The purposes of this study were to determine the effect of the exogenous polyamines, spermidine and spermidine, on growth of crown gall tumors, to assay levels of spermidine and spermine in normal and crown gall tumor tissue and to determine the effects of selected anti-tumor compounds on growth of crown gall tumors as well as on the endogenous levels of the polyamines in those tumors.Findings1. Spermidine and spermine are present in normal potato tissue and crown gall tumor tissue grown on potato discs.2. Exogenous spermidine and spermine do affect growth of crown gall tumors depending upon the concentration of the solutions added, the type of polyamine added and the number of times the solutions are applied.3. Spermidine (1 mM) has a positive growth effect on crown gall tumors.4. Crown gall tumor tissue contains more spermidine than corresponding normal potato tissue; spermine levels are consistently low compared to spermidine levels in both crown gall tissue and normal potato tissue.5. The level of spermidine in tumor tissue increases as the tumors develop; the level of spermine remains consistently low.6. Addition of methylglyoxal bis (guanylyhdrazone) inhibits tumor growth, but the inhibitory effect is decreased by concurrent administration of spermidine.7. Both novobiocin and nalidixic acid have an inhibitory effect on tumor growth, but the effects are decreased by concurrent administration of spermidine. Reversal of the nalidixic acid effect on growth by addition of spermidine is more pronounced than reversal of the novobiocin effect.8. Methylglyoxal bis (guanylhydrazone) decreases the endogenous levels of both spermidine and spermine in crown gall tumor tissue.Conclusions1. The polyamine, spermidine, plays an important role in the growth of crown gall tumors.2. Compounds which inhibit spermidine biosynthesis or the possible mode of action of spermidine inhibit tumor growth.3. Other polyamines appear to have no significant role in crown gall tumor growth.4. The growth of crown gall tumors involves the activation of a mechanism leading to the increased biosynthesis of spermidine.5. A specific inhibitor of polyamine biosynthesis in animals also reduces endogenous levels of spermidine and spermine in crown gall tumors.6. These data further support the hypothesis that crown gall tumors are similar to animal tumors.

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 23, 2010). Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--Lund University.

Thesis (Ph. D.)--Institute of Paper Science and Technology, Georgia Institute of Technology, 2004. / Includes bibliography.

Thèse (Ph. D.)--Université Laval, 2000. / Bibliogr.: f. 155-172. Publié aussi en version électronique.

Thesis (Ph. D.)--Lund University.

Abstract Ornithine decarboxylase (EC 4.1.1. 17) is the first and the rate-controlling enzyme in polyamine biosynthesis. It decarboxylates L-ornithine to form diamine putrescine. ODC activity in cells is strictly regulated and one of the central elements of ODC regulation is an inhibitory protein called antizyme. Antizyme binds to ODC, inhibits its activity and targets ODC for the proteasomal degradation. Essentiality of polyamines for the normal cell growth and proliferation is well known. Recently their roles in the regulation of several classes of cation channels have been discovered. Some of these channels are expressed abundantly in the brain, which has increased interest in the polyamine metabolism in the central nervous system. In this study guanosine 5'-triphosphate activatable ODC was detected in the rat brain lysates. This activation was more significant after antizyme was separated from ODC. GTP-activatable ODC was more resistant to heat and displayed higher Vmax than kidney ODC. Previously GTP-activatable ODC had been found in mammalian tissues only in some tumors. ODC and antizyme expression in brain was localized by in situ hybridization and immunocytochemistry. Both proteins displayed wide and largely overlapping expression patterns restricted to neurons. The proteins were localized predominantly to cytoplasm at the most brain regions, but antizyme had a main localization in nuclei in some regions of the brain. In addition, the role of one of the most highly conserved regions in eukaryotic ODCs was studied using site-directed mutagenesis. The aspartate-233 to valine mutation was made and detected to increase Km values for the cofactor PLP and the substrate L-ornithine as well as Ki value for the inhibitor DFMO. In another part of this study a transgenic mouse line expressing ODC under the control of viral promotor was generated. The most significant changes in ODC activity were detected in reproductive organs of male mice. The high number of infertile transgenic males supported earlier reports about the importance of balanced polyamine metabolism for spermatogenesis. Infertility of female mice was increased as well, but the involvement of polyamines remained unproven. Transgenic mice were prone to various pathological conditions such as inflammations and tumour formation, which may be due to deregulated polyamine metabolism.

