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Previous studies have shown that phosphorylation of the retinoid X receptor-α (RXRα) is associated with the development of hepatocellular carcinoma (HCC). However, these findings were revealed using HCC cell lines that express phosphorylated-RXRα (p-RXRα) proteins; therefore, it remains unclear whether p-RXRα affects hepatocarcinogenesis in vivo. Therefore, to investigate the biological function of p-RXRα in vivo, we developed a doxycycline-inducible ES cell line and transgenic mouse, both of which overexpress the phosphomimetic mutant form of RXRα, T82D/S260D, in a doxycycline-dependent manner. We found that the development of liver tumors, especially high-grade adenoma and HCC, was enhanced in diethylnitrosamine (DEN)-treated T82D/S260D-inducible mice. Moreover, the increased incidence of liver tumors in the transgenic mice was attributable to the promotion of cell cycle progression. Interestingly, the expression of β-catenin protein and its target gene cyclin D1 was elevated in the liver tumors of DEN-treated T82D/S260D-inducible mice, concurrent with increased cytoplasmic and nuclear β-catenin protein expression, indicating its stabilization and transcriptional activation. These results indicate that p-RXRα promotes DEN-induced hepatocarcinogenesis in mice through the activation of the β-catenin signaling pathway, suggesting that p-RXRα may serve as a possible therapeutic target for HCC.

The forced reduction of global DNA methylation suppresses tumor development in several cancer models in vivo. Nevertheless, the mechanisms underlying these suppressive effects remain unclear. In this report, we describe our findings showing that a genome-wide reduction in the DNA methylation levels induces cellular differentiation in association with decreased cell proliferation in Apc (Min/+) mouse colon tumor cells in vivo. Colon tumor-specific DNA methylation at Cdx1 is reduced in the DNA-hypomethylated tumors accompanied by Cdx1 derepression and an increased expression of intestinal differentiation-related genes. Furthermore, a histological analysis revealed that Cdx1 derepression in the DNA-hypomethylated tumors is correlated with the differentiation of colon tumor cells. Similarly, the treatment of human colon cancer cell lines with a hypomethylating agent induces differentiation-related genes, including CDX1. We herein propose that DNA demethylation exerts a tumor suppressive effect in the colon by inducing tumor cell differentiation.

The technology for generation of induced pluripotent stem cells (iPSCs) has made significant contributions to various scientific fields, and the field of cancer biology is no exception. Although cancer is generally believed to develop through accumulation of multiple genetic mutations, there is increasing evidence that cancer cells also acquire epigenetic abnormalities during development, maintenance, and progression. Because the epigenetic status of somatic cells changes dynamically through reprogramming, iPSC technology can be utilized to actively and globally alter the epigenetic status of differentiated cells. Using this technology, a recent study has revealed that some types of cancer can develop mainly through disruption of the epigenetic status triggered by dedifferentiation. In this paper, we outline the reprograming process and the epigenetic mechanism associated with the maintenance or conversion of cell identity. We then describe several observations suggesting that dedifferentiation can play an important role in cancer development. Finally, we introduce the system responsible for in vivo reprogramming to demonstrate the involvement of dedifferentiation-driven epigenetic disruption in cancer development, and propose that particular types of cancer can develop predominantly through epigenetic alterations.

P5, one of the protein disulphide isomerase (PDI) family members, catalyses disulphide bond formation in proteins and exhibits molecular chaperone and calcium binding activities in vitro, whereas its physiological significance remains controversial. Recently, we have reported that P5 localizes not only in the ER but also in mitochondria, although it remains unclear so far about its physiological significance(s) of its dual localization. Here we report that H(2)O(2)- or rotenone-induced cell death is suppressed in MTS-P5 cells, which stably express P5 in mitochondria. H(2)O(2)-induced cell death in Saos-2 cells occurred, in large part, through caspase-independent and poly(ADP-ribose) polymerase (PARP)-dependent manner. In MTS-P5 cells challenged with H(2)O(2) treatment, PARP was still activated, whereas release of cytochrome c or apoptosis-inducing factor and intramitochondrial superoxide generation were suppressed. We also found that mitochondrial P5 was in close contact with citrate synthase and maintained large parts of its activity under H(2)O(2) exposure. These results suggest that mitochondrial P5 may upregulate tricarboxylic acid cycle and possibly, other intramitochondrial metabolism.

