Inhibition of ornithine decarboxylase activity reduces polyamine levels and growth ofAgrobacterium tumefaciens

Flavins in different compartments of effective nodules fromGlycine max cv Maple Arrow xBradyrhizobium japonicum strains were studied by spectrophotometry and chromatographic techniques. Flavins in the peribacteroid space were riboflavin (80%) and FMN (20%), as identified by TLC and HPLC. Flavin concentrations in the soybean root nodule cytoplasm, in the symbiosome space (PBS) and in the cytosol of bacteroids were monitored between 20 and 40 days post infection (d.p.i.) Between the 20th and 29th d.p.i. an at least four times higher flavin/protein ratio was found in PBS of effective nodules compared with the nodule cytoplasm. Between nitrogenase activity in the free-living state and bacterial flavin accumulation, no correlation could be observed. Flavin accumulation is not restricted to an effective symbiosis, as indicated by the analysis of ineffective nodules with strainB. japonicum RH-31 Marburg. Flavin accumulation is absent in uninfected soybean root tissue and in free-living rhizobia, thus indicating that flavin accumulation is a result of symbiotic interaction. Flavin accumulation is also missing in nodules with a hypersensitive response against the bacteria.     

Regional changes in ornithine decarboxylase activity and polyamine levels during thyroxine-induced cardiac hypertrophy

Ornithine decarboxylase activities (ODC) and polyamine levels were determined in five cardiac regions of the rat heart, following daily administration of 1 mg/kg of thyroxine, in the right and left atria, the right and left ventricles and the septum. The thyroxine stimulated ODC activity in all five regions of the heart. Enzyme activity in the left atrium and the septum peaked a day earlier than in other regions and the decline of ODC activity was slower. Putrescine in control animals was present in all regions except the right atrium, where its content was below detectable levels. Following the administration of thyroxine, the putrescine content of the left atrium, the right ventricle and the septum declined, while spermidine and spermine levels remained unchanged. In direct contrast to the other regions of the heart, thyroxine stimulated an increase in polyamines, as well as in weight which occurred exclusively in the left ventricle. These findings suggest a causal relationship between increased polyamines and hypertrophy.     

Changes in ornithine decarboxylase activity and polyamine levels during the growth of Tetrahymena thermophila cultures

Ornithine decarboxylase activity and polyamine levels were determined at various growth phases of Tetrahymena thermophila cultures. Enzyme activity and intracellular polyamines increased in exponentially growing cells and peaked just before the stationary phase. Putrescine was the predominant polyamine and spermidine and spermine concentrations were low throughout. The increase in putrescine level can be totally accounted for by the enzyme activity detected, provided that there is an ample supply of the precursor, L-ornithine.     

Management of polyamine pools and the regulation of ornithine decarboxylase

The management of polyamine synthesis and polyamine pools differs fundamentally from that of most other small molecular-weight endproducts. The polyamines are vital to growth and important cellular functions, but they are toxic in excess. I argue here that their multivalent cationic character, leading to binding to cell constituents, precludes fluent feedback inhibition of synthesis. This has led to the development of elaborate alternative regulatory mechanisms controlling ornithine decarboxylase, the key initial enzyme of the pathway. Poorly regulated polyamine synthesis and the toxicity of polyamines impose upon cells a need to control uptake and to dispose of excess polyamines. Recent data on polyamine transport suggest unorthodox mechanisms of accomplishing these functions.     

Regional distribution of ornithine decarboxylase activity and polyamine levels in experimental cat brain tumors.

Biosynthesis of the polyamines putrescine, spermidine, and spermine, and activation of the first key enzyme ornithine decarboxylase (ODC) are closely associated with cellular proliferation. In the present study, the distribution of ODC activity and polyamine levels was investigated for the first time regionally in experimental brain tumors of the cat. Brain tumors were produced by stereotactic xenotransplantation of rat glioma cells. Twenty days after implantation, the brains were frozen in situ, cut into slices, and cryostat sections and tissue samples were taken to determine ODC activity and polyamine levels biochemically. The quantified data were color-coded to present the regional distribution of ODC activity and polyamine levels in the respective section. ODC activity significantly increased in some areas within the tumor, whereas peritumoral tissue showed no difference to the non-tumoral, contralateral hemisphere. This increase turned out in parallel to a high number of mitoses in the same tumor parts (r=0.861). Putrescine levels increased both, in the whole tumor and in the peritumoral edema. Regional differences in putrescine content did not correlate with solid and proliferative parts of the tumor. Spermidine and spermine levels were only slightly increased in some parts of the tumor. Thus, these experiments show the close correlation of a high mitotic rate and activation of ODC within experimental gliomas and underline the relevance of ODC as a biochemical marker of proliferation in brain tumors.     

