1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The α-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK_a 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 °C, catalyzed by Fe(II) and Cu(II) ions yielding NH₄⁺ ion, H₂O₂, and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other α-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml^? 1) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO–HO^?, and those with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB^? resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0–10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0–10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 μM) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity.     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.Abbreviations ODC ornithine decarboxylase - DAB 1,4-diamino butanone     

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products

Summary. A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonα (IFNα) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNα and the protein synthesis inhibitor fusion protein TGFα/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H₂O₂ and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.     

Cytotoxic effects of oxysterols produced during ozonolysis of cholesterol in murine GT1-7 hypothalamic neurons

Ozone present in the photochemical smog or generated at the inflammatory sites is known to oxidize cholesterol and its 3-acyl esters. The oxidation results in the formation of multiple “ozone-specific” oxysterols, some of which are known to cause abnormalities in the metabolism of cholesterol and exert cytotoxicity. The ozone-specific oxysterols have been shown to favor the formation of atherosclerotic plaques and amyloid fibrils involving pro-oxidant processes. In the present communication, cultured murine GT1-7 hypothalamic neurons were studied in the context of cholesterol metabolism, formation of reactive oxygen species, intracellular Ca^{2 +} levels and cytotoxicity using two most commonly occurring cholesterol ozonolysis products, 3β- hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco) and 5β, 6β-epoxy-cholesterol (ChEpo). It was found that ChSeco elicited cytotoxicity at lower concentration (IC₅₀ = 21 ± 2.4 μM) than did ChEpo (IC₅₀ = 43 ± 3.7 μM). When tested at their IC₅₀ concentrations in GT1-7 cells, both ChSeco and ChEpo resulted in the generation of ROS, the magnitude of which was comparable. N-acetyl-l-cysteine and Trolox attenuated the cytotoxic effects of ChSeco and ChEpo. The intracellular Ca^{2 +} levels were not altered by either ChSeco or ChEpo. Methyl-β-cyclodextrins, which cause depletion of cellular cholesterol, prevented ChSeco- but not ChEpo-induced cytotoxicity. The cell death caused by ChEpo, but not ChSeco, was prevented by exogenous cholesterol. Although oxidative stress plays a significant role, the results of the present study indicate differences in the pathways of cell death induced by ChSeco and ChEpo in murine GT1-7 hypothalamic neurons.     

Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells.

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.     

Growth inhibition by methionine analog inhibitors of S-adenosylmethionine biosynthesis in the absence of polyamine depletion

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cis-AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC50 levels, the analogs significantly reduced AdoMet pools by approximately 50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L-cis-AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.     

Xanthine oxidase-dependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells

Several studies have suggested that vitamin A (retinol, ROH) presents pro-oxidant properties in biological systems. Recent studies point out that xantine oxidase, a ROS-generating enzyme, catalyses ROH oxidation to RA in vitro. These works stimulated the authors to investigate whether xanthine oxidase could be involved on the ROH pro-oxidative effects reported in cultured Sertoli cells. In vitro, it was demonstrate that xanthine oxidase generates superoxide in the presence of ROH as assessed by superoxide mediated-NBT reduction. Superoxide production is potentiated in the presence of NADH and inhibited by allopurinol. In Sertoli cells, ROH treatment increased xanthine oxidase activity and inhibition of the enzyme with allopurinol attenuated ROH-induced ROS production, protein damage and cytotoxicity. Moreover, inhibition of ROH oxidation to RA by retinaldehyde dehydrogenase inhibitor potentiated both xanthine oxidase-dependent ROS production and cell damage in ROH-treated cells. The data show that xanthine oxidase may play a role on vitamin A pro-oxidant effects.     

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells

Summary. Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H₂O₂ and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H₂O₂. However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.     

Direct oxidation of lymphocytes by chromic acid does not induce blastogenesis

Blastogenic and cytotoxic effects of hexavalent chromium were evaluated by direct, 2 and 20 min oxidation of lymphocytes by 10.0 to 0.0005 mM CrO₃ at 0°C. Oxidized cells exhibited concentration-dependent cytotoxicity and the inhibition of tritiated thymidine incorporation rates. When lymphocytes were oxidized first by 1.0 mM periodic acid (H₅IO₆) and thereafter by 1.0 mM CrO₃, the viability and [³H]-TdR incorporation rates of sequentially oxidized cells were identical to the corresponding indicators of lymphocytes oxidized only by CrO₃. The reversal of the oxidation sequence restored [³H]-TdR incorporation to control levels and increased cell survival. It is therefore concluded that direct interaction of hexavalent CrO₃ with plasma membrane of lymphocytes results in concentration-dependent cytotoxicity and the inhibition of [³H]-TdR incorporation, but it does not induce blastogenesis.     

Sodium 5,6‐benzylidene‐L‐ascorbate induces oxidative stress,autophagy, and growth arrest in human colon cancer HT‐29 cells

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT‐29 cells with SAA and SBA, and found a significant exposure time‐dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC₅₀ = 5 mM) than SBA (IC₅₀ = 10 mM). A short‐term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase‐dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21^+/+ and p21^?/?), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co‐incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N‐acetyl‐L‐cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3‐II were found in SBA‐treated HT‐29 cells; implicating that SBA induced AKT phosphorylation‐autophagy and p21‐growth arrest in colon cancer HT‐29 cells through an NAC‐inhibitable oxidative stress pathway. J. Cell. Biochem. 111: 412–424, 2010. © 2010 Wiley‐Liss, Inc.     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.     

