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The state of the art in the area of polyamines is presented in this useful, two-volume publication. Basic information describing the role of polyamines in the processes of growth and differentiation is given. Also included are data on the regulation of polyamine biosynthesis and metabolism and their interactions with nucleic acids. Several chapters are devoted to the role of polyamines in various aspects of plant biology, with a special emphasis on their participation in the response of plants to extreme environments. Special attention is given to the use of inhibitors of polyamine biosynthesis as potential antitumor and antiproliferative agents. Additionally, recent progress in the molecular biology and genetic engineering of genes coding for polyamine biosynthetic enzymes is described. Cancer researchers, biologists, geneticists, biochemists, physiologists, and clinicians will find this volume indispensible. Contents VOLUME II: POLYAMINES IN MICROBES. Polyamines and the Growth of Bacteria and Viruses. Polyamines in Thermophiles. Propylamine Transfer Reactions in Thermophilic Archaebacteria: Enzymological Aspects and Comparative Biochemistry. Microbial Mutants Deficient in Polyamine Synthesis. Regulation of the Putrescine Biosynthetic Genes (SpeA, speB, and speC) in Escherichia coli. Utilization of L-Arginine, L-Ornithine, Agmatine, and Putrescine as the Major Source of Nitrogen and Its Control in Escherichia coli. POLYAMINES AND PLANTS. Polyamines and Plant Response to Stress. Polyamines and Plant Growth and Development. Polyamines and Plant Hormones. Polyamines in Plant Mutants. POLYAMINES AND DISEASES. Polyamines in Health and Disease. Polyamine Metabolism and Neoplastic Growth: A Programmed Deregulation? Polyamines as Markers of Malignancy. Polyamines as Indicators of Disease Activity and Response to Therapy in Cancer Patients. Inhibitors of Polyamine Biosynthesis as Therapeutic Agents. Clinical Studies of a- Difluoromethylornithine and a-Interferon in Cancer Patients. Changes in Polyamine Metabolism in Tumor-Bearing Hosts with Total Parenteral Nutrition and Intravenous Infusion of a-Difluoromethylornithine. Index. c. 328 pp., 7x10, due February 1989, ISBN-0-8493-6809-X.

SUMMARY The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. They have been implicated in growth and differentiation processes. In 1967, we reported that cancer cells are rich in polyamines. Subsequently, it has been shown that polyamines are released from cancer cells and may be detected in body fluids such as urine, blood and cerebrospinal fluids. It has also been demonstrated that the increase in cellular polyamine levels is an early and an obligatory event in the process of malignant transformation. This increase in cellular polyamine concentration is due to the activation of ornithine decarboxylase (ODC), which catalyses the rate limiting step in polyamine synthesis by converting ornithine to putrescine. Assays of urinary and blood polyamines have been used to detect cancer and to determine the success of therapy. A sensitive, rapid, chemiluminescence-based method for the determination of diamines and polyamines was developed and 2.000 urine samples were tested. An interesting "gene therapy" system for injecting amine oxidases into normal and transformed cells was developed as follows: serum amine oxidase and porcine kidney damine oxidase were trapped within reconstituted Sendai virus envelopes. Chick or rat fibroblasts, transformed by Rous sarcoma virus, were more susceptible to the injected enzymes, compared to the normal culture, when macromolecular synthesis was tested. An in vitro chemosensitivity assay for the testing of the sensitivity of cancer cells from individual patients ("tailored treatment") was also developed. All these studies stress the importance of polyamines in carcinogenesis.

The effect of the green tea polyphenol-(−)epigallocatechin-3-gallate (EGCG) was tested in cultures of normal and transformed NIH-pATMras fibroblasts. In this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of H-ras by activating the mammary tumor virus long terminal repeat (MMTV-LTR) promoter. This facilitates a reliable comparison of the susceptibility of normal and transformed cells to EGCG. It has been shown that EGCG inhibited the growth of transformed but not of the normal fibroblasts. In an attempt to elucidate the mode of the preferential inhibitory activity of EGCG, its effect on growth promoting factors has been examined. The level of ornithine decarboxylase (ODC, EC 4.1.1.17), which is a signal for cellular proliferation, was reduced by EGCG in the transformed but not in the normal cells. EGCG also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (MAPK) activities, without affecting the kinases in the normal cells. Similarly, EGCG also preferentially decreased the levels of the oncogenes Ras and Jun in transformed cell. EGCG preferentially induced apoptosis in the transformed fibroblasts. In vitro chemosensitivity tests demonstrated that EGCG inhibited the proliferation of leukemic cells. These findings suggest that EGCG has a therapeutic potential in the combat against cancer.

