Two-step purification of mouse kidney ornithine decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

Essential role for polyamine biosynthesis in thyroxine stimulated pancreatic development in neonatal rats.

Administration of thyroxine to rat pups leads to precocious development of the pancreas. The role of ornithine decarboxylase (ODC) and polyamines in thyroxine-induced pancreatic maturation was examined. Rat pups (aged 5 days) were given daily subcutaneous injection of thyroxine (0.1 micrograms/g body wt.) until the day before death. Serial ODC activities were measured in pancreatic homogenates after 1, 2, 3, 4, 5, 6, 7 and 10 days of thyroxine treatment. There was a biphasic induction of ODC activities by thyroxine: an early peak appeared on day 2 of treatment followed by a decrease on day 4; a second peak was evident on day 5 and then a decrease to control values by day 7. Significant increases in tissue concentrations of putrescine and spermidine were observed concomitant with two peaks of ODC activity. Pancreatic amylase concentration, DNA and protein also showed a significant increase after thyroxine treatment. Difluoromethyl ornithine (DFMO), a specific ODC inhibitor, given orally (8% in drinking water) to nursing dams at postnatal day 5 for 5 days caused an 83% inhibition of pancreatic ODC activity in thyroxine-treated pups when compared to thyroxine-treated pups not exposed to DFMO. Concomitantly, the thyroxine-induced increases in pancreatic weight, protein and amylase activity were suppressed. Our results suggest that increases in ODC activities and polyamine levels are critical intermediary steps in the precocious induction of pancreatic development by thyroxine.     

Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.     

Two-Step Purification of Mouse Kidney Ornithine Decarboxylase

Abstract

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4. 1. 1. 17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1. 0 × 10⁶?1. 4 × 10⁶ nmol/h. mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54, 000 and a minor band of Mr 51, 000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [¹⁴C)difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Ornithine decarboxylase activity in developing rat brain

—Total ornithine decarboxylase (ODC) (EC 4.1.1.17) activity per rat brain was elevated markedly from 14 days after conception to 12 days postnatum. ODC activity in the brainstem was very low and changed little during postnatal development. Activity in the cerebral hemispheres declined from a high level at birth to the low adult level by 8 days postnatum. Conversely activity in the cerebellum increased markedly from 3 days until 11 days postnatum, then suddenly decreased. Hence, the periods of greatest ODC activity paralleled those of maximal cell proliferation in each brain region. During perinatal brain development ODC activity changed considerably; it declined at about one day prior to term, and then increased rapidly to its highest level of activity at 4 h postnatum. Premature birth by caesarian section or lack of maternal care and nutrition did not affect this early postnatal response. The postnatal burst in ODC activity appears to be unique for brain tissue, since this response did not occur in heart, skeletal muscle or liver. Data from studies in which portions of fractions characterized by high or low enzymatic activity, respectively, were mixed or in which the supernatant enzyme fraction was dialysed are not consistent with the presence of direct inhibitors or activators of the enzyme. In addition, administration of cycloheximide to newborn rats abolished the 4-h postnatal burst in ODC activity. Our results suggest that the increase in ODC activity reflects enzyme synthesis de novo.     

Physiological consequences of early neonatal growth retardation: effects of alpha-difluoromethylornithine on renal growth and function in the rat

The physiological consequences of early neonatal growth retardation in the kidney were investigated using alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines. We administered by s.c. 500 mg/kg/day DFMO, or saline, to Sprague-Dawley rat pups from the day of birth through postnatal day (PD) 6 and evaluated renal function on PD 4, 7, 10, and 13 using tests of basal renal clearance and urinary concentrating ability. Kidney weights and gross pathology were also obtained. On PD 39, serum chemistries and organ weights were determined. In a second experiment, we evaluated concentrating ability on PD 7-10, and basal renal function, concentrating ability, diuretic response, serum chemistries, and organ weights on PD 132-140. DFMO selectively inhibited renal growth but did not inhibit glomerular and tubular functional maturation. In fact, the rates of filtration and reabsorption (per g renal tissue), and concentrating ability were increased in treated pups. These changes were associated with long-term effects on renal function, including uremia, glucosuria, and male-specific concentrating deficits in adulthood. Several hypotheses can be developed concerning the physiological mechanisms underlying these changes (e.g., altered renal urea metabolism), which in turn may reflect either a direct role of ODC in the regulation of maturation or secondary consequences of inhibition of ODC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of nucleic acid and protein synthesis in developing brain, kidney, and heart of the neonatal rat: effects of alpha-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase

