Methyl-acetylenicputrescine (MAP), an inhibitor of polyamine biosynthesis, reduces the frequency and cytolytic activity of alloantigen-induced LyT 2.2 positive lymphocytes in vivo

The objective of the present investigation was to examine the effect of (2R,5R)-6-heptyne-2,5-diamine (methyl-acetylenicputrescine; MAP), an irreversible inhibitor of ODC, on the induction of alloreactivity in vivo. Treatment of mice with MAP (0.5-0.01% in drinking water) inhibited CTL induction in a dose-dependent manner with an IC50 of approximately 144 mg/kg/day. MAP treatment reduced the frequency of LyT2+ (cytolytic/suppressor) splenic lymphocytes by greater than 75%. In contrast, MAP did not alter the number of L3T4+ (helper/inducer) lymphocytes. MAP treatment reduced lymphocyte putrescine and spermidine levels by 61 and 40%, respectively. The inhibitory effect of MAP on CTL induction could be reversed by simultaneous administration of putrescine (500 mg/kg). These data indicate that the observed inhibitory effect of MAP on CTL induction is mediated through inhibition of polyamine biosynthesis. Furthermore, results of the present investigation suggest that inhibition of polyamine biosynthesis may provide a unique target for immunosuppression.     

Immunosuppressive effects of (2R,5R)-6-heptyne-2,5-diamine, an inhibitor of polyamine synthesis: II. Beneficial effects on the development of a lupus-like disease in MRL-lpr/lpr mice.

(2R,5R)-6-heptyne-2,5-diamine (MAP; MDL 72175), a potent irreversible inhibitor of L-ornithine decarboxylase (ODC), possesses immunosuppressive activities in vitro as the result of inhibition of lymphocyte polyamine biosynthesis. The effects of MAP were now studied in vivo in MRL-lpr/lpr female mice, an animal model for human systemic lupus erythematosus (SLE). Administration of MAP (0.2% in drinking water; drug intake: 0.25-0.35 g/kg body weight/day) to female mice for 15 weeks, starting 8 weeks after birth, reduced by 47% the number of spleen cells, retarded development of lymphadenopathy and, at that time, markedly prolonged the survival of the mice. At week 23, MAP reduced plasma IgG concentrations by 50% whereas, in contrast, those of IgM were elevated 1.5-fold. No statistically significant effects of MAP were observed on plasma levels of anti-DNA autoantibodies although serum anti-RNP and anti-Sm titres tended downwards during treatment. Neither glomerular lesions nor proteinuria were improved by MAP administration. Finally chronic administration of MAP for 45 weeks prolonged the median survival time from 29.75 to 35.5 weeks.     

Identification of polymorphisms and balancing selection in the male infertility candidate gene, ornithine decarboxylase antizyme 3

Background  

The antizyme family is a group of small proteins that play a role in cell growth and division by regulating the biosynthesis of polyamines (putrescine, spermidine, spermine). Antizymes regulate polyamine levels primarily through binding ornithine decarboxylase (ODC), an enzyme key to polyamine production, and targeting ODC for destruction by the 26S proteosome. Ornithine decarboxylase antizyme 3 (OAZ3) is a testis-specific antizyme paralog and the only antizyme expressed in the mid to late stages of spermatogenesis.     

Spermidine synthase is required for virulence of Leishmania donovani

Genetic lesions in the polyamine biosynthetic pathway of Leishmania donovani, the causal agent of visceral leishmaniasis, are conditionally lethal mutations that render the insect vector form of the parasite auxotrophic for polyamines. Recently, we have demonstrated that a Δodc L. donovani null mutant lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was profoundly compromised in its ability to infect mice, indicating that ODC is essential for the infectious mammalian stage of the parasite and further validating the enzyme as a possible drug target. To assess whether other components of the polyamine biosynthetic pathway were also essential for parasite virulence, a cell line deficient in spermidine synthase (SPDSYN), the enzyme that converts putrescine to spermidine, was created by double-targeted gene replacement within a virulent L. donovani background. This Δspdsyn strain was auxotrophic for polyamines, required spermidine for growth in its insect vector form, and was adversely impacted in its ability to infect mice. These findings establish that SPDSYN, like ODC, is essential for maintaining a robust infection in mammals and indicate that pharmacologic inhibition of SPDSYN, and perhaps all components of the polyamine biosynthetic pathway, is a valid therapeutic strategy for the treatment of visceral and, potentially, other forms of leishmaniasis.     

Inhibition of polyamine biosynthesis in Crithidia fasciculata by D,L-alpha-difluoromethylornithine and D,L-alpha-difluoromethylarginine.

