The influence of poly(ester amide) on the structural and functional features of 3D additive manufactured poly(ε-caprolactone) scaffolds.

The current research reports for the first time the use of blends of poly(ε-caprolactone) (PCL) and poly(ester amide) (PEA) for the fabrication of 3D additive manufactured scaffolds. Tailor made PEA was synthesized to afford fully miscible blends of PCL and PEA using different percentages (5, 10, 15 and 20% w/w). Stability, characteristic temperatures and material's compatibility were studied through thermal analyses (i.e., TGA, DSC). Even though DMTA and static compression tests demonstrated the possibility to improve the storage modulus, Young's modulus and maximum stress by increasing the amount of PEA, a decrease of hardness was found beyond a threshold concentration of PEA as the lowest values were achieved for PCL/PEA (20% w/w) scaffolds (from 0.39 ±â€¯0.03 GPa to 0.21 ±â€¯0.02 GPa in the analysed load range). The scaffolds presented a controlled morphology and a fully interconnected network of internal channels. The water contact angle measurements showed a clear increase of hydrophilicity resulting from the addition of PEA. This result was further corroborated with the improved adhesion and proliferation of human mesenchymal stem cells (hMSCs). The presence of PEA also influenced the cell morphology. Better cell spreading and a much higher and homogenous number of cells were observed for PCL/PEA scaffolds when compared to PCL ones.

Conformationally restricted analogues of 1N,14N-bisethylhomospermine (BE-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cells.

Twelve analogues of 1N,14N-bisethylhomospermine (BE-4-4-4) with restricted conformations were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic activities and lower systemic toxicities than BE-4-4-4. The central butane segment of BE-4-4-4 was replaced with a 1,2-substituted cyclopropane ring, a 1,2-substituted cyclobutane ring, and a 2-butene residue. In each case, the cis/trans-isomeric pair was synthesized. Cis-monounsaturation(s) was also introduced at the outer butane segment(s) of BE-4-4-4. The two possible cis-dienes and a cis-triene formally derived from the tetraazaeicosane skeleton of BE-4-4-4 were also prepared. Four cultured human prostate cancer cell lines (LnCap, DU145, DuPro, and PC-3) were treated with the new tetramines to examine their effects on cell growth with a MTT assay. One representative cell line (DuPro) was selected to further study the cellular uptake of the novel tetramines, their effects on intracellular polyamine pools, and their cytotoxicity. All tetramines entered the cells, reduced cellular putrescine and spermidine pools while exerting only a small effect on the spermine pool, inhibited cell growth, and killed 2-3 logs of cells after 6 days of treatment at 10 microM. Four new tetramines, the two cyclopropyl isomers, the trans-cyclobutyl isomer, and the (5Z)-tetraazaeicosene, were more cytotoxic than their saturated counterpart (BE-4-4-4). Their cytotoxicity, however, could not be correlated either with their cellular uptake or with their ability to deplete intracellular polyamine pools. We attribute their cytotoxicity to their specific molecular structures. The cytotoxicity was markedly reduced when the central butane segment was deprived of its rotational freedom by replacing it with a double bond. Introduction of a triple bond or a benzene-1,2-dimethyl residue at the central segment of the polyamine chain, led to complete loss of biological activity. The conformationally restricted alicyclic derivatives were not only more cytotoxic than was the freely rotating BE-4-4-4 by several orders of magnitude but also had much lower systemic toxicities than the latter. Thus, we obtained new tetramines with a wider therapeutic window than BE-4-4-4.

cis-Unsaturated analogues of 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cell lines.

