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast. Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression. Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis. Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes. The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC. In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC. In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system. This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.
Keywords: Co-expression system; Cytochrome P450; UDP-glucuronosyltransferase; Xenobiotic; 7-ethoxycoumarin; Yeast microsome;

Structural characteristic of terminal dicarboxylic moiety required for apoptogenic activity of α-tocopheryl esters by Kentaro Kogure; Susumu Hama; Mayumi Kisaki; Hideaki Takemasa; Akira Tokumura; Ichiro Suzuki; Kenji Fukuzawa (93-99).
α-Tocopheryl succinate (TS) is known to induce apoptosis in various cells and has attracted attention as a chemotherapeutic agent. Recently, we reported the structural significance of the terminal dicarboxylic moiety for the action of TS [J. Nutr. Sci. Vitaminol. 49 (2003) 310–314]. In this study, to determine details of the relationship between the structure and the function of the terminal ester moiety of α-tocopherol (α-T), we synthesized four novel esters, α-tocopheryl oxalate (TO), α-tocopheryl malonate (TM), α-tocopheryl pimelate (TP) and α-tocopheryl succinate ethyl ester (TSE), and compared their apoptogenic activities with those of TS, α-T, γ-tocopherol (γ-T) and two commercially available α-T derivatives, α-tocopheryl nicotinate (TN) and α-tocopheryl acetate (TA), in vascular smooth muscle cells and a mouse breast cancer cell line C127I. TO and TM in addition to TS, but not the others, induced apoptosis in both cells. Particularly, TO was the most potent of all α-T derivatives used. The addition of exogenous superoxide dismutase (SOD) significantly prevented the apoptosis induced by TM as well as that by TS as reported previously, but did not affect TO-induced apoptosis. These results suggest that O2 generated exogenously participates in TM-induced apoptosis but not in TO-induced apoptosis. The difference in their apoptotic effects is attributed to structural properties of the terminal dicarboxylic moiety, which has an inflexible plane conformation in TO, while it is highly flexible in TM and TS.
Keywords: α-tocopheryl succinate; Novel α-tocopheryl ester; Apoptosis; Superoxide; Structural property;

Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo by S.Santosh Kumar; Bhavani Shankar; Krishna B. Sainis (100-111).
Chlorophyllin (CHL) has been examined as an antioxidant/radioprotector in splenic lymphocytes from BALB/c mice. CHL inhibited lipid peroxidation induced by 2,2′-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic stimulation of lymphocytes in vitro. Generation of intracellular reactive oxygen species (ROS) by radiation or AAPH was measured as oxidation of dichlorodihydrofluorescein diacetate (H2DCF-DA) using flow cytometry. Addition of CHL to lymphocytes in vitro significantly inhibited the increase in intracellular ROS. Further, lymphocytes from mice treated with CHL (100–400 μg/gbw i. p.) showed varying levels of ROS depending on the dose and the time (24 to 72 h) after injection. The extent of radiation-induced apoptosis and suppression of concanavalin A (con A)-induced mitogenesis ex vivo corresponded with changes in ROS levels in CHL-administered mice. Antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were also estimated in lymphocytes from CHL-treated mice. CHL offered protection against whole body irradiation (WBI)-induced lipid peroxidation and apoptosis in lymphocytes at all the time points studied. These results demonstrate antioxidant effect of CHL in vivo.
Keywords: Chlorophyllin; Radioprotection; Apoptosis; Lipid peroxidation; Lymphocyte; Antioxidant;

Targeted hepatic overexpression of human IRS-1: postnatal effects in the developing mouse by Peter J. Giannone; Barham K. Abu Dayyeh; Theresa C. Bienieki; Jack R. Wands; Philip A. Gruppuso (112-119).
Insulin receptor substrate-1 (IRS-1) is an intracellular docking protein involved in insulin and insulin-like growth factor (IGF) signaling. The present studies examine postnatal liver development in transgenic mice with targeted hepatic overexpression of human insulin receptor substrate-1 (hIRS-1). In mature animals, hIRS-1 overexpression augments liver growth. Based on our previous studies that have shown markedly attenuated insulin signaling in the late-gestation and early-postnatal rat, we hypothesized that the liver growth effect of overexpressed hIRS-1 would be attenuated in the neonatal period. Wild-type and heterozygous transgenic mice were studied at 1, 2, 4 or 8 weeks of age. Transgene expression was seen at all ages, albeit at a lower level in the youngest animals. Liver-to-carcass weight ratios were similar in hIRS-1 and wild-type mice at 1 and 2 weeks of age. At 4 and 8 weeks, transgenic mice had larger livers accounted for by increased hepatocyte number, not size. In addition, the transgenic mice had increased liver glycogen content at 8 weeks but not at 1 week. Relative to transgene mRNA expression, hIRS-1 protein levels were restricted in the younger animals. However, IRS-1-associated phosphatidylinositol-3 kinase (PI3K) activity was not similarly suppressed. Downstream from IRS-1, we found activation of the signaling kinase Akt in 8-week-old but not in 1-week-old animals. Our findings indicate that hepatic IRS-1-mediated signaling may be limited in neonatal mice at two levels, post-transcriptional down-regulation of IRS-1 content and attenuated signaling beyond the level of PI3K activation.
Keywords: Liver; Development; Insulin; IRS-1; Glycogen;

Enzymatic description of the anhydrofructose pathway of glycogen degradation by Shukun Yu; Charlotte Refdahl; Inge Lundt (120-129).
The anhydrofructose pathway describes the degradation of glycogen and starch to metabolites via 1,5-anhydro-d-fructose (1,5AnFru). The enzyme catalyzing the first reaction step of this pathway, i.e., α-1,4-glucan lyase (EC, has been purified, cloned and characterized from fungi and red algae in our laboratory earlier. In the present study, two 1,5AnFru metabolizing enzymes were discovered in the fungus Anthracobia melaloma for the formation of ascopyrone P (APP), a fungal secondary metabolite exhibiting antibacterial and antioxidant activity. These are 1,5AnFru dehydratase (AFDH) and ascopyrone tautomerase (APTM). AFDH catalyzed the conversion of 1,5AnFru to ascopyrone M (APM), a compound that has been earlier presumed to occur biologically, while APTM isomerized the APM formed to APP. Both enzymes were purified 400-fold by (NH4)2SO4 fractionation, hydrophobic interaction, ion-exchange and gel filtration chromatography. The purified AFDH showed a molecular mass of 98 kDa on SDS-PAGE and 230 kDa by gel filtration. The corresponding values for APTM was 60 and 140 kDa. Spectrophotometric and HPLC methods were developed for the assay of these two enzymes. To confirm that A. melaloma possessed all enzymes needed for conversion of glycogen to APP, an α-1,4-glucan lyase from this fungus was isolated and partially sequenced. Based on this work, a scheme of the enzymatic description of the anhydrofructose pathway in A. melaloma was proposed.
Keywords: Anhydrofructose pathway; Anthracobia; Ascopyrone; Dehydratase; Glucan lyase; Tautomerase;