The influence of Lactobacillus brevis on ornithine decarboxylase activity and polyamine profiles in Helicobacter pylori-infected gastric mucosa

Background. Functional probiotics may prevent Helicobacter pylori infection, and some evidence suggests that they also possess antitumor properties. Lactobacillus brevis (CD2) is a functional Lactobacillus strain with peculiar biochemical features, essentially related to the activity of arginine deiminase. This enzyme catalyzes the catabolism of arginine and affects the biosynthesis of polyamines (putrescine, spermidine, and spermine). Polyamines are polycations found in high concentrations in both normal and neoplastic cells. Our aims were: 1, to assess whether oral administration of L. brevis (CD2) affects H. pylori survival in the human gastric mucosa; 2, to evaluate the effects of L. brevis (CD2) on polyamine biosynthesis in gastric biopsies from H. pylori‐positive patients. Materials and Methods. For 3 weeks before endoscopy, 22 H. pylori‐positive dyspeptic patients randomly received (ratio 1 : 1) high oral doses of L. brevis (CD2) or placebo. Before and after treatment, H. pylori infection was determined by urea breath test (UBT). In gastric biopsies, ornithine decarboxylase activity and polyamine levels were, respectively, evaluated by a radiometric technique and high‐pressure liquid chromatography (HPLC). Results. L. brevis (CD2) treatment did not eradicate H. pylori. However, a reduction in the UBT delta values occurred, suggesting a decrease in intragastric bacterial load. Significantly, L. brevis (CD2) induced a decrease in gastric ornithine decarboxylase activity and polyamine levels. Conclusions. Our data support the hypothesis that L. brevis (CD2) treatment decreases H. pylori colonization, thus reducing polyamine biosynthesis. Alternatively, the arginine deiminase activity following L. brevis (CD2) administration might cause arginine deficiency, preventing polyamine generation from gastric cells.     

Changes in ornithine decarboxylase activity and polyamine levels in response to eight different forms of selenium.

The biological activity of selenium is known to depend on its chemical form. In this study, eight forms of selenium that differed in oxidation state or degree of methylation were studied for their acute effects on the activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMet DC) and on the concentrations of the polyamines putrescine, spermidine, and spermine in the liver. The polyamine pathway was studied because it is involved in the control of cell growth and in the cell's response to trophic, carcinogenic, and toxic stimuli, activities that selenium has been reported to affect. Female Sprague Dawley rats were administered 12 mumol Se/kg body weight via intraperitoneal injection and were sacrificed six hours later. Injection of sodium selenate, sodium selenite, selenomethionine, Se-methylselenocysteine, selenobetaine, and selenobetaine methyl ester resulted in significant increases in liver selenium, whereas injection of dimethylselenoxide and trimethylselenonium chloride did not. ODC activity and AdoMet DC activity were induced by those selenium compounds that also increased liver selenium content, but the magnitude of enzyme induction by those compounds was not correlated with the hepatic concentration of total selenium determined fluorometrically. Furthermore, the induction of ODC activity by the various forms of selenium did not result in concomitant increases in putrescine, spermidine, and spermine except in the case of selenite. Given that alterations in the metabolism of selenium are induced when the level of tissue selenium is elevated and that the relative abundance of various selenometabolites can be affected by the point of entry of selenium into intermediary metabolism, these data suggest that the changes that were observed in enzyme activities and polyamine levels are likely to be associated with the accumulation of a specific metabolite of selenium. The relevance of these findings to elucidation of the biological activities attributable to various forms of selenium is under investigation.     

Increase in ornithine decarboxylase activity and polyamine levels of the rat liver during an early period of hepatocarcinogenesis

The ornithine decarboxylase activity and the polyamine content in the fraction of plasma membranes and cytosol of the rat liver are studied in the early period (1-4 weeks) of nitrosodiethyl amine-induced hepatocarcinogenesis. The enzyme activity and polyamine levels in the cytosol of the rat liver cells are found to increase sharply during the first month of the disease, the maximum being observed the first-second week. By the end of the fourth week these indices become lower but they remain significantly higher than the normal levels. The polyamine level increases considerably in the fraction of plasma membranes with the maximum observed the second week.     

Changes of ornithine decarboxylase activity and polyamine content upon differentiation of mouse NB-15 neuroblastoma cells

The possible functions of ornithine decarboxylase (ODC) and polyamines in the differetiation of mouse NB-15 neuroblastoma cells were investigated by examining the changes of these parameters in the differentiaton and nondifferentiating NB-15 cells over a 5-day culture period. Differentiation of NB-15 cells was induced by the addition of dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthin (IBMX) to the growth medium and was monitored by neurite outgrowth, increase of acetylcholinesterase (AChE), and R_I cAMP-binding protein. Plating of NB-15 cells in fresh serum-containing growth medium was accompanied by rapid growth and a marked increase of ODC activity, this early increase of ODC activity was attenuated, both in duration and in magnitude, in the differentiating cells. The spermidine content of the differentiating neuroblastoma cell was significantly lower than that of the nondifferentiating cells. In the fully differentiated neuroblastoma cells, the ODC activity and spermidine content were lower than that of the undifferentiated cells by approximately 15-fold and five-fold, respectively. Based on these results it is proposed that changes of polyamine metabolism may be of significance in the differentiation of mouse neuroblastoma cells.     