Ornithine decarboxylase (ODC) and the polyamines are thought to play a role in maturation of mammalian tissues. Daily postnatal administration of alpha-difluoromethylornithine (DFMO, a specific inhibitor of ODC) to newborn rats caused organ-specific deficits in tissue weight gain, with brain and kidney as the major targets. Subnormal organ weights were associated with deficits in the levels of nucleic acids and proteins in the affected tissues, and examination of the synthetic rates of DNA ([3H]thymidine incorporation), RNA ([3H]uridine incorporation) and protein ([14C]leucine incorporation) confirmed that macromolecule synthesis was inhibited in DFMO-treated pups. The time of onset of effect of DFMO on the synthesis of nucleic acids and proteins was the same as that reported for depletion of polyamines by this treatment. Potential adverse effects of DFMO on cell survival were also assessed by labeling DNA with [3H]thymidine on day 3 and examining retention of label 12 days later; DFMO did not cause an increase in cell death. In contrast to the sensitivity of brain and kidney to postnatally administered DFMO, development of cardiac tissue was relatively resistant to growth inhibition despite polyamine depletion. The organ specificity of effect of DFMO results, in part, from the different timetables for cellular events in tissue development displayed by each organ type; administration of DFMO earlier in development (during days 15 to 17 of gestation) did produce deficiencies in cardiac growth and nucleic acid levels similar to those which had been seen for brain and kidney. These data support the view that polyamines play a key role in cell replication, differentiation and growth during critical periods of mammalian organ development through their regulation of DNA, RNA, and protein synthesis.     

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-α)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCδ) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKδ pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.     

Putative ornithine decarboxylase activity in Arabidopsis thaliana: inhibition and intracellular localisation

In this work we studied putative ornithine decarboxylase activity (ODC, EC 4.1.1.17) in leaves of Arabidopsis thaliana L. (ecotype Columbia) plants at non-flowering stage (about 21 d of culture). Putative ODC activity was higher in the particulate than in the soluble fraction and activity was pH-dependent, increasing linearly with the pH. Inclusion of 10 mM arginine in the assay showed that the incidence of ornithine transcarbamoylase activity (EC 2.1.3.3) accounted for about 35% in the particulate fraction, but that its contribution was negligible in the soluble fraction. Increasing concentrations of the irreversible inhibitor α-difluoromethylornithine (DFMO) progressively inhibited putative ODC activity with a 40% inhibition at 20 mM DFMO. Taking into consideration the incidence of ornithine transcarbamoylase activity, the total inhibition of putative ODC activity was of about 75%. Fractionation experiments permitted measurement of putative ODC activity in the nuclei- and chloroplast-enriched fractions. The assays performed on membranes and stromal fractions isolated from gradient purified chloroplasts showed that the enzyme activity was associated almost totally with the plastid membranes.     

Action of DL-alpha-difluoromethyl ornithine on Acanthamoeba culbertsoni.

The multiplication of A. culbertsoni in the peptone medium was not inhibited by 10-20 mM concentration of alpha-difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed by 10-20 mM DFMO in proteose peptone, yeast extract, glucose (PYG) medium. Ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO, excluding enzyme refractoriness and impermeability of cells for DFMO as the possible causes of DFMO resistance. The presence of polyamines in the peptone and PYG media as well as uptake of polyamines by the amoebae has been demonstrated. The growth and multiplication of A. culbertsoni in chemically defined medium was not affected by 1-5 mM DFMO while 10-20 mM DMFO yielded partial inhibition. A lowering of diaminopropane levels and enhancement of spermidine levels was observed in DFMO inhibited cells and level of ODC was drastically reduced in the inhibited cultures. Uptake of polyamines from the growth media may partly account for DFMO resistance of A. culbertsoni. Alternative mechanisms for DFMO resistance are indicated.     

