Probing the interaction between N(1),N(4)-dibenzylputrescine and tRNA through (15)N NMR: biological implications.

NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA. The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured. From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined. The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction. This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.

Antiproliferative effects of N1,N4-dibenzylputrescine in human and rodent tumor cells.

Polyamines (putrescine, spermidine and spermine) increase in proliferating tissues and are essential for cellular growth and cell division processes. We had previously shown that alkyl substituted putrescines can inhibit cell proliferation. We now tested the effects of the (N(alpha),N(omega)-dibenzyl derivatives of the simple diamines putrescine, cadaverine and 1,3-diaminopropane on the growth of three human squamous cell carcinoma (SCC) lines and a rat hepatoma (H-4-II-E) cell line. Survival assays were measured by treating exponentially-growing SCC cultures with N1,N4-dibenzylputrescine (DBP) (270 microM) or a rat hepatoma cell culture with DBP (100 microM) for 48 hrs. Inhibition of cell growth was measured either by the colony forming assay or by cell counting. DBP inhibited proliferation of the rat hepatoma (H-4-II-E) cell line and induced cytotoxicity when used at a concentration of 100 microM for >48 hrs. N1,N5-dibenzylcadaverine (DBC) also induced cytotoxicity at a similar concentration, while N1,N3-dibenzyl-1,3-diaminopropane (DBPr) was a much weaker inhibitor of cell growth. Inhibition of cell growth by DBP resulted in marked modifications of cell morphology, such as vacuole formation, decrease in size, pycnosis, change in staining behavior toward trypan blue and lack of adherence. DBP was also growth inhibitory in the three human SCC cell lines tested. The concentration of DBP required to achieve growth inhibition of SCC cells could be dramatically decreased in the presence of N1,N4-bis(buta-2,3-dienyl)butanediamine, a specific inhibitor of polyamine oxidase (PAOI). Moreover, although the presence of PAOI only prevented the oxidation (debenzylation) of approximately 20% of intracellular DBP over a 5-day period, it produced a 5-fold increase in the inhibition of cell proliferation by DBP. DBP (and DBC) inhibited putrescine uptake by rat hepatoma (H-4-II-E) cells in what appears to be a competitive reaction. A tenfold excess of putrescine over DBP did not inhibit the antiproliferative or cytotoxic effects of the latter. DBP administered for 72 hrs. depleted intracellular levels of putrescine, spermidine and spermine in the SCC lines by 50-100% of control values. It was found that DBP inhibited nucleic acid and protein synthesis at an early stage of cell proliferation, hence its growth inhibitory effect may be related to inhibition of the synthesis of macromolecules.

Structure/activity relationships in porphobilinogen oxygenase and horseradish peroxidase. An analysis using synthetic hemins.

The apo-enzymes of porphobilinogen oxygenase and horseradish peroxidase were reconstituted with hemin IX, deuterohemin IX, 2,4-diacetyldeuterohemin IX, 2-vinyl-4-deuterohemin IX and hemin I. The apoproteins did not reconstitute with the dimethyl or diethyl esters of hemin IX. The native enzymes and the synthetic hemoproteins showed similar oxygenase activities toward porphobilinogen in the presence of dithionite and oxygen. They also showed peroxidase activity in the presence of H2O2, which was affected by the side-chain substitution pattern of the hemes. Oxygenase activities, however, were not affected by the heme structure. Iron chelators completely inhibited the oxygenase, but not the peroxidase activities. The EPR spectra of the native and synthetic porphobilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high-spin heme-iron signal at g = 6.0. It reappeared 1 min later but the enzyme retained its catalytic activity. The changes in the EPR spectra could be correlated with the biphasic kinetics of the oxygenase reaction which was very fast during the first minute and then decreased to a half-value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate phase, but not during the slower phase. These results could be explained by the formation of a superoxide anion during the first minute of the oxygenase reaction, after which a protein-stabilized radical (g = 2.0) is generated (very likely a tyrosyl radical). The latter then oxidizes the substrate porphobilinogen and facilitates its reaction with O2 to give oxopyrrolenines.

Inhibition of ornithine decarboxylase by the isomers of 1,4-dimethylputrescine.

1,4-Dimethylputrescine (2,5-hexanediamine) was separated into its racemic and meso isomers by fractional crystallization of its dibenzoyl derivative. The racemic form was resolved into its (+)- and (-)-isomers with (+)- and (-)-dibenzoyltartaric acids. None of the three isomers (meso, +, and -) inhibited ornithine decarboxylase (ODC) activity in vitro, while all the three were strongly inhibitory of ODC when assayed in vivo in rats or in H-35 hepatoma cells. In rat liver the three isomers also decreased the putrescine pool while only the (+)-isomer decreased spermidine content. In the H-35 cells the (-)- and (+)-isomers decreased the spermidine and spermine content. When ODC was induced in the latter by insulin it was found that the (-)-isomer strongly inhibited protein and ODC synthesis, while the (+)-isomer and the meso isomer were less inhibitory. The meso isomer was a good inducer of ODC antizyme in rat liver, while the (+)- and (-)-isomers were poor inducers of the former.