Previous studies have demonstrated that polyamines accumulate in cancer cells and that overproduction of ornithine decarboxylase (ODC), which catalyzes polyamine synthesis, elicits the acquisition of the transformed phenotype. However, it was not clear whether the overexpression of ODC and the accumulation of polyamines are only innocent by-products of the transformation process. In this study we demonstrate that polyamines as such, may play a crucial role in malignant transformation. The system used consisted of NIH 3T3 fibroblasts transfected with a construct (pATMras) in which Ha-ras was under the transcriptional control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter (MMTVras cells). Dexamethasone, which activates the promoter, triggered phenotypic transformation. This was accompanied by an increase in ODC activity and polyamine accumulation. Cells, thus transformed, grew in soft agar and formed typical foci. alpha-Difluoromethylornithine (DFMO), which blocks polyamine synthesis, inhibited the dexamethasone-enhanced transformation. This inhibition was reversed by polyamines. Polyamines caused transformation of MMTVras cells in the absence of dexamethasone. Under these conditions, cells became anchorage independent. This phenomenon is not explained by the leakiness of ras, since normal, immortalized NIH 3T3 fibroblasts, also grew in soft agar in the presence of polyamines. Taken together, these observations suggest that polyamines may stimulate malignant transformation of immortalized cells, in cooperation with other factors, such as oncogenes or genetic defects.

We describe an ornithine decarboxylase (ODC) activity-based assay for the quantitation of multidrug resistance (MDR) and its reversal by MDR modulators in cultured mammalian cells. ODC catalyzes the first and rate-limiting step in polyamine biosynthesis. The activity of this enzyme rises rapidly after growth initiation, such as after addition of serum-containing medium to quiescent mammalian cells. This increase in enzyme activity is prevented when growth is arrested, such as after treatment with cytotoxic drugs. In this assay cultures of drug-sensitive animal and human carcinoma cells as well as their MDR sublines were exposed to various concentrations of different cytotoxic agents for 6-48 h. A dose-dependent decrease in ODC activity was obtained with a variety of chemotherapeutic agents including anthracyclines, vinca alkaloids, epipodophyllotoxins, actinomycin D, antifolates, and cisplatinum. Anticancer drug resistance levels were calculated as the 50% inhibitory concentration of ODC activity obtained with drug-resistant cells divided by that obtained with sensitive cells. These cytotoxicity determinations correlated favorably with those obtained by the well-established colony formation assay. The ODC assay also proved useful in the assessment of MDR reversal with modulators of the MDR phenotype. Therefore, these studies show that the ODC assay could be useful for the reliable determination of drug resistance levels in cultured mammalian cells and for the assessment of drug resistance reversal by various modulators of the MDR phenotype.

In 1678 Antonie van Leeuwenhoek identified crystalline substances in human semen. The structure of these crystals, named "spermine", was not elucidated by Rosenheim until 250 years later. Subsequently a triamine (spermidine) and a diamine (putrescine; 1,4-diaminobutane) were isolated from prokaryotic and eukaryotic systems. Soon it became apparent that polyamines can promote the growth of fastidious bacteria. Subsequently a group in Helsinki studied the accumulation of polyamines in regenerating rat liver, while Caldarera and his group studied polyamine synthesis in the developing chick embryo. These investigations led to metabolic studies. Ornithine decarboxylase was identified as a key enzyme in polyamine biosynthesis, while polyamine and diamine oxidations were studied by Mondovi. alpha-Diflouromethylornithine (DFMO) was synthesized by Merrell-Dow and became a potent inhibitor of ornithine decarboxylase. The findings of Russell that polyamines are excreted in the urine of cancer patients drew the attention of oncologists, who attempted the use new technologies for the detection of cancer and improving therapy. With the advance of molecular biology the structure of polyamine-biosynthetic enzymes was elaborated. Plants served as another important tool to study the physiological functions of polyamines. Bagni and his group at Bologna were pioneers in that field and for more than forty-six years set the foundation of a most interesting discipline.