Comparison of 3-isobutyl-1-methylxanthine-induced accumulation of putrescine in rat pancreas and liver

3-Isobutylmethylxanthine (IBMX), a potent phosphodiesterase inhibitor, causes accumulation of putrescine of same magnitude in rat pancreas and liver. IBMX produces increases of acetyl CoA: polyamine N'-acetyltransferase (PAT) and of ornithine decarboxylase (ODC) activities in both organs. However ODC activity is 300 times higher in liver than in pancreas. In the latter organ, there is a transient increase of N1-acetylspermidine, followed by a decrease of spermidine, alpha-Difluoromethylornithine (DFMO), a potent ODC inhibitor, impairs the accumulation of putrescine in liver but not in pancreas. These results suggest that in pancreas the accumulated putrescine is essentially formed from spermidine, via N1-acetylation and oxidation, while in liver it is formed from decarboxylation of ornithine. A possible involvement of cAMP in the stimulation of the polyamine interconversion pathway is discussed.     

Activities of ornithine aminotransferase and ornithine decarboxylase in chronically uremic rats

The activities of two enzymes mediating different pathways of ornithine catabolism were measured in liver and kidney of chronically uremic rats and their pair-fed controls. Two months following partial nephrectomy hepatic ornithine aminotransferase (OAT) activity tended to be lower in uremic rats and was correlated with urea clearance and with carbamoyl phosphate synthetase activity. Renal OAT activity in uremic rats was also correlated with urea clearance. When uremic rats were maintained for five months, OAT activity was significantly decreased in liver but not in kidney and the activity of ornithine decarboxylase (ODC), the enzyme regulating polyamine biosynthesis, was reduced in both liver and kidney. In cross-over experiments, evidence was obtained for a factor in uremic kidney cytosol which inhibited renal ODC activity.     

Maturation of growth hormone stimulation of kidney ornithine decarboxylase in the rat

The effect of ovine growth hormone (GH) on kidney ornithine decarboxylase (ODC) was studied in newborn, preweanling and young adult rats. Basal kidney ODC activity was very low from 4 to 22 days after birth but rose 20-fold by day 25; it remained elevated through day 45. GH failed to stimulate ODC in the first two weeks after birth. GH did however stimulate ODC markedly from 20 through 45 days. Kidney ODC was stimulated in the neonate by vasopressin and by isoproterenol, but not angiotensin II. Liver ODC remained relatively low and stable during development, and was responsive to GH at all ages studied. We conclude that a) the pattern of development of basal kidney ODC appears to be unique to this tissue and may be related to the postnatal maturation of renal morphology and/or function, b) neonatal kidney ODC is unresponsive to certain hormones but is not completely refractory to stimulation. These findings may have implications for the role of hormones in the maturation of the kidney and in the regulation of early renal function.     

Inhibition of the differentiation of 3T3-L1 cells by interferon-beta and difluoromethyl ornithine

Both mouse interferon-beta (MuIFN-beta) and the inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethyl ornithine (DFMO), inhibited the differentiation of mouse 3T3-L1 fibroblasts into adipocytes in a dose-dependent manner. DFMO and MuIFN-beta added together to cultures that were induced to differentiate produced an additive anti-differentiation effect. In contrast to this additive cellular effect, DFMO reduced the antiviral activity of MuIFN-beta in both undifferentiated and differentiated cells; DFMO alone had no detectable effect on replication of encephalomyocarditis virus. Putrescine, the product of ornithine decarboxylation, when added to 3T3-L1 cultures (i) enhanced differentiation, (ii) reversed completely the inhibition of differentiation by DFMO, but (iii) had little effect on the antidifferentiation effect of MuIFN-beta. Polyamine content changed four-fold or less in cultures treated with 0.5 mM DFMO and less than two-fold in cultures treated with 100 IU/ml MuIFN-beta for seven days. Thus, it appears not only that MuIFN-beta and DFMO inhibit differentiation of 3T3-L1 cells by different mechanisms but also that the antiviral action of IFN does not involve the regulation of polyamine metabolism by ornithine decarboxylase.     

Ovarian function in the rat following irreversible inhibition of L-ornithine decarboxylase

