Melanocyte-Stimulating Hormone Release-Inhibiting Factor-1 (MIF-1) Can Be Formed from Tyr-MIF-1 in Brain Mitochondria but Not in Brain Homogenate

Abstract: Two samples of the peptide tyrosine-melanocyte-stimulating hormone release-inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH₂) were tritiated on different amino acids (Tyr or Pro) and incubated together at 37°C with fractions of rat brain. The amount of intact tetrapeptide remaining was determined by HPLC. By 3 min, most of the Tyr-MIF-1 was degraded. Because similar amounts of [³H]Pro and [³H]Tyr appeared after incubation of the Tyr-MIF-1 peptides in brain homogenate, even as early as 30 s, examination of only this crude preparation would misleadingly indicate that Tyr-MIF-1 is not a precursor of melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1; Pro-Leu-Gly-NH₂) in brain tissue. However, incubation of the mitochondrial fractions of brain under the same conditions resulted in more than three times as much [³H]Tyr being formed as [³H]Pro, with accompanying accumulation of MIF-1. Addition of excess MIF-1 to the mitochondrial fraction completely suppressed the formation of MIF-1 and more than doubled the amount of Tyr-MIF-1 remaining intact. When Tyr-MIF-1 tritiated only on the Tyr was added to the mitochondrial fraction, the main peaks of radioactivity appeared only at the positions of Tyr and Tyr-MIF-1, not at the position of Tyr-Pro. The results indicate that Tyr-MIF-1 can serve as a precursor of MIF-1 in brain mitochondria, an effect not evident when crude brain homogenate is used.     

Regional differences in peptide degradation by rat cerebral microvessels: a potential novel regulatory mechanism for communication between blood and brain

The blood-brain barrier (BBB), composed of the microvessels of cerebral capillary endothelial cells, regulates the passage of peptides into the brain in several ways, mainly by saturable transport or passive diffusion. Here we describe an additional mechanism by which this regulatory function can occur. Cerebral microvessels were isolated from different regions of the brain and incubated with the mu-opiate selective endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) or the opiate-modulating Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), both tetrapeptides selectively tritiated at the Pro. Degradation was determined by HPLC. For both peptides, the metabolism by microvessels from the cerebral cortex was much greater than that by microvessels from the hypothalamus or pons. For endomorphin-1, the least degradation was in the pons; for Tyr-MIF-1 there was no difference in metabolism by microvessels from the pons or hypothalamus. The results show a novel mechanism at the BBB by which the BBB can selectively regulate the activity of different peptides in different regions of the brain.     

In vitro quantitative study of the degradation of endomorphins

The catabolism of the endomorphins was investigated in detail. The endomorphins were degraded relatively slowly in the rat brain homogenate (t1/2(endomorphin-1)=4.94 min; t1/2(endomorphin-2)=3.81 min). The inhibition of metalloproteases and aminopeptidases stabilised the endomorphins to the greatest extent. The digestion of endomorphins tritiated specifically on Tyr(1), Pro(2) or Phe(3) established also that only the aminopeptidase pathways were essential for inactivation of the endomorphins, and that the tetrapeptides were degraded by cleavage of the Pro(2)-Trp(3) or Pro(2)-Phe(3) bond. The end-products of the catabolism were amino acids; the fragments Tyr-Pro-OH and Pro-Trp-Phe-NH2 were present as intermediates. Metabolites produced by brain carboxypeptidases were not detected.     

The Metabolism of Serum Proteins in Neonatal Rabbits

1. Incorporation of S³⁵-labeled amino acids into serum proteins has been studied in neonatal and developing rabbits. It was found that, per unit weight, neonatal rabbits synthesized only about 1/36 of the gamma globulin, 1/7 of the beta globulin, ½ of the alpha globulin, and ⅛ of the albumin that an adult synthesized. The growing rabbit developed the ability to synthesize various serum proteins at different times. 2. Plasma volumes and serum protein concentrations were determined at different times during the growth period of the rabbit. Plasma volumes were found to be 1 and ½ times larger in newborn animals than in adults, with a gradual decline to the adult level. The total serum protein concentration at birth was about 60 to 65 per cent of the adult value and gradually increased with growth as the plasma volume decreased. 3. Half-lives of homologous albumin and gamma globulin were studied. The half-life of albumin in neonates was nearly twice as long as the half-life in adults, the latter value being reached at 1 month of age. The half-life of gamma globulin in neonates was more than twice as long as the half-life in adults and reached adult values at 2 to 3 months. 4. Attempts were made to alter serum protein metabolism. Gamma globulin synthesis early in life was augmented with antigen injections.     

