Detection of 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine in broiled beef.

2-Amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine (4'-OH-PhIP) showed mutagenicity in Salmonella typhimurium TA98 in the presence of S9 mix, inducing 180 revertants per 100 micrograms. Since creatinine, tyrosine and glucose, which are present in meat, are expected to be involved in the formation of 4'-OH-PhIP, its presence in broiled beef was examined. 4'-OH-PhIP was detected in broiled beef by high-performance liquid chromatography and UV-spectrometry after extraction with 0.1 N HCl and purification by blue cotton treatment and ion exchange column chromatography. Its level was estimated to be 21.0 ng per g of broiled beef, which is comparable to that of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).     

Liver cancer and precancerous changes in rats induced by the basic fraction of tryptophan pyrolysate

The basic fraction of a tryptophan pyrolysate (Trp-P-BF) was given orally to Wistar rats for about 2 years. In Experiment I, 5 male rats each were given 0.2, 0.4, 0.6 and 0.8% Trp-P-BF. Dose-dependent growth retardation was observed in these groups and neoplastic nodules were found in the liver of 1 rat given 0.2% Trp-P-BF and a hepatocellular carcinoma was found in 1 rat given 0.4% Trp-P-BF diet. In Experiment II, 25 rats of both sexes were fed 0.5% or 0.2% Trp-P-BF diet. Neoplastic nodules were induced in 2 of 22 males and 5 of 18 females given 0.2% Trp-P-BF diet. Mammary adenomas developed, but no neoplastic nodules were found in the liver of rats fed on 0.05% Trp-P-BF or control diet. Females were more sensitive to Trp-P-BF than males.     

Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity

Growth hormone (GH) transgenic amago salmon (Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F(2) and F(3) generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Delta6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F(2) and F(3) salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F(2) and F(3) fish were completely immature, but the expression pattern was changed when fish approached maturation. Genes showing enhanced expression in GH transgenic fish in F(2) and F(3) by array analysis were vitelline envelope protein, hemopexin-like protein, heme-oxygenase, inter alpha-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and bikunin. Reduced expression genes were lectin, Delta6-desaturase, apolipoprotein, and pentraxin. In particular, lectin was found to be highly suppressed in all F(2) and immature F(3) salmon. Further, serum lysozyme activity, one of innate immunity, was significantly (p<0.05) decreased in both F(2) and F(3) GH transgenic fish. These results indicate that the GH transgene fish had altered hepatic gene expression relating to iron-metabolism, innate immunity, reproduction, and growth.     

In vitro generation of insulin‐secreting cells from human pancreatic exocrine cells

Transplantation of surrogate β‐cells is a promising option for the treatment of insulin‐deficient diabetes mellitus in the future. Although pancreatic exocrine cells of rodents have been shown to transdifferentiate into insulin‐secreting cells, no studies are reported on human exocrine cells. Here, we report the generation of insulin‐secreting cells from exocrine cells of the human pancreas. When cultured in suspension with epidermal growth factor, human pancreatic exocrine cells readily formed spherical cell clusters. Expression of Pdx1 was induced in all 19 cases in which we successfully isolated exocrine cells, and insulin expression was induced in 11 cases. In addition, insulin secretion was evaluated in four cases, and the newly‐made cells were found to secrete insulin in response to various stimuli. Although further studies are required to improve both the quality and quantity of such insulin‐secreting cells, our data suggest that pancreatic exocrine cells represent a potential source of insulin‐secreting cells for treatment of type 1 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00095.x, 2011)     

Adduct Formation at C-8 of Guanine on in vitro Reaction f the Ultimate Form of 2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine with 2'-Deoxyguanosine and Its Phosphate Esters

We examined the reactivity of the N -hydroxyamino derivative of a carcinogenic heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP), after its O -acetylation with four 2'-deoxyribonucleoside 3'-monophosphates. ³²P-Postlabeling analysis demonstrated that the levels of adducts with 2'-deoxyguanosine 3'-moiiophosphate were much higher than those with the other three nucleotides. ¹H-NMR, mass spectral and UV absorption spectral analyses of the major adducts formed by N -acetoxy-PhIP with 2'-deoxyguanosine and with its phosphate esters indicated that PhIP bound at the C-8 position of guanine, as previously demonstrated with other heterocyclic amines.     

Furan fatty acid as an anti-inflammatory component from the green-lipped mussel Perna canaliculus

A lipid extract of Perna canaliculus (New Zealand green-lipped mussel) has reportedly displayed anti-inflammatory effects in animal models and in human controlled studies. However, the anti-inflammatory lipid components have not been investigated in detail due to the instability of the lipid extract, which has made the identification of the distinct active components a formidable task. Considering the instability of the active component, we carefully fractionated a lipid extract of Perna canaliculus (Lyprinol) and detected furan fatty acids (F-acids). These naturally but rarely detected fatty acids show potent radical-scavenging ability and are essential constituents of plants and algae. Based on these data, it has been proposed that F-acids could be potential antioxidants, which may contribute to the protective properties of fish and fish oil diets against chronic inflammatory diseases. However, to date, in vivo data to support the hypothesis have not been obtained, presumably due to the limited availability of F-acids. To confirm the in vivo anti-inflammatory effect of F-acids in comparison with that of eicosapentaenoic acid (EPA), we developed a semisynthetic preparation and examined its anti-inflammatory activity in a rat model of adjuvant-induced arthritis. Indeed, the F-acid ethyl ester exhibited more potent anti-inflammatory activity than that of the EPA ethyl ester. We report on the in vivo activity of F-acids, confirming that the lipid extract of the green-lipped mussel includes an unstable fatty acid that is more effective than EPA.     

Adduct formation at C-8 of guanine on in vitro reaction of the ultimate form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine with 2'-deoxyguanosine and its phosphate esters.

We examined the reactivity of the N-hydroxyamino derivative of a carcinogenic heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), after its O-acetylation with four 2'-deoxyribonucleoside 3'-monophosphates. 32P-Postlabeling analysis demonstrated that the levels of adducts with 2'-deoxyguanosine 3'-monophosphate were much higher than those with the other three nucleotides. 1H-NMR, mass spectral and UV absorption spectral analyses of the major adducts formed by N-acetyoxy-PhIP with 2'-deoxyguanosine and with its phosphate esters indicated that PhIP bound at the C-8 position of guanine, as previously demonstrated with other heterocyclic amines.     

T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice

Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor-reactive T-cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma-associated antigen (MAGE) family antigens, that are ideal targets for adoptive T-cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non-obese diabetic/SCID/γc(null) (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE-A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE-A4, then adoptively transferred into NOG mice inoculated with MAGE-A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen-specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE-A4-specific TCR is a promising strategy to treat patients with MAGE-A4-expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T-cell response during adoptive therapy with human lymphocytes.