The possibility that the transient rise in rat ovarian ornithine decarboxylase (ODC) activity and the associated increase in putrescine which occur under luteinizing hormone control late in proestrus have an essential role in ovarian function has been tested using DL-α-difluoromethylornithine (DFMO) an irreversible inhibitor of ODC. Treatment with DFMO, 500 mg/kg s.c. at 12:00 h on the day of proestrus and 200 mg/kg, 6, 12 and 18 h thereafter completely suppressed both the rise in ovarian ODC activity and the associated increase in putrescine concentrations. However, ovulation took place normally under these conditions and the course of the resulting pregnancies was also normal. Similarly, combined treatment with DFMO and an inhibitor of S-adenosyl-L-methionine decarboxylase, 1,1-((methylethanediylidine)-dinitrilo) bis (3-aminoguanidine), 25 mg/kg, given at 10:00 h on the morning of proestrus failed to influence either ovulation or the subsequent period of gestation. These data provide no support for a functional role of the pre-ovulatory rise of ODC in rat ovary in the major peri-ovulatory events of that particular cycle, although they do not exclude effects on systems (e.g. steroidogenesis) not directly examined with our experimental approach. On the other hand, inhibition of ODC resulted in an increase in the number of uterine implantation sites following mating at the next proestrusestrus 4 days later. These data would support the previously expressed view that ODC may be associated with the gonadotrophin-induced initiation of follicular development for ovulation in the succeeding cycle. Moreover, since inhibition of the enzyme resulted in facilitation of the process, the normal physiological function of ODC, and/or the putrescine generated through its action, would appear to be inhibitory.     

Ornithine decarboxylase, transglutaminase, diamine oxidase and total diamines and polyamines in maternal liver and kidney throughout rat pregnancy.

Ornithine decarboxylase (ODC; EC 4.1.1.17), transglutaminase (EC 2.3.2.13), diamine oxidase (DAO; EC 1.4.3.6) and total di- and poly-amines were studied in rat liver and kidney cortex throughout pregnancy. In liver, ODC activity exhibited two major peaks (4.5-5 times the control activities) on days 15 and 17. Also putrescine and spermidine increased biphasically (3-4-fold), but no variation in spermine content was observed. Transglutaminase activity showed slight variations only near the end of gestation. In kidney, ODC activity did not fluctuate significantly during pregnancy, whereas both transglutaminase activity and putrescine content showed three major increases, in very early, middle and late pregnancy. No significant variations in spermidine and spermine were observed. In both organs, DAO activity, very low or undetectable until day 10, dramatically increased (10- and 20-fold in kidney and liver respectively) in the second half of pregnancy, reaching maxima on days 16-17 and 19. The results obtained for transglutaminase, ODC and total di- and poly-amines are interpreted on the basis of hyperplastic and hypertrophic events in the liver and kidney respectively. The behaviour of DAO suggests that the enzyme plays an important role in the control of intracellular diamine concentration.     

Involvement of the ornithine decarboxylase pathway in macrophage collagenase production

Definition of the cellular events involved in the production of collagenase by macrophages following activation has revealed prostaglandin E2 (PGE2)- and cAMP-dependent steps. Since ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is regulated by cAMP and is associated with certain aspects of protein synthesis, the potential role of this enzyme and its polyamine product, putrescine, in collagenase synthesis was examined. Lipopolysaccharide (LPS) activation of macrophages resulted in a maximal ODC response after 6 to 9 h with a 10- to 12-fold elevation in enzyme activity. This elevation in ODC appeared to be regulated by PGE2 since indomethacin inhibited LPS-induced macrophage ODC levels by 70%. Associated with the indomethacin-mediated inhibition of ODC was a loss of collagenase synthesis. Furthermore, partial restoration of collagenase production in indomethacin-inhibited cultures could be achieved by the addition of putrescine. In additional studies alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, also inhibited collagenase production when added to LPS-treated macrophages. This inhibition by DFMO could be reversed by the exogenous addition of putrescine. These findings demonstrate that the ODC pathway is an important intracellular component in the sequence of events that lead to macrophage collagenase synthesis.     

Gastrin and epidermal growth factor induction of ornithine decarboxylase in rat colonic explants

An organ culture system was utilized to examine the effect of gastrin (G-17-I) and epidermal growth factor (EGF) on colonic mucosal ornithine decarboxylase (ODC) activity, and the expression of the ODC gene. Exposure of colonic mucosal explants to either gastrin or EGF (50-500 ng/ml) for only 4 h resulted in a profound stimulation (150-600%) in ODC activity over the basal level. These increases were essentially abolished by difluoromethylornithine (DFMO; 2 nmol/ml) or CaCl2 (2 umol/ml). Gastrin also activated the ODC gene in the colonic mucosa as evidenced by increased steady-state ODC mRNA levels in the colonic mucosal explants after 4 h exposure to the hormone, when compared with the controls. It is concluded that colonic mucosal ODC is responsive to both gastrin and EGF.