Using Crithidia fasciculata as a model organism for Trypanosoma cruzi, we have examined the effects of D,L-alpha-difluoromethylornithine (DFMO) and D,L-alpha-difluoromethylarginine (DFMA) on growth and polyamine synthesis. In a defined, polyamine-free medium growth was markedly inhibited by DFMO (94% at 50 mM; IC50 = 37 mM) and to a lesser extent by DFMA (65% at 50 mM). Addition of putrescine, but not agmatine, reverses inhibition of growth, suggesting that the site of inhibition is ornithine decarboxylase (ODC). Consistent with this conclusion, DFMO or DFMA results in a complete loss of putrescine and significant reductions in intracellular spermidine, glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione). In addition, significant concentrations of DFMO (0.8 mM) were present in DFMA-treated cells. However, in contrast to other organisms, conversion of DFMA to DFMO is probably not catalysed by arginase. Substantial ornithine decarboxylase activity (63.1 pmol min-1 mg-1; ODC) was observed in control cells, sufficient to account for polyamine synthesis during growth. In addition, a trace arginine decarboxylase (ADC) activity (1.19 pmol min-1 mg-1) was found. Evidence is presented showing that the apparent ADC activity is actually due to the concerted action of arginase (1.5 nmol min-1 mg-1) and ODC. Thus DFMA appears to inhibit growth of C. fasciculata via conversion to DFMO and subsequent inhibition of ODC.     

Polyamine N-acetyltransferase from Fasciola hepatica.

A cytosolic polyamine N-acetyltransferase which catalyses polyamine and diamine acetylation has been partially purified from the liver fluke Fasciola hepatica. The enzyme has an apparent Mr of 50,000 and unlike the corresponding mammalian liver counterpart is capable of putrescine acetylation. Among the substrates tested, spermidine had the highest reaction rate but putrescine had a lower Km value. The Km values for spermidine, spermine, norspermidine, putrescine, cadaverine and 1,3-diaminopropane were 20 microM, 1.30 mM, 20 microM, 7 microM, 10 microM and 50 microM, respectively. Acetylated polyamines were also substrates for the trematode acetylase, but histones were inactive. The partially purified enzyme had no deacetylase activity. The Km for acetyl-CoA was 4.4 microM. Coenzyme A was strongly inhibitory with a Ki value of 5.3 microM. Bis(benzyl)polyamine analogue MDL 27695 was a potent competitive inhibitor of the enzyme with a Ki of 22 microM. Inhibition by 1,4-dimethyl-putrescine was non-competitive and had a Ki value of 15 microM. The trematode acetylase is highly dependent on sulfhydryl groups for its activity. As had been reported in nematodes, polyamine acetylation could represent a process by which trematodes convert excess polyamines to forms suitable for transport and excretion. On the other hand, this could be the regulatory step of a functional interconversion pathway in these parasites.     

Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum

Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC50) values determined for both strains revealed different inhibition profiles. The IC50 value for the chloroquine-sensitive NF54 strain was 97 microM and 501 microM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a significant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental effect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition profiles with 1,7-diaminoheptane resulted in an IC50 value of 466 microM for the NF54 strain and 319 microM for the R strain. Spermidine pools changed significantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC50 value of 431 microM and an IC50 value of 340 microM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the first evidence of homospermidine in P. falciparum. The use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.     

Role of ornithine decarboxylase antizyme inhibitor in vivo

Ornithine decarboxylase (ODC) antizyme inhibitor (AZI) has been shown to regulate ODC activity in cell cultures. However, its biological functions in an organism remain unknown. An embryonic stem (ES) cell clone was established, in which the Azin1 gene was disrupted by the gene trap technique. To identify the function of Azin1 gene in vivo , a mutant mouse line was generated using these trapped ES cells. Homozygous mutant mice died at P0 with abnormal liver morphology. Further analysis indicated that the deletion of Azin1 in homozygous mice resulted in the degradation of ODC, and reduced the biosynthesis of putrescine and spermidine. Our results thus show that AZI plays an important role in regulating the levels of ODC, putrescine and spermidine in mice, and is essential for the survival of mice.     

