Inhibition of multiplication in Acanthamoeba castellanii by specific inhibitors of ornithine decarboxylase

Proliferation of Acanthamoeba castellanii (Neff strain) in either a broth medium or a defined medium was arrested by alpha-monofluoromethyldehydroornithine (delta-MFMOme), alpha-difluoromethylornithine (DFMO), and (R,R')-delta-methyl-alpha-acetylenic putrescine (MAP), three specific inhibitors of ornithine decarboxylase. Although all three inhibited the ameba enzyme, delta-MFMOme was the most effective inhibitor of multiplication. Growth inhibition was reversed by the addition of polyamines. The inhibitors did not induce differentiation by themselves although DFMO caused encystment when supplemented with CaCl2 or MgSO4.     

Inhibition of polyamine biosynthesis and growth in plant pathogenic fungi in vitro

Polyamine (PA) biosynthesis inhibitors, difluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and bis-(cyclohexylammonium) sulphate (BCHA) have been tested for their effects on colony diameters at different intervals after inoculation of four plant pathogenic fungi (Helminthosporium oryzae, Curvularia lunata, Pythium aphanidermatum and Colletotrichum capsici). All these inhibitors, except DFMA had strongly retarded the growth of four fungi in a dose- and species-dependent fashion, and H. oryzae and C. lunata were found to be most sensitive to the effects of PA inhibitors. P. aphanidermatum and C. capsici were relatively insensitive and required rather high concentrations of inhibitors to get greater inhibition of mycelial growth, except DFMA which had stimulatory effect on the growth of these two fungi. However DFMA had greatly suppressed the growth of H. oryzae and C. lunata. The effect was generally more pronounced with MGBG than with DFMO and BCHA, and 1 mM Put completely prevented the inhibitory effects of 1 and 5 mM DFMO. Analysis of free and conjugated PAs in two sensitive fungi (H. oryzae and C. lunata) revealed that Put was present in highest concentrations followed by Spd and Spm and their levels were greatly reduced by DFMO application, and such inhibitions were totally reversed by exogenously supplied Put; in fact, PA titers were considerably increased by 1 mM Put alone and in combination with 1 mM DFMO. These results suggest that PA inhibitors, particularly DFMO and MGBG may be useful as target-specific fungicides in plants.     

Growth inhibition of pathogenic yeast isolates by α-difluoromethylornithine: An inhibition of ornithine decarboxylase

A large body of evidence exists suggesting that polyamines can play essential roles in cellular growth and differentiation. We examined the ability of -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, the major rate-limiting enzyme in polyamine biosynthesis, to inhibit the growth of Candida albicans, C. tropicalis, and C. parapsilosis. Substantial growth-inhibition was observed for all three species at DFMO concentrations ranging from 1 to 100 mM. C. tropicalis was significantly more susceptible to DFMO than C. albicans or C. parapsilosis. Depletion of cellular polyamine pools was seen in all 3 species following exposure to DFMO and polyamine depletion enhanced the susceptibility of the organisms to DFMO. The action of DFMO was specifically antagonized by exogenous polyamines. These data suggest that polyamines are important in the growth of Candida spp. and that inhibitors of polyamine biosynthesis may be useful as antifungal agents.     

Changing root and shoot architecture with the rolA gene from Agrobacterium rhizogenes: Interactions with gibberellic acid and polyamine metabolism