From the results of our previous physicochemical studies of polyamine-nucleic acid interactions, we concluded that polyamine analogues in cisoidal conformation are capable of wrapping around the major groove of the double helix, of displacing natural polyamines from their nucleic acid binding sites, and of inhibiting cell division. On the basis of this hypothesis, nine unsaturated pentamines, formally derived from the cytotoxic pentamine 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4), were prepared in an attempt to increase antineoplastic activity. Cis-double bonds were introduced in all possible sites in the saturated pentaazapentacosane structure of BE-4-4-4-4 to yield two pentacosenes, four pentacosadienes, two pentacosatrienes, and one pentacosatetraene. Cis-double bonds should also provide good targets for mixed-function oxidases that might eliminate the accumulation of unsaturated pentamines in serum, thereby reducing systemic toxicity in animals. We determined the ability of these new pentamines to inhibit growth in four cultured human prostate cancer cell lines (LnCap, DU145, PC-3, and DuPro) using a MTT assay. LnCap and DU145 cells were very sensitive, PC-3 cells were relatively resistant, and DuPro cells were intermediate in sensitivity to most of these synthetic pentamines. In all cell lines, pentamines that had unsaturation(s) at the end of the chain showed the highest cell growth inhibitory effects. The cellular uptake, effects on cellular polyamine levels, and cytotoxicity of these pentamines on one representative prostate cancer cell line (DuPro) were further examined with a colony-forming efficiency (CFE) assay. The pentamines with unsaturation(s) at the end of the chain were once again the most cytotoxic among both the saturated (BE-4-4-4-4) and unsaturated analogues. Appreciable amounts of all pentamines entered DuPro cells and depleted cellular polyamine pools by day 6 of treatment. For most pentamines, however, cell growth inhibitory and cytotoxic effects could not be directly correlated either with their cellular uptake or with their ability to deplete cellular polyamine pools. The position of the double bonds in the aliphatic backbone seems to be the most important determinant of cytotoxicity. For some pentamines, however, depletion of cellular polyamines may add to their efficacy.

[(1)N,(12)N]Bis(Ethyl)-cis-6,7-dehydrospermine: a new drug for treatment and prevention of Cryptosporidium parvum infection of mice deficient in T-cell receptor alpha.

Cryptosporidium parvum infection of T-cell receptor alpha (TCR-alpha)-deficient mice results in a persistent infection. In this study, treatment with a polyamine analogue (SL-11047) prevented C. parvum infection in suckling TCR-alpha-deficient mice and cleared an existing infection in older mice. Treatment with putrescine, while capable of preventing infection, did not clear C. parvum from previously infected mice. These findings provide further evidence that polyamine metabolic pathways are targets for new anticryptosporidial chemotherapeutic agents.

Conformationally restricted analogues of 1N,12N-bisethylspermine: synthesis and growth inhibitory effects on human tumor cell lines.

Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain. An analogous pair of isomers was obtained when the butane segment was replaced with a 1, 2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne segment, was devoid of growth inhibitory activity against five of the six human cell lines studied (DU145 being the only exception), a clear indication of the importance of conformational mobility at the 4N, 9N-butane segment of BES for its biological activity. When the butane segment was replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not seem to be directly related to their uptake by the cells or to their effects on cellular polyamine levels. BES analogues with restricted conformations but which contained the equivalent of a two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as 1,2-diaminocyclopropyl or 1, 2-diaminocyclobutyl derivatives, were devoid of growth inhibitory effects at the concentrations studied. The development of conformationally restricted polyamine analogues appears to show promise in the further quest for polyamine-related therapeutic agents with specificity of action.

Effects of 1,2-naphthoquinones on human tumor cell growth and lack of cross-resistance with other anticancer agents.

The sensitivity of human tumor and rat prostate tumor cells to a series of naphthoquinones, including tricyclic compounds of the beta-lapachone and dunnione families as well as 4-alkoxy-1,2-naphthoquinones, was evaluated. To better understand the mechanism of cytotoxicity of 1,2-naphthoquinones, the roles of various resistance mechanisms including P-glycoprotein, multidrug resistant associated protein, glutathione (GSH) and related enzymes, altered topoisomerase activity, and overexpression of genes that control apoptosis (bcl-2 and bc-xL) were studied. MCF7 cells were most sensitive to the naphthoquinones with IC50 values ranging from 1.1 to 10.8 microM, as compared to 2.5 to >32 microM for HT29 human colon, A549 human lung, CEM leukemia and AT3.1 rat prostate cancer cells. MCF7 ADR cells, selected for resistance to adriamycin (ADR), displayed cross-resistance to the tricyclic 1,2-naphthoquinones. Drug efflux via a P-glycoprotein mechanism was ruled out as a mechanism of resistance to 1,2-naphthoquinones, since KB-V1 cells expressing high levels of P-glycoprotein and the KB-3.1 parent line were equally sensitive to these compounds. Any resistance of the tricyclic naphthoquinones noted in ADR-resistant cells appeared to relate to the GSH redox cycle and could be circumvented by exposure to buthionine sulfoximine or by changing the structure from a tricyclic derivative to a 4-alkoxy-1,2-naphthoquinone. The 1,2-naphthoquinones were found to be cytotoxic against CEM/VM-1 and CEM/M70-B1 cells that were selected for resistance to teniposide or merbarone, respectively. In addition, cells overexpressing bcl-2 or bcl-xL proteins were as sensitive to 1,2-naphthoquinones as were control cells. Because of their effectiveness in drug-resistant cells, these agents appear to hold promise as effective chemotherapeutic agents.