Effects of hyperprolactinemia on ornithine decarboxylase activity and polyamine levels in seminal vesicles of genetically prolactin-deficient adult dwarf mice

Prolactin (PRL) has been shown to exert many different actions in various biological systems. Polyamines are known to influence the growth and function of the seminal vesicles (SV). Furthermore, ornithine decarboxylase (ODC) is considered a key enzyme in the biosynthesis of polyamines and is regulated by PRL in certain target tissues. Adult Ames dwarf mice (df/df), genetically deficient in PRL, were used for this study. The experimental groups were as follows: Group 1, pituitary-grafted; Group 2, sham-operated; Group 3, castrated + testosterone propionate (TP)-treated (25 micrograms/mouse, 3 times/wk, s.c.) + grafted; and Group 4, castrated + TP as above. The animals were killed 40 days later, and polyamines and ODC activity in SV and liver were determined. Serum PRL, FSH, and testosterone (T) were also measured. In the grafted groups, there were significant elevations in serum PRL and FSH levels. In the gonad-intact, pituitary-grafted group, animals exhibited an elevation in plasma T levels, and similar levels were achieved in the castrated, androgen-replaced groups. In hyperprolactinemic mice, the weights of SV were significantly greater than in the corresponding control groups. The relative weights of the SV showed a similar pattern. An increase in ODC activity was observed in both SV and liver in hyperprolactinemic groups. In those animals in which serum T levels were held constant, an increase in the enzyme activity in SV was detected in hyperprolactinemic group whereas in liver, no significant difference was observed. Concentrations of polyamines in the SV were increased in hyperprolactinemic, castrated, TP-treated mice. The present results indicate that PRL can exert a direct stimulatory effect on the growth, ODC activity, and polyamine levels in the SV.     

Steroid regulation of kidney histidine decarboxylase and ornithine decarboxylase levels in mouse kidney: effects of the mutation testicular feminization, Tfm

1. Testosterone represses kidney histidine decarboxylase levels in both normal male and female mice. Tfm/Y mutant mice lack an androgen receptor and are phenotypically female. It has been suggested that the testosterone induction of HDC levels in these animals is a result of aromatisation to oestrogens in the absence of the androgen receptor; the oestrogens then induce the enzyme. 2. It is shown that the induction of HDC in Tfm/Y mice is specific to testosterone and not other androgens and can be mimiced by low doses of beta-oestradiol in normal female mice. 3. Analysis of Tfm/+ mice indicates that the testosterone induction effect is a function of individual kidney cells.     

Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

A new perspective on ornithine decarboxylase regulation: prevention of polyamine toxicity is the overriding theme

The polyamines are essential cellular components for growth. Control of a key regulated enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), as a function of growth, is an area of intense interest. A unique regulatory property of ODC is the short half-life of the protein, which has been suggested to be an important factor in rapid activation of polyamine biosynthesis after cells are mitogenically stimulated. In this paper, it is argued that the biological significance of the short half-life of ODC is unrelated to the rate of its induction to a new steady state by growth factors, which is in fact limited by the relatively long half-life of the ODC mRNA. Instead, I suggest that the rapid turnover of ODC protein becomes of significance when cells cease growth and expeditious downregulation of the enzyme is important in preventing polyamine overproduction, which would result in cytotoxicity in the arrested cells. Although mitogenic activation of ODC expression has been studied extensively, there is very little known about the mechanisms controlling downregulation of polyamine biosynthesis during the arrest of animal cell growth. These considerations suggest that this would be a fertile area of future inquiry.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Effect of lithium chloride on ornithine decarboxylase activity in rat adrenal.

The effects of lithium chloride on ornithine decarboxylase (ODC) activity were compared in the adrenal and kidney of control (saline treated) and prolactin-treated rats. ODC activity was decreased in kidney of both groups of animals, the magnitude of the effect of lithium in the hormone-treated group varying with the time of administering the lithium relative to prolactin. The response in the adrenal was quite different. Following treatment with LiCl, there was a gradual increase in ODC activity from a low of 10-35 pmol CO2 x 30 min-1.mg protein-1 in control animals to values 20- to 30-fold greater at 5 h. In rats treated simultaneously with LiCl and prolactin, ODC activity was greater at 5 h than that observed in animals receiving either compound alone, indicating that their effects were additive. When LiCl was given 4 h after prolactin, i.e., 1 h before sacrifice, ODC activity decreased to a very low level at 5 h, as in other tissues. The increase in ODC activity in the adrenal following LiCl is of the same magnitude as the changes observed in tissues stimulated to undergo alterations in proliferation, differentiation, or metabolic or membrane activity by hormones and other external stimuli.