鸟氨酸脱羧酶/多胺系统在大鼠缺血预适应心肌保护中的作用

目的：探讨鸟氨酸脱羧酶（ODC）/多胺系统在缺血预适应（IPC）心肌保护中的作用。方法:应用离体灌流大鼠心脏复制模拟心肌缺血/再灌注模型。心脏随机分为6组：对照组 (control)、缺血/再灌注组 (IR)、弱缺血预适应组 (IPCw)、强缺血预适应组 (IPCs)、IPCw+多胺抑制剂组 (DF-EG-IPCw)和IPCs+多胺抑制剂组(DF-EG-IPCs)。免疫印迹法定量分析多胺合成限速酶鸟氨酸脱羧酶 (ODC)的表达;高效液相色谱测定心肌组织中的多胺（腐胺、精脒、精胺）含量;Powerlab多导生理记录系统记录心脏功能;氯化三苯基四氮唑 (TTC) 染色检测心肌梗死面积;TUNEL方法检测细胞凋亡率,比较其中的差异性。结果:（1）与对照组比,IR组ODC表达下调,腐胺含量增加,精胺含量减少,总多胺池减少（P<0.05）,此时心功能下降（LVDP、HR、CF均低于对照组,P<0.05）,心肌梗死面积及心肌细胞凋亡率增加（P<0.05）;（2）与IR组比,弱及强缺血预处理组（IPCw、IPCs）心肌ODC表达上调,腐胺含量减少,精胺含量增加,总多胺池增加（P<0.05或P<0.01）,此时大鼠心功能有明显改善（LVDP、HR、CF与IR组比,P<0.05）,心肌梗死面积及心肌细胞凋亡率均明显降低（P<0.01）;（3）给予多胺抑制剂后,心肌ODC表达,腐胺、精脒、精胺及总多胺池含量均明显降低（DF-EG-IPCw组 vs IPCw组;DF-EG-IPCs组vs IPCs组,P<0.05或P<0.01）,心功能明显下降（P<0.05）,心肌梗死面积及细胞凋亡率均明显增加（P<0.05）。结论:缺血预适应能明显上调大鼠心肌ODC/多胺系统,减轻缺血/再灌注心肌损伤;多胺合成代谢抑制剂取消了预适应介导的心功能改善、缩小心肌梗死面积及减少心肌细胞凋亡的作用,提示ODC/多胺系统可能参与了缺血预适应介导的心肌保护作用。     

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.     

Polyamines reduce oxidative stress in Escherichia coli cells exposed to bactericidal antibiotics

Bactericidal antibiotics (fluoroquinolones, aminoglycosides and cephalosporins) at their sublethal concentrations were able to produce hydroxyl radicals, hydrogen peroxide and superoxide anions (ROS) in Escherichia coli cells, which resulted in damage to proteins and DNA. The cells responded to oxidative stress by a 2-3-fold increase in cell polyamines (putrescine, spermidine) produced as a consequence of upregulation of ornithine decarboxylase (ODC). Relief of oxidative stress by cessation of culture aeration or addition of antioxidants substantially diminished or even completely abolished polyamine accumulation observed in response to antibiotics. Alternatively, inhibition of polyamine synthesis resulted in enhancement of oxidative stress in antibiotic-processed cells. When added to antibiotic-inhibited culture, polyamines reduced intracellular ROS production and thereby prevented damage to proteins and DNA. These effects eventually resulted in a substantial increase in cell viability, growth recovery and antibiotic resistance that were more strongly expressed in polyamine-deficient mutants.     

Attenuation of isoproterenol-mediated myocardial injury in rat by an inhibitor of polyamine synthesis.

OBJECTIVE: Ornithine decarboxylase (ODC) is an initial rate-limiting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine) that play a role in cell growth and differentiation. Recent studies have shown that spermidine and spermine cause injury to a variety of cells including myocytes in vitro. In this investigation, we used alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis to test the hypothesis that polyamines contribute to myocardial injury in rat. METHODS: Male Sprague Dawley rats were treated with (i) saline (0.2 ml/day, s.c.), (ii) isoproterenol (ISO) (5 mg/kg/day for 8 days, s.c.) to produce necrotizing myocardial injury, or with (iii) DFMO + ISO. DFMO was started 2 days before the initiation of ISO and both ISO and DFMO were continued until the end of the experimental period. Myocardial injury was assessed by determining the increased release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) into the plasma, and by morphometric analysis of the lesion area in heart sections stained with Gomori trichrome. RESULTS: ISO induced the release of CPK and LDH by 6 hr and 24 hr, respectively, and produced subendocardial necrosis, which was both acute and resolving following 8 days of ISO. DFMO treatment inhibited ISO-induced increases in (i) ODC activity and putrescine and spermidine levels in heart, (ii) CPK and LDH activity in plasma, and (iii) the area of subendocardial lesions. CONCLUSIONS: These observations suggest that polyamines are one of the intracellular factors that contribute to ISO-mediated cardiac injury in the rat.     