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.

Reaction of beta-lapachone and related naphthoquinones with 2-mercaptoethanol: a biomimetic model of topoisomerase II poisoning by quinones.

1,2-Naphthoquinones, such as beta-lapachone, 4-alkoxy-1,2-naphthoquinones, and tetrahydrofuran-1,2-naphthoquinones, react rapidly with 2-mercaptoethanol in benzene to give 1,4-, 1,2-, 1,3- and 1,6-Michael-type adducts that are formed by the addition of the thiol group to the quinone ring. Menadione (2-methyl-1,4-naphthoquinone) reacts with the thiol reagent very slowly under the same reaction conditions. Although the formation of the adducts can be followed by 1H-NMR, attempts to isolate the adducts failed due to their retroconversion to the starting products. On addition of a Lewis acid, however, the adducts undergo cyclization reactions that give stable derivatives that can be isolated and characterized. Determination of the structures of the derivatives allowed for the identification of the adducts from which they originated. Thus, beta-lapachone and 2,3-dinordunnione underwent 1,4- and 1,2-Michael type additions to the quinone ring, while 4-pentyloxy-1,2-naphthoquinone underwent two simultaneous Michael additions to the quinone ring of the naphthoquinone. Menadione underwent a single 1,3-addition. The alkylation rates of the thiol group of 2-mercaptoethanol by the naphthoquinones parallel the naphthoquinones efficiencies in inducing DNA cleavage through DNA-bound topoisomerase II. These results support our hypothesis that the cytotoxic effect of the naphthoquinones derive, at least in part, from their alkylation of exposed thiol residues on the topoisomerase II-DNA complex.

Induction of DNA topoisomerase II-mediated DNA cleavage by beta-lapachone and related naphthoquinones.

Recent studies have suggested that 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione (beta-lapachone) inhibits DNA topoisomerase I by a mechanism distinct from that of camptothecin. To study the mechanism of action of beta-lapachone, a series of beta-lapachone and related naphthoquinones were synthesized, and their activity against drug-sensitive and -resistant cell lines and purified human DNA topoisomerases as evaluated. Consistent with the previous report, beta-lapachone does not induce topoisomerase I-mediated DNA breaks. However, beta-lapachone and related naphthoquinones, like menadione, induce protein-linked DNA breaks in the presence of purified human DNA topoisomerase IIalpha. Poisoning of topoisomerase IIalpha by beta-lapachone and related naphthoquinones is independent of ATP and involves the formation of reversible cleavable complexes. The structural similarity between menadione, a para-quinone, and beta-lapachone, an ortho-quinone, together with their similar activity in poisoning topoisomerase IIalpha, suggests a common mechanism of action involving chemical reactivity of these quinones. Indeed, both quinones form adducts with mercaptoethanol, and beta-lapachone is 10-fold more reactive. There is an apparent correlation between the rates of the adduct formation with thiols and of the topoisomerase II-poisoning activity of the aforementioned quinones. In preliminary studies, beta-lapachone and related naphthoquinones are found to be cytotoxic against a panel of drug-sensitive and drug-resistant tumor cell lines, including MDR1-overexpressing cell lines, camptothecin-resistant cell lines, and the atypical multidrug-resistant CEM/V-1 cell line.

Synthesis of isoornithines and methylputrescines. An evaluation of their inhibitory effects on ornithine decarboxylase.

2-(Aminomethyl)-4-aminobutyric acid (isoornithine), 3-methylisoornithine, and 2,3-dimethylisoornithine were not decarboxylated by liver ornithine decarboxylase (ODC, EC 4.1.1.17) of thioacetamide-treated rats but were good competitive inhibitors of the enzyme (Ki ranged from 0.72 to 1.79 mM). When assayed in vivo in the treated rats, the above mentioned isoornithines were also found to inhibit liver ODC when administered 1 h before sacrifice. When the methylputrescines formally derived from the decarboxylation of several isoornithines were assayed on rat liver ODC, it was found that only 2,3-dimethylputrescine decreased the enzymatic activity. When assayed in vivo, it was found to decrease ODC activity by 60%, when the latter was measured 1 h after administration. The effect was reverted 4 h after administration of the drug. Isoornithines were not taken up by H-35 hepatoma cells; hence they did not affect their ODC activity. 2,3-Dimethylputrescine however, was transported into the cells and significantly decreased its ODC activity.

