Koichi Takao

Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

Effects of 3-styrylchromones on metabolic profiles and cell death in oral squamous cell carcinoma cells

4H-1-benzopyran-4-ones (chromones) are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E)-3-(4-hydroxystyryl)-6-methoxy-4H-chromen-4-one (compound 11), (E)-6-methoxy-3-(4-methoxystyryl)-4H-chromen-4-one (compound 4), (E)-6-methoxy-3-(3,4,5-trimethoxystyryl)-4H-chromen-4-one (compound 6) showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC) lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

Measurement of spermidine/spermine-N1-acetyltransferase activity by high-performance liquid chromatography with N1-dansylnorspermine as the substrate.

We developed a nonradioactive assay to measure spermidine/spermine N(1)-acetyltransferase (SSAT) activity by high-performance liquid chromatography (HPLC). N(1)-dansylnorspermine was prepared and evaluated as a substrate of acetylation with acetyl-CoA by SSAT in rat hepatoma (HTC) cells. Kinetic studies revealed that the K(m) values of N(1)-dansylnorspermine and acetyl-CoA were approximately 11 and 13 microM, respectively. When the assay method was applied to HTC cell samples, the SSAT activity, even at the control level, could easily be detected in as few as 20 microg protein of cell extract corresponding to 1 x 10(5) cells per determination, and 100 samples could be analyzed overnight. Thus, our HPLC method is a rapid and sensitive assay for the measurement of SSAT activity.

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N(1)-acetylspermidine (N(1)AcSpd), N(8)-acetylspermidine (N(8)AcSpd), N(1)-acetylspermine, N(1),N(8)-diacetylspermidine, and N(1),N(12)-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N(1)AcSpd and N(8)AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with (13)C(2)-N(1)AcSpd and (13)C(2)-N(8)AcSpd which have the (13)C(2)-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N(1)-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N(1)-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-(15)N(3)]-N(1)-acetylspermine and [1,4,8-(15)N(3)]spermidine ((15)N(3)-Spd), respectively; for SMO, [1,4,8,12-(15)N(4)]spermine and (15)N(3)-Spd, respectively; and for SSAT, (15)N(3)-Spd and [1,4,8-(15)N(3)]-N(1)-acetylspermidine, respectively.

Synthesis and Biological Evaluation of 3-Benzylidene-4-chromanone Derivatives as Free Radical Scavengers and α-Glucosidase Inhibitors.

A series of 3-benzylidene-4-chromanone derivatives (3-20) were synthesized and the structure-activity relationships for antioxidant and α-glucosidase inhibitory activities were evaluated. Among synthesized compounds, compounds 5, 13, 18, which contain catechol moiety, showed the potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity (5: EC50 13 µM; 13: EC50 14 µM; 18: EC50 13 µM). The compounds 12, 14, 18 showed higher α-glucosidase inhibitory activity (12: IC50 15 µM; 14: IC50 25 µM; 18: IC50 28 µM). The compound 18 showed both of potent DPPH radical scavenging and α-glucosidase inhibitory activities. These data suggest that 3-benzylidene-4-chromanone derivatives, such as compound 18, may serve as the lead compound for the development of novel α-glucosidase inhibitors with antioxidant activity.

A conceptual model of the polyamine binding site of N1-acetylpolyamine oxidase developed from a study of polyamine derivatives

We used various polyamine derivatives to study the substrate binding site of N1-acetylpolyamine oxidase (PAO) that was partially purified from rat liver. The substrate activities of acetylpolyamines indicated the presence of two anionic centers corresponding to the 1,3-diaminopropane (1,3-DAP) structure and a hydrophobic region in addition to the cleavage site of the acetamidopropyl group. Based on the results of the inhibitory activities of 1,3-DAP derivatives, we developed a conceptual model of the polyamine binding site of PAO. We used this model to identify a potent competitive inhibitor, N1,N7-dihexyl-1,7-diamino-4-azaheptane, and to develop an affinity column, 1,16-diamino4,13-diazahexadecane–linked Sepharose, which was useful for the purification of PAO.

Synthesis and Evaluation of Fatty Acid Amides on the N-Oleoylethanolamide-Like Activation of Peroxisome Proliferator Activated Receptor α

A series of fatty acid amides were synthesized and their peroxisome proliferator-activated receptor α (PPAR-α) agonistic activities were evaluated in a normal rat liver cell line, clone 9. The mRNAs of the PPAR-α downstream genes, carnitine-palmitoyltransferase-1 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) as PPAR-α agonistic activities. We prepared nine oleic acid amides. Their PPAR-α agonistic activities were, in decreasing order, N-oleoylhistamine (OLHA), N-oleoylglycine, Oleamide, N-oleoyltyramine, N-oleoylsertonin, and Olvanil. The highest activity was found with OLHA. We prepared and evaluated nine N-acylhistamines (N-acyl-HAs). Of these, OLHA, C16:0-HA, and C18:1Δ(9)-trans-HA showed similar activity. Activity due to the different chain length of the saturated fatty acid peaked at C16:0-HA. The PPAR-α antagonist, GW6471, inhibited the induction of the PPAR-α downstream genes by OLHA and N-oleoylethanolamide (OEA). These data suggest that N-acyl-HAs could be considered new PPAR-α agonists.

Fluorescent substrates for polyamine catabolic enzymes and transport.

The most widely used methods for measuring polyamine enzyme activities are radioisotope methods that measure the radioactivity of compounds produced from radiolabeled substrate by the enzyme reaction. Several fluorescent polyamines have been developed for the measurement of the polyamine transport system (PTS) or transglutaminase. Although fluorophores in the fluorescent polyamines may affect the affinity of the polyamine moiety to the enzyme protein, the assays that use fluorescent substrate are sensitive and simple for common laboratory usage.In this chapter, the uses of dansyl polyamines with a simple high-performance liquid chromatography system for the measurement of the PTS and polyamine catabolic enzymes including spermidine/spermine N¹-acetyltransferase and N¹-acetylpolyamine oxidase are described.

Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives

Background and Purpose— We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. Methods— Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. Results— NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. Conclusions— The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

N1-Nonyl-1,4-diaminobutane ameliorates brain infarction size in photochemically induced thrombosis model mice

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.