The effects of rolA on root and shoot architecture have been ascribed to a deficiency in gibberellic acid (GA₃) and to changes in polyamine metabolism. Using tobacco, we examined interactions among GA₃, a polyamine accumulation inhibitor (α-DL-difluoromethylornithine or DFMO) and the rolA gene controlled by the 35S CaMV promoter. We measured the effects of these three agents on architecture and polyamine accumulation in excised roots and whole plants grown in vitro. Previous work showed that DFMO or genetic transformation with the rolA gene from Agrobacterium rhizogenes, controlled by the 35S promoter (P_35S-rolA), caused excised tobacco roots to grow faster with altered root system architecture. We show that gibberellic acid (GA₃) reversed the effects of DFMO on the architecture of excised root systems, but neither reversed the effects of DFMO on growth, nor the changes in growth and architecture associated with P_35S-rolA. GA₃ treatment alone resulted in increased agmatine levels, suggesting that the inhibition of the effects of DFMO on architecture was through a stimulation of the arginine decarboxylase (ADC) pathway, GA₃ alone also inhibited the accumulation of putrescine and tyramine conjugates in excised roots. In tobacco plants growing in vitro DFMO and P_35S-rolA were associated with reduced shoot height, which was partially restored by GA₃ treatment; however, GA₃ also stimulated shoot height in the controls. GA₃ did not lessen the leaf wrinkling associated with P_35S-rolA. P_35S-rolA increased root number in young seedlings in vitro, and increased root system length in seedlings grown in soil. As in excised roots, the developmental changes linked to DFMO and P_35S-rolA were accompanied by reductions in putrescine titers. GA₃ treatment stimulated putrescine accumulation in stems and leaves, and partially reversed the negative effects of DFMO and P_35S-rolA on putrescine accumulation in roots, stems and leaves. Again, the restoration of putrescine pools appeared to be through a stimulation of the ADC pathway, since agmatine accumulated in plants exposed to GA₃. In general, the effects of DFMO and P_35S-rolA on phenotype and polyamine metabolism were coordinated, and in many cases these effects were similarly modulated by GA₃, reinforcing the previous conclusion that the phenotypic effects of rolA in roots and shoots occur through interference with polyamine metabolism and that the putrescine conjugates are particularly important in regulating root system growth and architecture. We were unable, however, to discem consistent evidence for a direct role for GA₃ in establishing the RolA phenotype.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (≤5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-α-difluoromethylornithine (DFMO) anddl-α-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult-to-root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin-induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50-500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal-bis-guanyl-hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4-¹⁴C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5-fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis.     

The effect of combination treatment with alpha-difluoromethylornithine and Corynebacterium parvum on B16 melanoma growth and tumoricidal effector cell generation in vivo

Summary The objective of the present investigation was to establish whether a known lymphoreticular-stimulating agent Corynebacterium parvum would augment the established antitumor activity of -difluoromethylornithine in vivo. Furthermore, since C. parvum is known to boost cell mediated cytotoxicity, the effect of DFMO (DL--difluoromethylornithine·HCl·H₂O) treatment was evaluated on macrophage and natural killer (NK) cell tumoricidal activity. DFMO administered alone, 1% or 2% in drinking water, inhibited 49.4% or 88.0% of B16 melanoma growth in vivo, respectively. Administration of C. parvum alone, three doses of 300 g each, inhibited tumor growth 57.4%. When administered together, DFMO and C. parvum treatment resulted in 89.8% (1% DFMO) or 97.4% (2% DFMO) inhibition of melanoma growth depending upon the dose of DFMO. C. parvum-treated animals had increased levels of macrophage-mediated tumoricidal activity directed against B16 melanoma cells in vitro, however, NK cell activity was reduced. DFMO treatment alone had no effect on macrophage or NK cell tumoricidal activity. In animals receiving both C. parvum and DFMO treatments macrophage-mediated tumoricidal activity was augmented. These results demonstrate that C. parvum can augment the antitumor activity of DFMO in vivo, possibly through macrophage activation. Furthermore, in contrast to many other cancer chemotherapeutic drugs, DFMO is apparently not immunosuppressive regarding tumoricidal effector cells.     

Polyamine depletion and growth inhibition ofCryptococcus neoformans by α-difluoromethylornithine and cyclohexylamine

The ability of two known inhibitors of polyamine synthesis,-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and cyclohexylamine, an inhibitor of spermidine synthase, to inhibit thein vitro growth and polyamine synthesis of clinical isolates ofCryptococcus neoformans was examined. Treatment ofC. neoformans with either DFMO or cyclohexylamine resulted in depletion of cellular polyamines and inhibition of growth.Cryptococcus neoformans was shown to lack detectable spermine and to require high concentrations of spermidine, but not putrescine, for growth. The growth inhibition by DFMO and cyclohexylamine was reversed by exogenous polyamines. These findings document the ability of cyclohexylamine and DFMO to inhibit polyamine synthesis and growth in clinically important isolates ofC. neoformans.     