Degradation of cholecystokinin octapeptide,related fragments and analogs by human and rat plasma in vitro

The rate of degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma was investigated, using high pressure liquid chromatography for the separation and identification of the degradation products.CCK tetrapeptide showed a half-life of 13 min in human plasma while its cleavage in rat plasma occurred at a very high rate (half-life of less than 1 min).The kinetics of disappearance of both sulphated and unsulphated CCK-8 indicated more than a single rate of degradation; the faster degrading system showed a half-life of 18 min for unsulphated CCK-8 and of 50 min for the sulphated peptide in human plasma as compared respectively with 5 and 17 min in rat plasma. The cleavage of CCK-8 was inhibited by bestatin and puromycin, suggesting that aminopeptidases play a major role in the breakdown of this peptide.CCK-9 analogs were rapidly converted into their corresponding octapeptides (half-life of 2.7 min in human plasma). This conversion was inhibited by puromycin and bestatin, suggesting the participation of aminopeptidase(s) probably specific for basic amino acids.CCK decapeptide exhibited a greater stability than the nonapeptides (half-life of 30 and 45 min in human and rat plasma respectively) and also gave rise to CCK-8 as degradation product. This cleavage was not affected by aminopeptidase inhibitors but was decreased by aprotinin (Trasylol®), suggesting that trypsin-like and/or kallikrein-like enzyme(s) were involved in the plasma metabolism of this peptide.     

Effect of diphenylhydramine on the Tyr-MIF-1 antinociception in rats.

Tyr-MIF-1 is a representative of the MIF's family of endogenous peptides. It has been isolated from bovine hypothalamus and human parietal cortex that suggests its involvement in nociception. Tyr-MIF-1 can bind to the mu-receptors as well as to its specific non-opiate receptors in the brain. Data in the literature rise the idea that histamine (HA), a well known nociceptive agent, and Tyr-MIF-1 might have a common pathway in their effects on nociception. We tested that possibility by investigation of the combined action of diphenhydramine (DPH, an H (1) -antagonist) and Tyr-MIF-1 on nociception. The changes in the nociceptive effects were examined in the male Wistar rats by the Randall-Sellito paw-pressure (PP) and the tail-flick (TF) tests. Tyr-MIF-1 in a dose of 1 mg/kg exerted strong naloxone-reversible analgesic effects. DPH (100 microg/kg, i.p.) had an antinociceptive action, too. The co-administration of Tyr-MIF-1 and DPH enhanced the antinociceptive effect, as compared to DPH (PP) and to TYR-MIF-1 alone (TF). These effects were reversed when methylene blue (MB, 500 microg/rat) was applied 1h before the combination. However, naloxone (1 mg/kg, i.p.) only slightly affected the antinociceptive effect of DPH and TYR-MIF-1, compared to that of MB. The results obtained confirmed the hypothesis that cyclic nucleotides are involved in the realization of nociceptive effects of both HA and Tyr-MIF-1.     

Effects of neonatal treatment with Tyr-MIF-1 and naloxone on the long-term body weight gain induced by repeated postnatal stressful stimuli

Stressful stimuli repeatedly applied during the first postnatal weeks can induce body weight gain in the mouse during adulthood. This effect can be prevented by injecting naloxone concomitantly with stress. The peptides belonging to the Tyr-MIF-1 family have a great modulating activity on numerous stress-induced phenomena. The aim of the present work was to compare the effect of repeated neonatal injections of Tyr-MIF-1 or naloxone on the long-term body weight gain induced by a stressing procedure applied daily during the first three weeks of life. The results indicate that although naloxone blocked the development of the stress-induced effects, Tyr-MIF-1 potentiated them.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Developmental regulation of rat lung Cu,Zn-superoxide dismutase.

In the present investigation we found that lung Cu,Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu,Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu,Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu,Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu,Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu,Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu,Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.     

Ontogeny of B-lymphocyte function. VII. Effect of cyclic AMP on the heterogeneity of the antibody produced by B lymphocytes from immature donors.

Liver cells from neonatal mice were incubated for 1 hr with or without cyclic AMP before they were used as the source of B lymphocytes to reconstitute lethally irradiated mice. All recipients also received 1 × 10⁸ adult thymus cells and were immunized with DNP-BGG. Mice reconstituted with neonatal B cells which had been incubated with cyclic AMP produced an anti-DNP response which was of greater magnitude, higher affinity and greater heterogeneity of affinity than that produced by mice reconstituted with neonatal B cells incubated without cyclic AMP. The possibility that cyclic AMP plays a role in the differentiation of B-cell function is suggested.     