Polyamines, oxidized by serum amine oxidase, yield aminoaldehydes and hydrogen peroxide. Acrolein may be formed from the aminoaldehydes by a spontaneous beta-elimination process. These oxidation products "oxidized polyamines" inhibit bacterial growth and exhibit anticancer activity. The antimicrobial activity of oxidized polyamines is not limited to bacteria; and the inactivation of bacterial viruses, plant viruses and animal viruses, was also reported. Bacteriophages of the T-odd series are permeable and were inactivated by oxidized polyamines. The inactive phages absorb to their bacterial host and injected their DNA, which formed a stable inactive complex with the aminoaldehydes. Aminoaldehydes, synthesized chemically, also inactivated viruses. The growth of the plant viruses: Tobacco mosaic virus, Potato virus X and Alfalfa mosaic virus was also inhibited by oxidized polyamines. The animal viruses, which were inactivated by oxidized polyamines included Myxoviruses (influenza and Newcastle disease viruses), West Nile, vaccinia and Sindbis viruses. These findings may have practical implications.

The naturally occurring polyamines, spermine [NH2(CH2)3NH(CH2)4NH(CH2)3NH2] and spermidine [NH2(CH2)3NH(CH2)4NH2], as well as the diamine putrescine [NH2(CH2)4NH2], are widely spread in nature. They occur in plants, micro-organisms and animal tissues and fulfil many important physiological functions. Due to their cationic nature they interact with negatively charged macromolecules such nucleic acids, phospholipids and proteins. This ionic interaction, which is reversible, leads to the stabilization of DNA, tRNA, membranes and some proteins. Early studies demonstrated that polyamines stimulate the growth of pro- and eukaryotic cells and that they play an important role in carcinogenesis and in malignant transformation processes. As a result of these studies various inhibitors of polyamine biosynthesis have been synthesized and are used to combat cancer and parasitic diseases (e.g., African sleeping sickness).

The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. They have been implicated in growth and differentiation processes. Polyamines accumulate in cancerous tissues and their concentration is elevated in body fluids of cancer patients. Assays of urinary and blood polyamines have been used to detect cancer and to determine the success of therapy. Drugs which inhibit the synthesis of polyamines can prevent cancer and may also be used for therapeutic purposes. Ornithine decarboxylase, which catalyzes the rate limiting step in polyamine synthesis, can serve as a marker of proliferation. Recently, a new in vitro chemosensitivity test, based on the disappearance of ornithine decarboxylase in drug-treated cancer cells has been developed. The increasing interest in polyamines and their physiological functions may lead to a more extensive application of these compounds or their derivatives in cancer diagnosis and treatment.

Green tea which is widely consumed in China, Japan and India, contains polyphenolic compounds, which account for 30% of the dry weight of the leaves. Most of the polyphenols are flavanols, of which (-)-epigallocatechin-3-gallate (EGCG) is most abundant. Epidemiological studies revealed that the incidences of stomach and prostate cancers are the lowest in the world among a population that consumes green tea on a regular basis. It has also been reported that the quantity of green tea consumed, plays an important role in reducing cancer risk and in delaying cancer outbreak and recurrence. Various systems were used to confirm anti-cancer activities of green tea and/or EGCG. These included experimental animals in which cancer was induced chemically. Cultured cells transformed chemically or by oncogenes were also used. These studies clearly demonstrated that green tea or EGCG have anticancer and cancer preventive properties. The mechanisms of these activities have also been studied in details. It has been shown that green tea and its active components interfere with signal transduction pathways. Thus the activities of various protein kinases are inhibited, the expression of nuclear proto-oncogenes declines and the activity of ornithine decarboxylase (ODC) is reduced. ODC, which catalyzes the rate-limiting step in the biosynthesis of polyamines is closely linked with cellular proliferation and carcinogenesis. Inhibitors of ODC, like alpha-difluoromethylornithine (DFMO) have long been used for cancer prevention and therapy. It has been suggested that polyamine depletion by green tea could offer one explanation for its anti-cancer activities.

A sensitive chemiluminescence-based method for the assay of ornithine decarboxylase (ODC) has been developed. This method, which permits the detection of putrescine (the product of ODC) at a picomolar range, can be used to determine ODC activity in cellular extracts. Extracts are incubated with ornithine and spotted onto p81 phosphocellulose paper strips. After drying, the papers are washed with ammonium hydroxide to remove contaminants, which may interfere with the assay. Putrescine is next eluted from the paper by shaking in an elution buffer containing magnesium sulfate. Partially purified hog kidney diamine oxidase is then used to oxidize putrescine in the eluate. The hydrogen peroxide formed during the oxidation is determined by chemiluminescence using luminol and peroxidase. This simple analytical method has the sensitivity of conventional assays based on the use of radioactive ornithine.