Evidence for a role of neosynthetized putrescine in the increase of glial fibrillary acidic protein immunoreactivity induced by a mechanical lesion in the rat brain

The effect of alpha-difluoromethylornithine (alpha-DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) the rate limiting enzyme of polyamine biosynthesis, was studied on the astroglial reaction in a model of mechanical brain injury. alpha-DFMO markedly decreased the astroglial activation induced by the microdialysis probe implantation in the striatum of the male rat, as studied by glial fibrillary acidic protein (GFAP) immunocytochemistry. This response was restored by putrescine (20 nmol/ml) administered via the microdialysis probe. These results suggest that the astroglial reaction and the polyamine biosynthesis activation induced by a localized mechanical lesion are causally linked phenomena.     

Polyamine metabolism in prostaglandin E2-treated human T lymphocytes

The effects of Prostaglandin (PG) E2 treatment of human T lymphocytes on polyamine metabolism were investigated. PGE2 is known to inhibit lymphocyte proliferation, while polyamines play an important role in several biochemical processes leading to increased cell growth. Preincubation of T lymphocytes with PGE2 (10^-6 M) for 10 min. was able to increase ornithine decarboxylase (ODC) activity and putrescine as well as spermine levels, while spermidine concentration was drastically reduced. After 30 and 60 min of treatment, a decrease in ODC activity and putrescine concentration was observed. On the contrary, the initial inhibition of sperrnine-NI-acetyl-transferase (SAT) activity was followed by a progressive increase of this catabolic enzyme. These changes were related to modifications of cAMP concentrations. Our data may help clarify the mechanisms underlying the biphasic effect of PGE2, which ultimately leads to inibition of cell proliferation.     

On the role of polyamines in bone resorption induced by parathyroid hormone

In order to elucidate the possible role of polyamines in the mobilization of mineral from long-term bone cultures stimulated with parathyroid hormone we have measured the activity of ornithine decarboxylase in osteoblasts, the levels of polyamines in calvarial bone and determined the effect of added polyamines and inhibitors of polyamine biosynthesis on calcium mobilization. Parathyroid hormone (10 nmol l-1) stimulated omithine decarboxylase activity by approximately 50% in both cultured bone cells of osteoblastic phenotype, UMR 106 and in mouse calvarial osteoblast-like cells. In mouse calvaria the levels of putrescine and spermidine were increased by parathyroid hormone after 24 hours. The levels of spermine were very low and were unchanged by parathyroid hormone. The two polyamine synthesis inhibitors alpha-difluoromethylornithine (DFMO; 2 mmol l-1) and methylglyoxal-bis-guanylhydrazone (MGBG; 50 mu mol l-1) did not significantly affect the mobilization of 45Ca from parathyroid hormone-stimulated bones. All three polyamines, putrescine, spermidine and spermine, inhibited the mobilization of 45Ca induced by parathyroid hormone in a dose-dependent manner. The inhibition induced by putrescine was reversible. In summary, we have shown that parathyroid hormone increases the accumulation of polyamines in bone, but the effect is small. Furthermore, inhibition of polyamine biosynthesis does not reduce parathyroid hormone-induced mineral mobilization and the addition of polyamines leads to a reduced rather than a stimulated mineral mobilization. Thus, polyamines do not seem to be critically involved in the changes in bone resorption induced by parathyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL-4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL-4-dependent E-cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL-4-induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1-deficient macrophages display rather enhanced IL-4-induced polyamine production, suggesting that an Arg1-independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS-induced expression of several proinflammatory genes was increased significantly in polyamine-depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL-4-induced expression of several AAM mediators, they inhibit the LPS-mediated expression of proinflammatory genes in CAMs.     

Effects of luminal stimuli on polyamine metabolism in the small intestine of the rat: the role of enteric nerves

The aim of this study was to investigate to what extent polyamine metabolism in the small intestine of the rat is controlled by the enteric nervous system. Polyamine metabolism was followed by measuring the activity of ornithine decarboxylase (ODC) and in some instances also the content of polyamines (putrescine, spermidine and spermine). ODC activity in the intestine was increased when intraluminal pressure was increased and 3 h after placing cholera toxin in the intestinal lumen. Cholera toxin also increased the tissue putrescine content. Atropine or hexamethonium given i.v. did not influence the evoked changes of ODC activity. The pressure induced changes were not decreased by placing lidocaine on the serosal surface. On the other hand, the ODC activity of control segments were decreased by hexamethonium or atropine. The presence of glucose in the intestinal perfusate did not augment tissue ODC activity, neither did the heat stable enterotoxin from Escherichia coli (STa). It is concluded that the effect on polyamine metabolism evoked by luminal pressure or cholera toxin seems not to be mediated via nerves, while nerves seem to influence ODC activity during control conditions. The experiments with enterotoxins suggest that cAMP is the intracellular second messenger controlling intestinal ODC activity.     