Brain peptide reverses effect of morphine on human lymphocytes

E-rosette formation by human lymphocytes incubated with sheep red blood cells (sRBC) is inhibited by morphine. We studied the ability of the opiate antagonists naloxone and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) to block this action. Active E-rosette formation by lymphocytes incubated with morphine was reduced from the control of 35.7±1.7% to 23.7±1.5% (p<0.001). Similarly, total E-rosette formation was reduced by morphine from the control of 65.8±1.3% to 53.2±2.9% (p<0.001). These effects were blocked by co-incubation of the lymphocytes with either Tyr-MIF-1 or naloxone (p<0.05). Tyr-MIF-1 was active (p<0.05) at concentrations as dilute as 10⁻¹³M. These results indicate that the neuropeptide Tyr-MIF-1 exerts an antiopiate effect at the human T-lymphocyte.     

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80-150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.     

Developmental regulation of RNA transcript destabilization by A + U-rich elements is AUF1-dependent

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t((1)/(2))) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t((1)/(2)) of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3'-untranslated region (3'-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitro decay of the GM-CSF 3'-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3'-UTR are generated that are truncated at either end of the ARE. Furthermore, the t((1)/(2)) of the ARE-containing 3'-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3'-UTR in vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.     

Retardation of fetal brain cell growth during maternal starvation: Circulating factors versus altered cellular response

Maternal starvation inhibits fetal brain development during late gestation in the rat. To determine whether intrinsic or extrinsic factors might be the principal contributor to altered growth, brain cells from 20 day fetuses were cultured in a 96 well plate with MEM and 10% adult rat serum. Tissue growth was monitored by spectrophotometric measurement of the mitochondrial reduction of a chromagen 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT). After 1, 4 or 6 days incubation, MTT activity in non confluent cultures was shown to be directly related to tissue mass. When fetal brain cell cultures were incubated with 1% and 10% concentrations of adult rat serum, an 11-fold increase in MTT activity paralleled a 15-fold increase in tritiated thymidine incorporation. The impact of maternal starvation on fetal brain cell growth was examined by measuring MTT activity in fetal brain cells from fed and starved mothers. When cultures were incubated for 6 days with graded concentrations of fed adult serum (1.25–10%), the MTT response was slightly but consistently lower in cells from starved when compared with cells from fed mothers. By contrast, a marked difference in MTT activity which was paralleled by a lower DNA content became apparent when fetal rat brain cells were incubated with starved adult serum. Fetal serum and adult male serum were found to support growth equally well, while incubation of fetal brain cells with maternal sera resulted in lower MIT values than with the corresponding fetal sera. When cells were incubated with fetal sera pooled from starved mothers, MTT activity was decreased by 42 to 45%. A relative decrease in MTT activity was also apparent when cells were exposed to sera from starved mothers. Graded concentrations of starved fetal serum (2.5–10%) produced an increase in MTT activity that was consistently lower than similar concentrations of fed fetal serum, a finding suggesting a decrease in growth factors. Mixing fasted with fed serum did not correct the diminished growth, and indicated that an inhibitor might also be functioning to restrict growth. These findings therefore suggest that the principal determinants of diminished fetal brain growth during maternal starvation are not only intrinsic to the cells but are importantly related to the altered extrinsic factors in the fetal circulation.     

Stimulus interval, rate and direction differentially regulate phosphorylation for mechanotransduction in neonatal cardiac myocytes

The effect of interval, direction and rate of strain on mechanotransduction in neonatal rat cardiomyocytes is determined for focal adhesion kinase (Y397pFAK), extracellular signal-regulated kinase ERK1/2 (Thr(183)/Tyr(185)) and paxillin (pY31) and phosphorylation time courses to 10% strain assessed. Cells are non-responsive at 5 min but recover at 15 min (P<0.03) with FAK nuclear translocation by 30 min. Cyclic biaxial strain increased phosphorylation from slower to faster rates (P<0.05). Uniaxial strain to groove-aligned myocytes increased FAK and ERK1/2 phosphorylation transversely more than longitudinally (P<0.05). Mechanotransduction may have a refractory period of 5 min and differentiate directions and rates of strain.     

Pharmacokinetics of Hexobarbital,Sulphadimidine and Chloramphenicol in Neonatal and Young Pigs