Ornithine decarboxylase (ODC) and diamine oxidase (DAO) are important enzymes involved in the metabolism of polyamines (putrescine, spermidine and spermine). The influence of testosterone (T) and 17, beta-estradiol (E2) on the activity of ODC and DAO was examined in cultivated normal rat kidney (NRK) epithelial cells. The results showed an increase in enzyme activities 4 hours or 12 hours after hormonal treatment. Both T and E2 led to a significant increase (1.6-fold) in ODC protein level as compared to the controls. Cellular concentration of spermidine and spermine increased (2.2- and 2.6-fold respectively) 4 hours after T addition. A higher levels in concentrations of putrescine (1.4-fold) and spermine (1.5-fold) 12 hours after E2 treatment were observed. These results suggest that the biosynthesis and terminal oxidation of the polyamines in NRK epithelial cells are androgen- and estrogen-mediated and depend on the hormonal sensitivity of the cells.

Previous studies have demonstrated that polyamines accumulate in cancer cells and that overproduction of ornithine decarboxylase (ODC), which catalyzes polyamine synthesis, elicits the acquisition of the transformed phenotype. However, it was not clear whether the expression of ODC and the accumulation of polyamines are only innocent by-products of the transformation process. In this study we confirm previous findings what polyamines can trigger the transformation of immortalized cultured cells. In addition to NIH 3T3 fibroblasts, studied previously, rat kidney epithelial cells or fibroblasts also grew in soft agar in the presence of polyamines. It has also been demonstrated that spermidine, preferentially stimulated the transcription and the expression of c-myc while those of c-fos were preferentially stimulated by putrescine. These findings suggest that the effect of polyamines on cellular transformation, could be explained, at least partially, by stimulation of proto-oncogene expression.

In the present study developmental patterns of renal polyamineoxidizing enzymes polyamine oxidase (PAO) and diamine oxidase (DAO) in male and female ICR mice were demonstrated. The effects of testosterone (10μg/100g body weight) on renal PAO and DAO activities were also studied. The differences between sexes in both PAO and DAO activities were most clearly expressed in the immature kidney. At the age of 20 days PAO and DAO activities were 1.52 fold (p < 0.01) and 1.75 (p < 0.02) respectively higher in male mouse kidney than in female. Maturational processes reflected in significant increases in polyamine- oxidizing enzyme activities mainly in female mouse kidney, comparable with the gain in the kidney wet weight. Our data show that testosterone is able to influence renal PAO and DAO activities in addition to the well-known stimulation of polyamine biosynthesis. The hormonal effects were sex and age dependent. The influence of testosterone on renal PAO activity was mainly age dependent. The slight stimulation of renal PAO activity observed in 20- and 50-day old mice, 24h after testosterone administration, change with a decrease in the enzyme activity at the age of 70 days. The effects of testosterone on renal DAO activity were mainly sex dependent. Testosterone caused stimulation of DAO activity with a very close magnitude (nearly twice) in female mouse kidney, independently of the age of mice. In contrast, in male mice the hormone treatment resulted in a statistically significant increase in renal DAO activity at the age of 70 days (.1.3 fold, p < 0.05) only. It could be suggested that our data indicate the different contribution of renal PAO and DAO in androgen regulation of polyamine levels, depending on sex and the stage of the postnatal development.

DNA polymerase from the malarial parasite Plasmodium falciparum required Mg2+ for activity, Putrescine (1 mM) caused a twofold increase in enzyme activity in the presence of a suboptimal concentration of MgCl2 (2 mM). Spermidine (1.5-2.0 mM) or spermine (0.1-0.3 mM) increased the activity of malarial DNA polymerase, in the presence of 2 mM MgCl2, by factors of 6 and 3-5, respectively. The activity of DNA polymerase from calf thymus or from NIH 3T3 cells transformed by the ras oncogene were not stimulated by these polyamines to the same extent. These findings suggest that in malaria-infected erythrocytes, polyamines, at physiological concentrations, serve as a cofactor for the parasitic alpha-like DNA polymerase. Malarial parasites grown in cultured human erythrocytes did not synthesize DNA after treatment with alpha-difluoromethylornithine, which caused polyamine depletion in the infected cells. DNA synthesis was resumed after adding putrescine to the polyamine-depleted cultures. DNA synthesis was also initiated when actinomycin D was added along with putrescine to polyamine-depleted cells. It thus appears that polyamines are essential for the translation of the DNA polymerase mRNA and that polyamines play an important role in regulating the cell cycle of the malarial parasite.