Global DNA hypomethylation and concomitant site-specific gene hypermethylation are among the most common molecular alterations in human neoplasia. Although site-specific DNA hypermethylation has been shown to be associated with the development of various tumors accompanied by transcriptional silencing of target genes, the functional significance of global DNA hypomethylation in tumorigenesis remains unclear. Previous studies have revealed that a genetic reduction of the DNA methylation levels leads to opposing effects on tumor development, depending on the tumor cell type and the stage of tumorigenesis. In the present study, we investigated the effect of DNA hypomethylation on gastric carcinogenesis in mice. The genetic reduction of DNA methylation levels suppressed the incidence, number and size of gastric tumors in two different mouse models for gastric tumorigenesis: the N-methyl-N-nitrosourea-induced model and the Apc(Min/+) mouse model that spontaneously develops gastric tumors with aging. Histological analyses revealed DNA hypomethylation to completely inhibit the development of invasive gastric tumors. These findings indicate that the reduction of DNA methylation levels suppresses gastric carcinogenesis and suggest that DNA methylation is closely associated with gastric tumorigenesis.

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 microM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.

We report here that Streptomyces coelicolor A3(2) produces at least seven butyrolactone autoregulators: two of the IM-2 type, four virginiae butanolide type, and one A-factor type. The most abundant one corresponds to virginiae butanolide-C9 having a C2 side chain of nine carbons. Model butyrolactone compounds as well as extracts of S. coelicolor mycelia showed clear induction of morphological differentiation, implying that S. coelicolor A3(2) probably possessed butyrolactone-type autoregulator(s) controlling the morphological differentiation.

Activating mutations in the beta-catenin gene is thought to be responsible for the excessive beta-catenin signaling involved in the majority of colon carcinomas in rodent models. Our recent study which indicated that beta-catenin mutations are present frequently in early dysplastic lesions of rat colon induced by a colon-specific carcinogen, azoxymethane led us to perform more specifically a comparative study regarding types of the beta-catenin mutation as well as K-ras mutations between such early appearing lesions and colon tumors. Male F344 rats, 6 weeks old, received s.c. injections of azoxymethane (15 mg/kg body weight) once a week for 3 weeks, and were killed at 16 and 46 weeks of age. Colons of animals killed at 16 weeks of age were processed for early altered lesions. Colon tumors from animals killed at 46 weeks of age were evaluated histopathologically. Laser capture microdissection system was used to obtain DNA of epithelial cells in both intramucosal lesions and colon tumors. After amplification of exon 3 of the beta-catenin gene and exon 1 of the K-ras gene, the products were then sequenced directly in both directions. Mutations in the exon 3 of beta-catenin gene were detected in 22 of 56 early lesions (39.3%) and 21 of 37 colon cancers (56.8%). Remarkably, all beta-catenin mutations detected in the colon tumors converged at codons encoding functionally important residues that may directly mediate beta-catenin degradation, whereas mutations in the early appearing lesions were found to be scattered in the exon 3 of the gene. K-ras mutations were also detected in 24 of 56 early lesions (42.9%) and 11 of 37 colon cancers (29.7%). All K-ras mutations converged at codon 12 and codon 13, even in the early lesions. The results of this study provide evidence for the first time that beta-catenin mutation is selected during the colon carcinogenesis. Our results also suggest that the activation of beta-catenin signaling pathway is not only an initiating event, but also plays a pivotal role in the promotion stage of colorectal carcinogenesis.

Pseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation-factor (LAF) of unknown structure. From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL.

Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydro VB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydro VB-A reductase was found to require NADPH, not NADH, as a coenzyme. The K(m) values of the enzyme for NADPH and (+/-)-6-dehydro VB-A were determined to be 50 +/- 2 microM and 100 +/- 5 microM, respectively. Ultracentrifugation experiments revealed that 6-dehydro VB-A reductase was present almost exclusively in the 100,000 x g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The M(r) of the native 6-dehydro VB-A reductase was estimated to be 82,000 +/- 3000 by molecular sieve HPLC. The optimal pH was found to be 6.7 +/- 0.2.

LipL of Pseudomonas sp. strain 109 is a unique lipase capable of catalyzing macrocyclic lactone synthesis using omega-hydroxyfatty acid esters as substrates. Several fatty acid esters were tested as inducers of LipL production. The addition of either soybean oil or a non-ionic detergent (Noigen HC) resulted in a 44 to 45-fold increase in extracellular LipL, and the presence of both resulted in a further 56-fold increase. Among the triglycerides tested, triolein was the most effective, with a 50-fold increase in LipL production. A Northern blot hybridization analysis found that the lipL transcript increased in the presence of soybean oil or Noigen HC, indicating that the production of LipL is regulated at the transcriptional level.

IM-2, one of the butyrolactone autoregulators of Streptomyces, triggers blue pigment production in Streptomyces sp. FRI-5 at a concentration of 0.6 ng/ml. Two IM-2-induced metabolites were identified by comparison of the HPLC profiles of extracellular metabolites produced in the absence and presence of IM-2. The two compounds were purified to homogeneity from the culture broth, and assigned as tryptophol and guanosine, respectively. With respect to the IM-2-induced production of guanosine, the intracellular and extracellular nucleotide contents were investigated in the absence and presence of IM-2. No difference in nucleotide content, including that of ppGpp, was observed, indicating that ppGpp does not participate in the induction pathway of IM-2. It was therefore concluded that IM-2 is an independent regulator of Streptomyces metabolism, which not only induces blue pigment but also affects metabolism of tryptophan and guanosine.

The presence of the vbrA gene encoding the transcriptional antiterminator NusG equivalent protein of Streptomyces virginiae was tested for in 73 Streptomyces species by Southern hybridization. Fifty-five strains (75%) including S. griseus, S. lividans TK-21 and S. coelicolor A3(2) showed clear hybridization signals, indicating wide distribution of vbrA or vbrA homologs in Streptomyces species. With hybridization patterns against 3 different probes, i.e., probes covering vbrA alone, the downstream gene rplK alone, and both vbrA-rplK, the 55 strains were classified into 4 groups. In the groups I, II and III (total 50 strains) vbrA was found to be adjacent to rplK, indicating that the gene arrangement vbrA-rplK is common in Streptomyces and that these genes may constitute a part of gene cluster encoding several components of the transcription and translation apparatus, as in Escherichia coli.

Allosamidin (1) was converted to demethylallosamidin (2) and didemethylallosamidin (3) by a reaction with methylamine and ammonia, respectively. In the same way, a series of N-monoalkyl derivatives was prepared from 1 and 2. A variety of 6- and 6”-O-acyl derivatives of 1 was also prepared by chemically selective acylation. The inhibitory activity of each of these derivatives on chitinases originating from insects and fungi showed that a monomethylaminooxazoline or dimethylaminooxazoline structure was important for strong activity, and that the 6”-O-acyl derivatives of 1 each retained relatively high activity.

A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.

Resting cells and acetone-dried cells of Corynebacterium sp. S-401 catalyzed the fatty acid ester synthesis of various alcohols and fatty acids. These reactions were carried out in phosphate buffer and/or organic solvents. In some cases synthetic reactions of esters in nonpolar solvents, such as n-hexane and benzene, gave better results compared with those obtained in phosphate buffer.

Corynebacterium sp. S-401 was isolated from soil using squalene as carbon source. Microbiological properties of this bacterium are described. The five metabolic products of this bacterium from squalene were identified as mono-, di-, tri-, tetra-, and pentahydrated squalene. The resting cells of the bacterium catalyzed the hydration of oleic acid to give (—)-10R- hydroxyoctadecanoic acid and further oxidized product, 10-oxooctadecanoic acid, was also isolated from the reaction mixture.