Role of auxins, polyamines and ethylene in root formation and growth in sweet orange

The primary objective of this work was to investigate the role of polyamines (PAs) on root formation and growth in two sweet orange (Citrus sinensis L. Osb.) cultivars Pineapple and Pêra. Adventitious shoots (30-d-old) derived from epicotyl explants were transferred to root induction medium containing Murashige and Skoog salts at different strengths and supplemented with different concentrations and combinations of auxins. Root formation and development decreased in both sweet orange cultivars concomitant with the reduction of medium strength. The α-naphtaleneacetic acid was important during the root differentiation phase, but its combination with indole-3-butyric acid was essential for root elongation. The addition of PAs significantly improved root formation and/or growth, depending on their concentration, whereas the presence of inhibitor of PAs biosynthesis α-difluoromethylornithine (DFMO) inhibited these processes. The rooting impairment caused by DFMO was partially reversed by the supplementation of putrescine. Aminoethoxyvinylglycine AVG and AgNO₃ also inhibited in vitro rooting in both sweet orange cultivars, indicating that ethylene was likewise important for rhizogenesis in sweet orange.     

The effects of some polyamine biosynthetic inhibitors on growth and morphology of phytopathogenic fungi

We have studied the effects of two polyamine biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA), and of polyamines (PAs), alone and in combination, on mycelial growth and morphology of four phytopathogenic fungi: Botrytis sp, B. cinerea, Rhizoctonia solani and Monilinia fructicola. The inhibitors were added to a Czapek agar medium to get final concentrations of 0.1, 0.5 and 1.0 mM. DFMO and DFMA, suicide inhibitors of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) respectively, inhibited mycelial growth strongly; the effect was generally more pronounced with DFMA than with DFMO, but each fungus had its own response pattern. The addition of the PAs putrescine (Put) and spermidine (Spd) to the culture medium resulted in a promotion of growth. In Botrytis sp and Monilinia fructicola exposed to inhibitors plus PAs, mycelial growth was actually increased above control values. Mycelial morphology was altered and cell size dramatically reduced in plates containing inhibitors alone, whereas with PAs alone, or in combination with inhibitors, morphology was normal, but cell length and diameters increased considerably. These results suggest that PAs are essential for growth in fungal mycelia. The inhibition caused by DFMA may be due to its arginase-mediated conversion to DFMO.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Properties of brain L-glutamate decarboxylase: inhibition studies

—l -Glutamate decarboxylase purified from mouse brain was found to be highly sensitive to the sulfhydryl reagents, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and p-chloromerburibenzoate (PCMB), which were competitive inhibitors (K_i for DTNB is 1·1 · 10^?8m ). Iodoacetamide and iodoacetic acid were less effective inhibitors than DTNB and PCMB. The mercapto acids, 3-mercaptopropionic, 2-mercaptopropionic, and 2-mercaptoacetic acids were potent competitive inhibitors with K_i values of 1·8, 53 and 300 μm , respectively. 2-Mercaptoethanol was less effective. Aminooxyacetic acid was the most potent carbonyl-trapping reagent tested inhibiting the enzyme activity completely at 1·6 μm , followed by hydroxylamine, hydrazine, semicarbazide, and d -penicillamine. Carboxylic acids with a net negative charge were strong competitive inhibitors e.g. d -glutamate (K_i 0·9 mm ), α-ketoglutarate (K_i, l·2mm ), fumarate (K_i,1·8 mm ), dl -β-hydroxyglutamate (K_i, 2·8 mm ), l -aspartate (k_i, 3·1 mm ) and glutarate (K_i, 3·5 mm ). 2-Aminophosphonobutyric and 2-aminophosphonopropionic acids, phosphonic analogs of glutamate and aspartate, respectively, had no effect at l0mm . γ-Aminobutyric acid, l -glutamine, l -γ-methylene-glutamine, and α,γ-diaminoglutaric acid, amino acids with no net negative charge at neutral pH, had no effect at 5 mm . Glutaric and α-ketoglutaric acids were the most potent inhibitors among the various dicarboxylic and α-keto-dicarboxylic acids tested (K_i, 3·5 and 1·2 mm , respectively). Compounds with one carbon less, succinic and oxalacetic acids, or with one carbon more, adipic and α-ketoadipic acids, were less inhibitory. The monovalent cations, Li⁺, Na⁺, NH₄⁺, and Cs⁺ had no effect on l -glutamate decarboxylase activity in concentrations up to 10mm . Divalent cations, on the other hand, were very potent inhibitors. Among eleven divalent cations tested, Zn²⁺ was the most potent inhibitor, inhibiting to the extent of 50 per cent at 10μm . The decreasing order of inhibitory potency was: Zn²⁺ > Cd²⁺, Hg²⁺, Cu²⁺ > Ni²⁺ > Mn²⁺ Co²⁺ > Ba²⁺ > Ca²⁺ > Mg²⁺ > Sr⁺², The anions, I^?, Br^?, Cl^? and F^? were only weak inhibitors. The K_i value for Cl^? was 17mm . The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme. Intermediates of glycolysis had little effect on l -glutamate decarboxylase activity, but intermediates of the tricarboxylic acid cycle, e.g. α-ketoglutarate (K_i= 1·2 mm ) and fumarate (K_i= 1·8 mm ) were relatively potent inhibitors. The nucleotides, ATP, ADP, AMP, cyclic AMP, GTP, GDP, GMP, and cyclic GMP were weak inhibitors. l -Norepinephrine (K_i= 1·3 mm ) and serotonin were potent inhibitors, while acetylcholine, dopamine and histamine were less effective. Ethanol and dioxane inhibited the enzyme activity to the extent of 20-50 per cent at 10 per cent (v/v), while slight activation was observed at low concentrations (0·1-1 per cent) of both solvents. The possible role of Zn²⁺ and some metabolites in the regulation of steady-state levels of γ-aminobutyric acid also was discussed.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.     