Evidence for the occurrence of polyamine oxidase in the dicotyledonous plant Medicago sativa L. (alfalfa)

Polyamine oxidase (EC 1.5.3.3) activity has not been detected previously in cells of dicotyledonous plants, although it has been characterized extensively in monocotyledonous plants. Evidence is presented in this report for the occurrence of polyamine oxidase in dialyzed crude extracts of the dicotyledonous plant, Medicago sativa L. (alfalfa). Three enzyme assays were used to quantitate the formation of the three products of the reaction catalyzed by polyamine oxidase. 1-Pyrroline formation was measured colorimetrically as a yellow quinazolinium complex with o-aminobenzaldehyde. Hydrogen peroxide formation was measured spectrophotometrically with a coupled peroxidase assay system by peroxidative oxidation of guaiacol. [³H]1,3-Diaminopropane formation was measured by using [1,8-³H]spermidine as the substrate and separating the radiolabelled reaction product from the substrate by paper electrophoresis. This latter assay provided evidence that a polyamine oxidase of type [EC 1.5.3.3] catalyzed the cleavage reaction between a secondary nitrogen atom and an adjacent carbon of the butyl moiety of spermidine. Significant polyamine oxidase activity was detected in floral tissues, cortex tissues of the root, young leaves, and young germinated seedlings of alfalfa. The occurrence of polyamine oxidase in alfalfa accounts for the formation of the essential substrate, 1,3-diaminopropane, required for the biosynthesis of the uncommon polyamines, norspermidine and norspermine, which we have recently detected in alfalfa.Abbreviations PAO polyamine oxidase - MOPS [3-(N-morpholino)propanesulfonic acid] - MES [2-(N-morpholino)ethanesulfonic acid] - TES [N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid] - BICINE [N,N-bis (2-hydroxyethyl)glycine] - DTC diethyldithiocarbamic acid - R_m the distance of migration of a polyamine relative to putrescine after electrophoresis on paper     

Oxidation of diaminobenzidine in the heterocysts ofAnabaena cylindrica

Hemoproteins were localized in the cyanobacteriumAnabaena cylindrica with diaminobenzidine (DAB). Incubation of whole cells in the light with DAB resulted in deposition of oxidized DAB on the lamellae of the vegetative cells and central heterocyst region. This reaction was greatest at pH 7.5, light-dependent, insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, and abolished by glutaraldehyde fixation. A light-independent oxidation of DAB was also observed with light and electron microscopy in the honeycomb region and periphery of heterocysts. This reaction was greatest at pH 7.5, enhanced by H₂O₂, and active in glutaraldehyde-fixed frozen sections. Inhibitors such as sodium cyanide, sulfide, and hydroxylamine severely reduced DAB oxidation and nitrogenase activity under aerobic but not anaerobic conditions. These results indicate that the heme proteins, localized in heterocysts by light-independent DAB oxidation, are involved in the oxygen-protection mechanism of the O₂-labile nitrogenase.     

Cytotoxicity of polyamines to Amoeba proteus: Role of polyamine oxidase

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H₂O₂, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N ¹-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N ¹-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3–20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid ( 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity ( 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.Abbreviations DAO diamine oxidase - DFMO DL--difluoromethylornithine - DP 1-3-diaminopropane - IC₅₀ 50% inhibition concentration - MAO monoamine oxidase - N ¹-ACSP; N ¹-acetylspermine - N¹-ACSPD N ¹-acetylspermidine - N ⁸-ACSPD N ⁸-acetylspermidine - ODC ornithine decarboxylase - PAO(s) polyamine oxidase(s) - PUT putrescine - SP spermine - SPD spermidine     

Does diaminobenzidine demonstrate prostaglandin synthetase?

Summary A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as interstitial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H₂O₂ — in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.     

Ornithine decarboxylase activity is associated with proliferation but not with T3-induced differentiation of Caco-2 cells

Omithine decarboxylase (ODC) activity and polyamine (putrescine, spermidine, spermine) concentrations were measured in parallel in enterocyte-like Caco-2 cells maintained under various culture conditions. ODC activity was maximal at the begining of the exponential growth phase, decreasing dramatically thereafter to a negligible level at confluency (day 9). Kinetic studies performed on day 3 revealed the presence of a single enzyme with a K_m around 200 μM and a V_max of about 2 nmol CO₂ released/h/mg protein. Similar values were obtained in both serum-supplemented and transferrin/selenium (TS)-defined culture media, indicating that ODC kinetic parameters are not affected by any factors present in serum. Polymine concentrations were maximal on day 5. By day 9, they returned to initial levels and remained at these fairly high values until day 21. Since we have previously shown (Jumarie and Malo, 1994, In Vitro Cell. Dev. Biol., 30A:753–760) that triiodothyronine (T₃) stimulates differentiation but not proliferation of Caco-2 cells maintained in TS-defined medium, we investigated if it induces differentiation by a polyamine-dependent mechanism. Short- and long-term measurements revealed similar ODC activity and polyamine levels whether T₃ was present or not in the culture medium. These results clearly demonstrate that polyamine synthesis is more likely to be associated with Caco-2 cell proliferation, and that the T₃ effect on Caco-2 cell differentiation does not involve polyamine biosynthesis. Moreover, our data show that ODC activity is not solely regulated by intracellular polyamine concentration. © 1995 Wiley-Liss Inc.