An alpha-like DNA polymerase has been identified and characterized in the extracts from the malarial parasite Plasmodium falciparum. The enzyme is sensitive to the specific inhibitors of alpha-DNA polymerase, N-ethylmaleimide and aphidicolin, and is cell-cycle specific. High activity has been found in the schizont, is lower in trophozoites, and has only negligible activity in the ring form. The enzyme has a molecular weight of about Mr 100,000-103,000 estimated by detecting activity in SDS-polyacrylamide electrophoresis and by Bio-Gel filtration. Another active band of a molecular Mr 68,000 was detected by SDS electrophoresis when the enzyme was stored for 2 months at -20 degrees C. The catalytic activity of parasite enzyme was optimal between pH 8 and pH 9. The apparent Michaelis constant for dTTP was 4.3 microM.

The activities of cAMP-dependent and independent protein kinases were determined after feeding confluent glioma C6-BU-1 cultures. It has been shown that the activity of both enzymes rose considerably after feeding, and that the ratio of 32 P incorporation into histone, in the absence and the presence of cAMP, was maximal 4 hours after feeding. This increase in protein kinase activity was followed by the activation of ornithine decarboxylase and accumulation of putrescine. Spermine, at millimolar concentrations, inhibited protein kinase, apparently by inactivating the catalytic subunit. It is suggested that this inhibition of protein kinase by polyamines is another regulatory mechanism, which controls cellular growth.

The incorporation of [ 14 C]arginine and [ 14 C]ornithine into various polyamines was studied in mung bean ( Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation. Both [ 14 C]arginine and [ 14 C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio. It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum-containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post-stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detected 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post-stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serum-containing medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.

A cell-free extract of Daphnia magna was found to lyse Escherichia coli cells as shown by leakage of the enzymes alkaline phosphatase and β-galactosidase from the bacteria. The cell-free extract was separated on Sephadex G-200, and the fractions showing an ability to lyse E. coli cels were isolated. The factor which was responsible for the lysis of the bacterial cells was probably a protein with a molecular weight of several thousands. Mg 2+ and Ca 2+ ions augmented the activity of the Daphnia extract on E. coli cells.

57Fe Mössbauer spectra of normal and Rous sarcoma virus-infected cultured chick embryo fibroblasts and rat glioma cells have been measured between 0.08 and 318 K. Ferritin-like iron and bacterio-ferritin-like iron have been found in these cells, in various relative amounts, indicating a close relationship between the two storage materials. The bacterio-ferritin-like iron was found to be predominantly membrane-bound. Above 260 K very wide lines were observed in the Mössbauer spectra, yielding an effective viscosity of about 1 poise in the normal chick embryo fibroblasts and about 0.5 poise in the virus-infected chick embryo fibroblasts.

N-Acetylputrescine and N1-acetylspermidine were synthesized from putrescine in cultured human lymphocytes. They were identified by paper chromatography and t.l.c. Acetylputrescine was also identified by mass spectrometry. N-Acetylputrescine and N1-acetylspermidine were formed in untreated cells and in lectin-transformed cells.

N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescine were assayed as substrates of diamine oxidases from pea seedling and pig kidney. With the exception of N-methylputrescine they were found to be oxidized to the corresponding aminoaldehydes. 1-Methyl-, 2-methyl-, 1-ethyl- and 1-propylputrescine were oxidized by the oxidases at lower rates than the N-alkylderivatives. 1,3-Dimethylputrescine had negligible oxidation rates while 1,4-dimethylputrescine (2,5-diaminohexane) was not a substrate. The oxidation of putrescine by the kidney oxidase was inhibited by 1,4-dimethylputrescine, while the pea oxidase was strongly inhibited by the former as well as by 2-methylputrescine and 1,3-dimethylputrescine. Serum amine oxidase did not oxidize the substituted putrescines although several of the latter inhibited spermidine oxidation by this oxidase.