Half-life and apparent specific volume of distribution of hexobarbital, sulphadimidine and chloramphenicol were investigated in newborn, 1, 3, 5 and 8 weeks old pigs. Hexobarbital sleeping time and plasma concentration of hexobarbital at recovery were measured in the same age groups. The half-life of hexobarbital and chloramphenicol was long in newborn pigs but decreased fast during the first week after birth. From 1 to 8 weeks after birth the decrease was less pronounced. The half-life of sulphadimidine increased during the first 3 weeks of life, but in 1 and 3 weeks old pigs the amount of N4-acetylated sulphadimidine in plasma at 200 min. after the injection was higher than in the newborn pigs. The apparent specific volume of distribution of hexobarbital, sulphadimidine and chloramphenicol was changed in different ways from birth to 8 weeks of age. The hexobarbital sleeping time was very long in the newborn pigs and decreased until 3 weeks of age. The concentration of hexobarbital in plasma at recovery was unchanged from birth to 8 weeks of age. The concentration of chloramphenicol metabolites in plasma 100 min. after the injection increased very fast during the 8 weeks of observation. The concentration of N4-acetylated sulphadimidine in plasma at 200 min. after the injection increased from birth to 1 week of age, then it decreased. The data are stressing that the neonatal pig is a convenient model for pharmacokinetic testing of drugs used as pharmacotherapeutics in neonatal life.     

Leucine aminopeptidase-like activity in Aplysia hemolymph rapidly degrades biologically active alpha-bag cell peptide fragments

We have investigated the role that proteolytic enzymes in Aplysia hemolymph play in the inactivation of the neurotransmitter alpha-bag cell peptide (alpha-BCP(1-9), Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu). alpha-BCP fragments containing Pro in positions 1 or 2, or Tyr in position 1, were degraded relatively slowly (half-life, t1/2 = 10-64 min), whereas fragments lacking these residues were degraded relatively rapidly (t1/2 = 0.5-2.7 min). Of 12 peptidase inhibitors tested, only bestatin, amastatin, and phenanthroline significantly inhibited alpha-BCP(3-9) degradation. alpha-BCP(3-9) yielded only four observable cleavage products (in order of decreasing abundance at early time points): alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), and alpha-BCP(7-9). Degradation of alpha-BCP(3-9), alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), or alpha-BCP(7-9) was strongly inhibited by bestatin, moderately inhibited by amastatin, and not inhibited by arphramenine B. The rates of degradation of eight alpha-BCP fragments and three other peptides in plasma were well correlated with their rates of degradation in mammalian leucine aminopeptidase (LAP, EC 3.4.11.1). Collectively our data support the following ideas. 1) In hemolymph one or more LAP-like enzymes rapidly and sequentially cleave alpha-BCP(3-9) or other small peptides lacking Pro at positions 1 or 2 or Tyr at position 1. 2) LAP-like peptidases in hemolymph may act in concert with previously described ganglionic peptidases to degrade neurally released alpha-BCP(1-9) and alpha-BCP(1-8) into inactive fragments.     

Cardiovascular actions of lungfish bradykinin in the unanaesthetised African lungfish, Protopterus annectens

Bradykinin (BK) isolated from plasma of the African lungfish, Protopterus annectens, contains four amino acid substitutions compared with BK from mammals (Arg(1)-->Tyr, Pro(2)-->Gly, Pro(7)-->Ala, Phe(8)-->Pro). Bolus intra-arterial injections of synthetic lungfish BK (1-1000 pmol/kg body wt.) into unanaesthetised, juvenile lungfish (n=5) produced a dose-dependent increase in arterial blood pressure and pulse pressure. The maximum pressor response occurred 2-3 min after injection and persisted for up to 15 min. The threshold dose producing a significant (P<0.01) rise in pressure was 50 pmol/kg and the maximum increase, following injection of 300 pmol/kg, was 9.3 +/- 2.3 mmHg. Injection of the higher doses of lungfish BK produced a significant (P<0.05) increase in heart rate (2.8 +/- 0.8 beats/min at 100 pmol/kg). In contrast, bolus intra-arterial injections of mammalian BK, in doses up to 1000 pmol/kg, produced no significant cardiovascular effects in the lungfish. The data support the existence of a functioning kallikrein-kinin system in the lungfish and demonstrate that the ligand-binding properties of the receptor(s) mediating the cardiovascular actions of lungfish BK are appreciably different from mammalian B1 and B2 receptors.     

Localization of the thiorphan-sensitive endopeptidase, termed enkephalinase A, on glial cells

Degradation of tritiated Leu-enkephalin was studied in cultures of primary astrocytes from rat brain. The incubation experiments with a cell suspension revealed Tyr as the main tritiated metabolite; however, Tyr-Gly-Gly and Tyr-Gly were detectable as well. Using a crude membrane preparation of the astrocytes, we found about equal amounts of Tyr and Tyr-Gly-Gly but only trace quantities of Tyr-Gly. The production of Tyr was completely inhibited by bestatin, an inhibitor of aminopeptidases, that of Tyr-Gly-Gly by thiorphan, a specific inhibitor of enkephalinase A. The results prove the ability of glial cells to degrade enkephalin by aminopeptidase and a membrane-bound enkephalinase A.     

A human neuroblastoma cell line with a stable ornithine decarboxylase in vivo and in vitro

A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.