Beta-lapachone-mediated apoptosis in human promyelocytic leukemia (HL-60) and human prostate cancer cells: a p53-independent response.

beta-Lapachone and certain of its derivatives directly bind and inhibit topoisomerase I (Topo I) DNA unwinding activity and form DNA-Topo I complexes, which are not resolvable by SDS-K+ assays. We show that beta-lapachone can induce apoptosis in certain cells, such as in human promyelocytic leukemia (HL-60) and human prostate cancer (DU-145, PC-3, and LNCaP) cells, as also described by Li et al. (Cancer Res., 55: 0000-0000, 1995). Characteristic 180-200-bp oligonucleosome DNA laddering and fragmented DNA-containing apoptotic cells via flow cytometry and morphological examinations were observed in 4 h in HL-60 cells after a 4-h, > or = 0.5 microM beta-lapachone exposure. HL-60 cells treated with camptothecin or topotecan resulted in greater apoptotic DNA laddering and apoptotic cell populations than comparable equitoxic concentrations of beta-lapachone, although beta-lapachone was a more effective Topo I inhibitor. beta-Lapachone treatment (4 h, 1-5 microM) resulted in a block at G0/G1, with decreases in S and G2/M phases and increases in apoptotic cell populations over time in HL-60 and three separate human prostate cancer (DU-145, PC-3, and LNCaP) cells. Similar treatments with topotecan or camptothecin (4 h, 1-5 microM) resulted in blockage of cells in S and apoptosis. Thus, beta-lapachone causes a block in G0/G1 of the cell cycle and induces apoptosis in cells before, or at early times during, DNA synthesis. These events are p53 independent, since PC-3 and HL-60 cells are null cells, LNCaP are wild-type, and DU-145 contain mutant p53, yet all undergo apoptosis after beta-lapachone treatment. Interestingly, beta-lapachone treatment of p53 wild type-containing prostate cancer cells (i.e., LNCaP) did not result in the induction of nuclear levels of p53 protein, as did camptothecin-treated cells. Like other Topo I inhibitors, beta-lapachone may induce apoptosis by locking Topo I onto DNA, blocking replication fork movement, and inducing apoptosis in a p53-independent fashion. beta-Lapachone and its derivatives, as well as other Topo I inhibitors, have potential clinical utility alone against human leukemia and prostate cancers.

Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis.

N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities. 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA. When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced. From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA. These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding). The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2. The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA. A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities.

The interplay between basicity, conformation, and enzymatic reduction in biliverdins.

Biliverdins with extended conformations are reduced by biliverdin reductase (BvR) at higher rates than biliverdins with helical conformations. To find out the molecular basis for this important feature of BvR mechanism, helical and extended biliverdins were titrated for their acid-base equilibria in a protic solvent (methanol). It was found that the basicity of biliverdins increases with the stretching of the conformation. Biliverdin IX gamma (all-syn) has a pKa = 3.6; 5,10,15-syn,syn,anti-biliverdin has a pKa = 3.7; 5,10,15-syn,anti,syn-biliverdin has a pKa = 6.1; 5,10,15-syn,anti,anti-biliverdin has a pKa = 6.4; and 5,10,15-all-anti-biliverdin has a pKa = 7.9. The increase in basicity with progressive stretching of conformations closely parallels the increase in the reduction rates by BvR. A biliverdin constrained by a four carbon chain to a helical conformation and which is a very weak base (pKa = 0.4) is not reduced by BvR. Nucleophilic additions of 2-mercaptoethanol at the C10 in biliverdins closely parallel their basicities, as can be expected if the formation of a positive mesomeric species at C10 is linked to the basicity (i.e., the ease of protonation) of the N23 on the pyrrolenine ring.

Interactions between natural polyamines and tRNA: an 15N NMR analysis.

15N NMR spectroscopy was used to explore the interactions between natural polyamines and Escherichia coli tRNA. It was found that when tRNA is added to solutions of 15N-labeled spermine or spermidine, there is a considerable decrease in the relative heights of the -NH(2+)--resonances with respect to the signals arising from the -NH3+ groups. The presence of tRNA was also found to reduce the longitudinal relaxation times T1 of the nitrogens, mainly those of the -NH(2+)- groups. The longitudinal relaxation times of the nitrogens were used to characterize the temperature dependence of the binding, and they allowed us to calculate the activation energies that determine the correlation times of amino groups in the presence of tRNA. Both the thermodynamic and the relaxation results indicate that (i) spermine binds more strongly to tRNA than spermidine does and (ii) within each of these molecules the -NH(2+)- groups bind more strongly to tRNA than the more electropositive -NH3+ moieties. This specificity suggests that the interaction between polyamines and tRNA cannot be described exclusively in terms of electrostatic forces and that other interactions (most likely, hydrogen bonding) are very important for establishing the polyamine-tRNA link. Some of the factors that may conspire against the binding of -NH3+ groups to tRNA are briefly discussed.