A microorganism, strain KI 72 capable of utilizing e-aminocaproic acid cyclic dimer as sole carbon and nitrogen sources was isolated from sludge and identified as Achromobacter guttatus. This bacteria utilized 1% of the cyclic dimer in a day and was not inhibited by the higher concentration of the dimer. The growth rate was independent of the cyclic dimer concentration in the medium, but the maximum cell concentration increased with the increase of substrate concentration. The cell yield was 0.7 mg dry cell/mg e-aminocaproic acid cyclic dimer. Bacterial growth with the cyclic dimer as substrate was significantly stimulated by the addition of yeast extract. Ferric chloride was also stimulatory. Maximal growth was obtained in cultures incubated at pH 6 and at 33°C. Synthesized nylon oligomers, ranging from e-aminocaproic acid up to its linear hexamer, were found to be catabolized by this organism.

Pure (+)-trans-chrysanthemic acid was synthesized from (+)-⊿3-carene.Ozonolysis of (+)-⊿3-carene affords 2,2-dimethyl-3-(2-oxo-propyl)-cyclopropyl-acetaldehyde which was cyclized to (+)-2-acety1-6,6-dimethyl-bicyclo-[3,1,0]-2-hexene. The bicyclo compound was transformed into (+)-cis-homocaronic acid by ozonolysis followed by oxidation. Diester or anhydride of the acid and methyl magnesium iodide gave (-)-dihydrochrysanthemolactone which afforded finally (+)-trans-chrysanthemic acid via ethyl (-)cis-chrysanthemate.

The structure of aeruginoic acid which was isolated from the culture medium of P. aeruginosa was detremined and confirmed synthetically.

2-Methylpiperazino[1, 2-a]indole-6, 9-diones were synthesized as the analogues of mitomycin antibiotics. The compounds prepared from 2-carbethoxy-5-methoxyindole showed antibacterial activities.

(±)-Azetidine-2-carboxylic acid and 3-substituted-2-pyrrolidinones were synthesized from 2-pyrrolidinone via 3-bromo-2-methoxy-1-pyrroline (IIIa). The bromide (IIIa) was obtained by the bromination of 2-methoxy-l-pyrroline using NBS.

(±) -cis and trans-2,2-dimethyl-3- (2′-cyano-l′-propenyl) cyclopropane carboxylic acids and their optically active forms were synthesized starting from chrysanthemic acids via oximes of 2,2-dimethyl3- (2′-formyl-l′-propenyl) cyclopropane carboxylates. Their allethrolone esters were also prepared which showed insecticidal activity.

The 2,3-Dihydro-lH-pyrrolo[l,2-a]indole ring system was synthesised by the condensation reaction of toluquinone and 2-cyanomethylenepyrrolidine.

Pyrrolo[1,2-a]indoloquinones, IX and X, were synthesized and their antimicrobial activities as mitomycin analogues were bioassayed.

Mitomycin A (C16H19O6N3) and mitomycin C (C15H18O5N4) are pigments which have the quinoid structure. When treated with aqueous ammonia, mitomycin A is converted to mitomycin C. Acid hydrolysis of mitomycin C gave three degradation products, namely, C14H15O5N4, C14H15O6N3 and C13H14O5N2. Acetylation with acetic anhydride and pyridine and methylation with methyl iodide gave monoacetyl and monomethyl derivatives of mitomycin C respectively, though diacetate of demethyl derivatives were obtained when boiled with acetic anhydride.

The absolute configuration of allosamizoline (1), a constituent aminocyclitol derivative of allosamidin (2), was elucidated by the exciton chirality method using its 3,4-bis[p-(dimethylamino)benzoyl]-6-trityl derivative (7).

(1981). Asymmetric Synthesis of (S)-(—)-Squalene-2,3-epoxide. Agricultural and Biological Chemistry: Vol. 45, No. 7, pp. 1741-1742.