Inhibition of polyamine biosynthesis by alpha-difluoromethyl ornithine potentiates the cytotoxic effects of arabinosyl cytosine in HeLa cells

The object of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of HeLa cells using α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMO inhibits HeLa cell growth by causing a decrease in the intracellular levels of putrescine and spermidine without any significant effect on concentration of spermine. The inhibition is readily reversible by exogenous supply of putrescine to the medium. The DFMO treatment also results in an accumulation of cells in S phase. Further, the use of an S phase-specific drug like Ara-C following DFMO treatment results in a synergistic killing of the tumor cells as revealed by the inhibition of cell growth. These observations suggest that exploitation of regulation of the cell cycle by the depletion of polyamines with the use of inhibitors like DFMO might help in designing better therapeutic regimes in combination with other cytotoxic drugs.     

Polyamine analogues – an update

Summary. The polyamines are growth factors in both normal and cancer cells. As the intracellular polyamine content correlates positively with the growth potential of that cell, the idea that depletion of polyamine content will result in inhibition of cell growth and, particularly tumour cell growth, has been developed over the last 15 years. The polyamine pathway is therefore a target for development of rationally designed, antiproliferative agents. Following the lessons from the single enzyme inhibitors (α-difluoromethylornithine DFMO), three generations of polyamine analogues have been synthesised and tested in vitro and in vivo. The analogues are multi-site inhibitors affecting multiple reactions in the pathway and thus prevent the up-regulation of compensatory reactions that have been the downfall of DFMO in anticancer chemotherapy. Although the initial concept was that the analogues may provide novel anticancer drugs, it now seems likely that the analogues will have wider applications in diseases involving hyperplasia.     

A Convenient Method for Rapid Determination of Cereal Proteins: A Device to Eliminate the Effect of Color Substances on the Biuret Procedure

At least, four kinds of amylase inhibitors are found in culture of Streptomyces sp. No.280.¹⁾ A large amount of amylase inhibitors were produced by Streptomyces sp. No. 280 when cultivated on 3% oatmeal medium and it was found that the molecular weight of the inhibitors were transformed to smaller molecules during the cultivation time. The transformation of the amylase inhibitor was found to result from degradation of its carbohydrate moiety by α-amylase in the culture broth. The amylase inhibitor was hydrolyzed partially by the action of taka-amylase A or hog pancreatic α-amylase. With hydrolyzation of amylase inhibitor by α-amylase, neutral sugars (mainly maltose) were liberated from the amylase inhibitor and a modified inhibitor was newly formed, but amylase inhibitory activity against glucoamylase was not changed. The inhibitory activity against muscle Phosphorylase a, however, was almost completely lost.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.