The enantioselective participation of (S)- and (R)-diaminovaleric acids in the formation of delta-aminolevulinic acid in cyanobacteria.

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.

Reconstitution of apomyoglobin with extended biliverdins.

An analysis of the reconstitution of biliverdins with extended conformations and horse heart apomyoglobin was carried out. Biliverdins with the 5Z-syn, 10Z-syn, 15Z-anti and 5Z-anti, 10Z-syn, 15Z-anti conformations, as well as biliverdins with the Z,Z,Z, all-syn conformation recombined with apomyoglobin. In every case the P enantiomers were bound in excess to the M enantiomers, with exception of the 5-syn, 10-syn, 15-anti biliverdin where the M enantiomer bound preferentially to the protein. Biliverdins with an anti conformation at the C-10 meso bridge did not recombine with the protein. It was concluded that the presence of a syn conformation at the C-10 methine conferred to the biliverdin the necessary helicity to fit into the apomyoglobin heme pocket. This regioselectivity is of importance in view of the well known analogy between the ligand domains of myoglobin and the C-phycocyanins.

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules.

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.

Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Trypanosoma cruzi. Evidence of the presence of two alanine pools and of two CO2 fixation reactions.

The non-invasive technique of 13C-nuclear magnetic resonance was applied to study glucose metabolism in vivo in Trypanosoma cruzi, the causative agent of American trypanosomiasis (Chagas' disease). It was found that under anaerobic conditions [1-13C]glucose undergoes a glycolytic pathway whose main metabolic products were identified as [3-13C]alanine, [2-13C]succinate and phosphoryl[1-13C]choline; [2-13C]alanine was also a minor metabolite. The addition of 70% 2H2O to the incubation mixture led to the formation of [3-13C, 3-2H]alanine derived from the prior incorporation of 2H+ into pyruvate. The existence of a [3-13C, 3-2H]pyruvate precursor, although not isolated, could be inferred from the formation of [2-13C, 2-2H]succinate in the same experiment. The latter derives from the CO2 fixation reaction on pyruvate or phosphoenolpyruvate to give malate, which is then converted to succinate through the fumarate intermediate step. The presence of [2-13C]alanine must be traced to a randomization of label at the malate-fumarate stage. Both [3-13C, 3-2H]alanine and [2-13C, 2-2H]succinate were excreted from the cells into the supernatant. When the cell pellet was lysed with perchloric acid it released [3-13C]alanine which was devoid of 2H+. Hence, T. cruzi has two alanine pools: one which incorporates 2H+ from the 2H2O present in the medium and excretes alanine into the latter, and another which is impervious to 2H+ exchange. The fixation of CO2 on a C3 precursor was confirmed by incubation of the T. cruzi cells with [1-13C]glucose and sodium [13C]bicarbonate which led to the formation of [1,2-13C2]succinate (Jcc = 51.8 Hz). Incubation with sodium [13C]bicarbonate and [13C]glucose led to the formation of [1-13C]succinate (182.5 ppm) derived from the 13CO2 fixation on the C3 precursor, and of phosphoryl[1-13C]choline (59.39 ppm) which revealed the presence in T. cruzi of a reductive pathway of CO2 which is independent of the CO2 fixation reaction. The formation of phosphoryl[1-13C]choline from [1-13C]glucose should be attributed to 13CO2 liberated from the former by glucose-6-phosphate dehydrogenase.

The enzymatic and chemical reduction of extended biliverdins.

The substrate specificity of rat liver biliverdin reductase was probed using helical and extended biliverdins. The former were the ZZZ-all-syn biliverdins IX alpha and IX gamma, and the latter were the 5Z-syn, 10Z-syn, 15Z-anti; 5Z-anti, 10E-anti, 15E-anti biliverdins. It was found that the reduction rates of the biliverdins increased with the progressive stretching of their conformations. The most extended biliverdin was reduced at a higher rate than biliverdin IX alpha. The chemical reduction rates to bilirubins followed a similar pattern. Nucleophilic addition of 2-mercaptoethanol to the C10 methine was also favored in the extended biliverdins.