La citrulline est un acide aminé dont les propriétés anaboliques n’ont été mises en évidence que très récemment. Ces propriétés pourraient relever en particulier de son rôle de précurseur direct de l’arginine. L’arginine est un acide aminé central du métabolisme des mammifères, notamment comme précurseur de nombreuses molécules exerçant des fonctions physiologiques essentielles, en particulier les polyamines, que ce soit les polyamines « classiques » putrescine, spermidine et spermine, dérivés de décarboxylation de l’ornithine impliquées entre autres dans la prolifération cellulaire, ou l’agmatine, dérivé de la décarboxylation de l’arginine. Toutefois, les interactions métaboliques entre citrulline et polyamines restent encore peu connues. Le but de ce travail a été d’explorer le lien métabolique entre citrulline et polyamines, selon deux démarches distinctes.La première a été d’évaluer l’influence d’une complémentation prolongée (3 mois) en citrulline sur le métabolisme tissulaire des polyamines « classiques » chez le rat âgé. La citrulline n’affecte que de manière modérée le métabolisme des polyamines, principalement en corrigeant les altérations liées au vieillissement.La seconde, par analogie avec la putrescine et l’agmatine, a exploré l’implication du dérivé décarboxylé de la citrulline, la N-carbamoyl-putrescine (NCP) dans le métabolisme des mammifères après avoir mis au point une technique de dosage de celle-ci dans les milieux biologiques. Nous n’avons pas pu mettre en évidence la présence de NCP dans des tissus de rats ayant reçu de la citrulline par voie orale ou par voie parentérale suggèrant soit une absence de synthèse soit un métabolisme extrêmement rapide de la NCP. Par ailleurs, la NCP administrée par voie orale à des doses de 5 et 50 mg/kg/j ne semble pas induire de toxicité ni d’effets biologiques majeurs mais la démonstration de son absorption digestive reste à faire. Cependant, la NCP exerce quelques effets modérés sur le métabolisme des acides aminés et des polyamines, l’évaluation de doses plus élevées ou l’administration par voie parentérale permettrait de confirmer ces observations. / The anabolic properties of the amino acid citrulline have only been recently demonstrated. These properties may result at least in part from its role as an arginine precursor. Arginine plays a central role in mammalian amino acid metabolism, notably as the precusor of many molecules involved in essential physiological functions, such as polyamines either “classical” ones, namely putrescine, spermidine, and spermine, derived from ornithine decarboxylation and notably involved in cell proliferation, or more recently-discovered molecules such as agmatine, the decarboxylation derivative of arginine. Metabolic interactions between citrulline and polyamines are still mostly unknown.The aim of this work was to explore the metabolic link between citrulline and polyamines using two different strategies.First, the influence of a long term citrulline supplementation on tissue “classical” polyamine metabolism in aged rats has been evaluated. Polyamine metabolism is only moderately affected by citrulline supplementation, mostly correcting aging-related alterations.Second, by analogy with putrescine and agmatine, the involvement of the decarboxylated derivative of citrulline, N-carbamoyl putrescine (NCP), in mammalian metabolism was investigated after having developped an HPLC assay for the determination of NCP in biological media. We have not been able to demonstrate the presence of NCP in tissues of rats having received citrulline orally or parenterally suggesting either a lack of synthesis or an extremely rapid metabolism of NCP. In addition, oral administration of NCP 5 or 50 mg/kg/d did not appear to induce toxicity or major biological effects but the demonstration of its gastrointestinal absorption remains to be done. However, NCP has some moderate effects on amino acid and polyamine metabolism. The evaluation of higher doses of NCP or of its parenteral administration is required to confirm these observations.

Abstract The polyamines putrescine, spermidine and spermine are highly regulated polycations present in virtually all cells of higher eukaryotes. They are essential for proper cell growth and differentiation by participating in various physiological processes including DNA, RNA and protein synthesis, apoptosis and interactions with ion-channels. The complexity of polyamine metabolism and the multitude of compensatory mechanisms that are invoked to maintain polyamine homeostasis argue that these molecules are critical for cell survival. The primary aim of this study was to gain a better understanding of the mode of action of polyamines and the regulatory mechanisms in which they are involved. Transgenic mice overexpressing the polyamine biosynthetic enzymes S-AdoMetDC and ODC were found to maintain their polyamine pools by acetylation of spermidine and spermine and an increased export of these acetylated compounds. The expression of various genes was studied as a response to polyamine deprivation in cell- and kidney organ culture. Among these genes acetyl-CoA synthetase and ornithine decarboxylase were demonstrated to be developmentally regulated. Changes in gene expression patterns, with most of the transcripts upregulated in the polyamine-depleted samples, indicated selective stabilization of mRNAs. Polyamines were shown to play an important role in kidney organogenesis as their depletion results in a reduction of ureteric branching and retardation of tubule formation. The selective changes of various genes in the ureteric bud and mesenchyme indicate that polyamines might have a role in the regulation of epithelial-mesenchymal interactions during mouse kidney development.

This study provides information about the effects of polyamines on the restriction enzymatic cleavage of bacteriophage lambda DNA. The polyamines studied were spermine, spermidine, Nl-acetylspermidine and N8- acetylspermidine. The restriction enzymes studied were Xhoi, BamHI, EcoRI, and Hindiii. The electrophoretic pattern of lambda DNA digests by these enzymes were recorded photographically. These results were further analyzed by spectrographic digitization and replotting. Polyamines affect the electrophoretic pattern of restriction fragments in two ways: by causing DNA streaking and by decreasing ethidium bromide binding to DNA, which in turn affects DNA staining properties. The polyamines studied have effects which are increasingly dependent on the charge of the polyamine. The concentration necssary to alter the electrophoretic pattern decreases with increased positive charge of the polyamines. Spermine, the most highly charged polyamine studied, resulted in alterations at a lower concentration than any other polyamines studied. Following spermine was spermidine, and then the two acetylated polyamines, Nl-acetylspermidine, and N8- acetylspermidine.