JAL2: an integrated tool to enhance communication and autonomy of disabled people.

JAL2 is an integrated tool which enhances communication skills for people--mainly those with cerebral palsy--who suffer from any kind of disability that prevents speech. JAL2 can be used as a portable communicator and has an editor which can construct any text. It can also substitute for the keyboard of a personal computer, allowing the disabled the use of these machines. Furthermore, it is used to control one's personal environment by means of an infra-red transmitter. All of these options, scanned on a display, are accessed through a push-button.

Interaction of alkylputrescines with ornithine decarboxylase from rat liver and Escherichia coli: an in vitro and in vivo study.

The inhibitory effect of a series of 2-alkylputrescines on rat liver and Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. At 2.5 mM concentrations, 2-methyl-, 2-propyl-, 2-butyl-, 2-pentyl- and 2-hexylputrescines were stronger inhibitors of the mammalian enzyme than putrescine. Only the higher homologues (from 2-propyl- to 2-hexylputrescine) were inhibitors of the E. coli enzyme. An analysis of the effect of increasing concentrations of the 2-alkylputrescines showed that the main difference in the behaviour of the mammalian and E. coli decarboxylases toward 2-alkylputrescines was that the former was strongly inhibited by 2-methylputrescine whereas the latter was not. 2-Alkylputrescines were found to be competitive inhibitors of both the bacterial and mammalian enzyme. The smallest Ki values (0.1 and 0.5 mM) were found for the 2-hexyl- and 2-pentylputresciens. N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescines (50 mumol per 100 g body weight) were assayed as inhibitors of thioacetamide-induced rat liver ornithine decarboxylase. N-Propylputrescine was found to be the most inhibitory (66% inhibition) and although the N-alkylputrescines were taken up by the liver, they did not inhibit the liver polyamine pools. Both putrescine and N-methylputrescine were found to stabilize the thioacetamide-induced ornithine decarboxylase at the onset of the enzyme's degradation, while 2-alkylputrescines were inhibitory under similar conditions. N-Methylputrescine induced antizyme in thioacetamide-treated rats. In thioacetamide- or dexamethasone-treated rats, 2-methylputrescine was found to be the strongest in vivo inhibitor of the liver decarboxylase. Although 2-alkylputrescines were efficiently taken up by the liver, they did not noticeably inhibit its polyamine pools. 2-methylputrescine decreased the putrescine concentration of the liver, but not its spermidine and spermine content. No induction of ornithine decarboxylase antizyme by 2-methylputrescine could be detected. The intrahepatic concentration of the latter decreased with time, very likely due to its degradation by a diamine oxidase, since the decrease was inhibited by aminoguanidine.

Effect of N-alkyl and C-alkylputrescines on the activity of ornithine decarboxylase from rat liver and E. coli.

N-Methyl-, N-ethyl-, N-propyl-, N-butyl-, N,N-dimethyl- and N,N'-dimethylputrescines were assayed as inhibitors of ornithine decarboxylase (EC 4.1.1.17) from rat liver and from Escherichia coli. They were found to be poor inhibitors, with the exception of N-propylputrescine and N,N-dimethylputrescine, which were inhibitory at 25 mM. A homologous series of 1-alkylputrescines ranging from 1-methylputrescine (1,4-diaminopentane) to 1-heptylputrescine (1,4-diaminoundecane) was assayed for effect on the activity of ornithine decarboxylase from the same sources. 1-Methylputrescine (5 mM) inhibited the mammalian enzyme, while the higher homologues showed significantly less inhibitory activity. When assayed on the bacterial enzyme, 1-methylputrescine (5 mM) was not inhibitory, while the higher homologues showed inhibitory effects. At higher concentrations, 1-methylputrescine and 1-heptylputrescine were the best inhibitors of these series of rat liver ornithine decarboxylase. When 1-methylputrescine, 2-methylputrescine, 1,2-dimethylputrescine, 1,3-dimethylputrescine and 1,4-dimethylputrescine were assayed as inhibitors of the decarboxylase, 2-methylputrescine was found to be the best inhibitor of the rat liver enzyme, while 1,3-dimethylputrescine was the best inhibitor of the bacterial enzyme. 1,4-Dimethylputrescine (2,5-diaminohexane) did not inhibit the enzyme from either source. Both, 2-methylputrescine and 1-methylputrescine, as well as the 1,2- and 1,3-dimethylputrescines were competitive inhibitors of the enzyme, and a Ki of 1 mM was obtained for 2-methylputrescine when the rat liver decarboxylase was used. N-Methyl, 1-methyl and 2-methylputrescines were found to inhibit in vivo the activity of rat liver ornithine decarboxylase which had been previously induced by thioacetamide treatment. 2-Methylputrescine (50 mumol/100 g body weight) was found to be the best in vivo inhibitor (93% inhibition), while putrescine under similar conditions inhibited 56% of the enzymatic activity.