Hormonal regulation of ornithine decarboxylase and polyamines in primary cultured rat hepatocytes--differences in hormonal response between adult and fetal hepatocytes

Polyamines are polycationic substances which are widely distributed in living organisms and have a close relation to rapid growth phenomena. We examined ornithine decarboxylase (ODC), which is the rate limiting enzyme of polyamine biosynthesis, and polyamine induction in primary cultured rat hepatocytes by various hormones which increase during pregnancy, and revealed differences in hormonal responses between adult and fetal rat hepatocytes. Thirteen hormones, including estrone, estradiol, progesterone, testosterone, human chorionic gonadotropin (HCG), cortisol, dexamethasone, insulin, glucagon, epinephrine and epidermal growth factor (EGF), were tested. Among these hormones, only insulin, dexamethasone and EGF induced ODC activity and polyamine biosynthesis, especially that of putrescine, in both adult and fetal hepatocytes. The effects of EGF were the most significant. The combined effect of insulin and dexamethasone was additive, while that of insulin and EGF was synergistic. The rate of ODC induction was higher in adult hepatocytes than in fetal hepatocytes, however, the reaction was earlier in fetal hepatocytes. These observations suggest that ODC and polyamines in the fetal stage of development are regulated by a mechanism different from that in the adult liver.     

Polyamine transport and ornithine decarboxylase activity in hypoxic pulmonary artery smooth muscle cells.

Hypoxia causes remodeling of the pulmonary circulation that is dependent on increases in lungs polyamine contents. Mechanisms by which polyamines are regulated in hypoxic lung cells are unknown, but ornithine decarboxylase (ODC) activity, the initial enzyme in de novo biosynthesis, is depressed and polyamine transport is augmented in lungs from hypoxic rats (R.-T. Shiao et al. 1990. Am. J. Physiol. 259:L351-L358). To determine if hypoxia directly influences polyamine regulatory mechanisms in pulmonary vascular cells, we examined [14C]spermidine (SPD) transport and ODC activity in bovine main pulmonary artery smooth muscle cells (PASMCs) cultured under standard (culture medium Po2: greater than 100 mm Hg), "normoxic" (culture medium Po2: 50 to 70 mm Hg), or "hypoxic" (culture medium Po2: 18 to 30 mm Hg) conditions. Uptake of [14C]SPD in cells cultured under standard conditions was temperature- and concentration-dependent, exhibited saturation kinetics, and was abolished by metabolic inhibition. Modeling of transport according to Michaelis-Menten kinetics revealed that [14C]SPD uptake in cells cultured under standard conditions was characterized by Km and Vmax values of 0.78 microM and 4.5 pmol/min/10(6) cells, respectively. In comparison to cells cultured under standard conditions, Km was unaffected by culture under normoxic or hypoxic conditions while Vmax was increased to 18 pmol/min/10(6) cells in normoxic cells and to 33 pmol/min/10(6) cells in preparations cultured under hypoxic conditions. Inhibition of ODC with alpha-difluoromethylornithine (DFMO) also induced SPD transport, as evidenced by an increase in the Vmax to 65 pmol/min/10(6) cells. Both hypoxia- and DFMO-induced increases in [14C]SPD transport were suppressed by cycloheximide and actinomycin D, thus highlighting the importance of protein and RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of spermidine synthase from Trypanosoma brucei brucei

Spermidine synthase from Trypanosoma brucei brucei was characterized and found to be similar to spermidine synthase from other sources. The Km for putrescine was found to be 0.2 mM and the Km for decarboxylated S-adenosylmethionine 0.1 microM. The approximate molecular weight of the enzyme was 74 000 as determined by a combination of molecular sieve chromatography and sucrose density gradient centrifugation. Spermidine synthase activity was markedly inhibited in vitro by dicyclohexylamine (50% inhibition at 3 microM) and cyclohexylamine (50% inhibition at 15 microM); both being competitive inhibitors with respect to putrescine. S-Adenosyl-1,8-diamino-3-thiooctane, a nucleoside bisubstrate analog, was also a potent inhibitor of enzyme activity (50% inhibition at 25 microM). Administration of dicyclohexylamine to mice with trypanosomiasis resulted in no increase in survival time probably due to the lack of effect on trypanosome spermidine concentrations. Other possible inhibitors remain to be tested in vivo.