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IX alpha as the sole product of the enzymatic oxidation.

Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1% overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IX gamma 8a,13a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IX beta 13a,18a-dimethyl ether (1.9%), the dimethyl ester of hematobiliverdin IX delta 8a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IX alpha 18a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IX alpha was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IX alpha was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is alpha-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.

Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Crithidia fasciculata. Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase.

The non-invasive technique of 13C nuclear magnetic resonance was applied to study glucose metabolism in vivo in the insect parasite Crithidia fasciculata. It was found that under anaerobic conditions [1-13C]glucose underwent a glycolytic pathway whose main metabolic products were identified as [2-13C]ethanol, [2-13C]succinate and [1,3-13C2]glycerol. These metabolites were excreted by C. fasciculata into the incubation medium, while in the cells [3-13C]phosphoenolpyruvate was also detected in addition to the aforementioned compounds. The C3 acid is apparently the acceptor of the primary CO2 fixation reaction, which leads in Trypanosomatids to the synthesis of succinate. By addition of sodium bicarbonate to the incubation mixture L-[3-13C]malate was detected among the excretion products, while the ethanol:succinate ratio of 2.0 in the absence of bicarbonate changed to a ratio of 0.6 in the presence of the latter. This was due to a shift of the balance between carboxylation of phosphoenolpyruvate, leading to succinate, and pyruvate decarboxylation leading to ethanol. The addition of 25% 2H2O to the incubation mixture led to the formation of [2-13C, 2-2H]ethanol derived from the prior incorporation of 2H+ into pyruvate in the reactions mediated by either pyruvate kinase or malic enzyme. However, no 2H+ incorporation into L-malate was detected, excluding the possibility that the latter was formed by carboxylation of pyruvate, and lending support to the idea that L-malate results from the carboxylation of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxykinase. The formation of [2-13C, 2-2H]-succinate under the same conditions reflected the uptake of 2H+ during the reduction of fumarate. When the incubations were carried out in the presence of 100% 2H2O, several [1-13C, 1-2H]ethanol species were detected, as well as [2-13C, 2-2H]malate and [1,3-13C2, 1,3-2H2]glycerol. The former deuterated compounds reflect the existence of NAD2H species when the incubations were carried out in 100% 2H2O, while the incorporation of 2H+ into [1,3-13C2]glycerol must be attributed to the phosphoglucose-isomerase-mediated reaction during glycolysis.

Specific alpha-bridge cleavage by heme oxygenase of [alpha-14C]deuterohemin IX, [alpha-14C]hematohemin IX and 2,4-diacetyl[alpha-14C]deuterohemin IX.

The enzymatic oxidations of [alpha-14C]deuterohemin IX, [alpha-14C]hematohemin IX and 2,4-diacetyl[alpha-14C]deuterohemin IX were carried out by using a microsomal heme oxygenase system from rat liver in combination with the biliverdin reductase from the same origin. In every case the bilirubins formed were devoid of radioactivity, indicating the alpha-selective oxidation of the three hemins. Hematohemin IX was oxidized at the highest rate, followed by deuterohemin IX and 2,4-diacetyldeuterohemin. When the three hemins were preincubated with microsomal heme oxygenase in the absence of NADPH, and the latter was added after the preincubation period, it was found that the enzymatic oxidation of the hemins was inhibited. Therefore, for the maximal rate of oxidation both hemin and NADPH must be present simultaneously. In the presence of hemin IX (the natural substrate), the enzymatic oxidation of the synthetic hemins was inhibited. The oxidation of 2,4-diacetyldeuterohemin IX was the most inhibited, while the oxidation of hematohemin IX was affected to a much lesser degree. These results are in agreement with the higher affinity (Km = 150 microM) of hematohemin IX for the enzyme, as compared to 2,4-diacetyldeuterohemin IX (Km = 660 microM) and deuterohemin IX (Km = 330 microM).

Specificity of heme oxygenase: a study with synthetic hemins.

A large number of synthetic iron porphyrins were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. They all had in common two vicinal propionic acid residues at C6 and C7. Iron porphyrins of type I were not substrates of the enzyme. Iron porphyrins that carried electron-withdrawing substituents (acyl residues) at C2 and C4 were substrates of heme oxygenase, although the product yields were reduced. Several iron porphyrins, such as hemin XIII (4) and hem III (5), were better substrates of heme oxygenase than the natural substrate hemin IX (1). The enzymatic oxidation was selective for the alpha-methine bridge, and the alpha-biliverdins obtained were reduced by Biliverdin reductase to the corresponding alpha-bilirubins. Preincubation of the enzymatic system with hemin IX (1) and hemin XIII (4) in the absence of NADPH resulted in an inhibition of their oxidation. The iron-free porphyrins which carried two vicinal propionic acid residues at C6 and C7 were also found to be inhibitors of the enzymatic system when preincubated with the latter. The presence of hematochemin IX (18) suppressed the enzymatic oxidation of hemin IX (1).