DL-alpha-difluoromethylornithine (DFMO) inhibited ornithine decarboxylase (ODC) activity and arrested the growth of Plasmodium falciparum at the early trophozoite stage. The inhibition of ODC activity did not result in the formation of an alternative diamine such as cadaverine. When putrescine or spermidine were added to the parasites grown in culture, the arrest was reversed, and normal schizogony was completed even in the presence of DFMO. Some reversal of the inhibition was achieved with cadaverine at high concentrations, while 1,3-diaminopropane and spermine failed to restore the development. Resumption of growth could be detected when putrescine was added even after 67 h of DFMO treatment. Electron microscopy did not reveal any changes in the morphology of parasites treated for 47 h, while 73 h of treatment with DFMO induced massive accumulation of pigment. Death was observed a few hours later. These results suggest that DFMO acts as a cytostatic rather than as a cytocidal agent. The four carbon diamine restored cell growth while the shorter or the longer homologous compounds showed little activity.

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.

Polyamines were extracted from five different leishmanial strains (Leishmania sp., L. tropica major, L. mexicana, and two L. donovani isolates) and identified as pulrescine, spermidine, and spermine by thin-layer chromatography and mass spectrometry. These sensitive methods were also used to demonstrate the conversion of radioactive putrescine into spermidine and spermine. As in other types of cells, polyamine levels fluctuated during the growth cycle, maximal levels being attained during the logarithmic growth phase. In the five leishmanial strains, which were members of four different serotypes, the spermidine—putrescine ratios also varied, and in two strains of the same serotype, polyamine ratios were practically identical, suggesting that polyamine characteristics might serve as a further criterion for strain identification and classification.

This paper describes a sensitive enzymic micromethod for the quantitative assay of spermine and spermidine. The method is based on the oxidation of the polyamines by serum amine oxidase. The aminoaldehydes produced during the oxidation are assayed by N -methyl-2-benzothiazolone hydrazone hydrochloride, at 660 mμ. The molar absorbancy index for spermine is 12.5 × 10 3 liter mole −1 cm −1 and for spermidine 6.25 × 10 3 liter mole −1 cm −1 . When both spermine and spermidine are present in the biological material the value of the “combined polyamines” is obtained by the enzymic method. Spermidine is assayed by another method that uses dried Serratia marcescens cells. This permits the calculation of spermine by subtracting the amount of spermidine from that of the “combined polyamines.” The enzymic method has been applied to extracts from yeast, bacteria, and mouse liver.

1. 1. Oxidized spermine, an iminodialdehyde, obtained by the enzymic oxidation of spermine, forms complexes with deoxynucleotides, deoxynucleosides, purine bases and synthetic polyribonucleotides. 2. 2. The interaction of oxidized spermine with deoxynucleotides and deoxynucleosides has been demonstrated by spectrophotometric studies. The hyperchromic changes obtained are proportional to the amount of oxidized spermine added and are maximal with guanosine and guanosine 5′-phosphate. 3. 3. Electrophoretic studies indicate that guanine, cytosine and adenine, but not uracil, bind oxidized spermine. 4. 4. The transition from the coiled to the helical structure of polyriboadenylic acid is prevented by oxidized spermine. 5. 5. Complexes between polyribouridylic and polyriboadenylic acid are stabilized by oxidized spermine; the strands do not separate under acid conditions. Hydrogen bonding between complementary polyribonucleotide chains is prevented by pre-incubating the double-stranded structure with oxidized spermine. 6. 6. The binding of oxidized spermine to DNA decreases after deaminating the purine and pyrimidine bases. It has been concluded that the amino groups of the bases are responsible for the irreversible binding of oxidized spermine. The bases bind oxidized spermine in the decreasing order of reactivity: guanine, cytosine, adenine, thymine.

1. The effects of oxidized spermine on the biological activity of Bacillus subtilis DNA were studied by examining the transforming capacity of purified DNA treated in vitro, and of DNA spontaneously released from cells treated with oxidized spermine. 2. Transformation to tryptophan, histidine and methionine independence was affected significantly. The histidine marker was more sensitive to inactivation by oxidized spermine than the linked tryptophan and unlinked methionine markers. 3. NaCl, at high concentrations, antagonized the inhibitory effect of oxidized spermine, indicating the involvement of ionic factors in the binding of oxidized spermine to the DNA. 4. Transforming DNA after treatment with oxidized spermine, was more resistant to heating at 100° than untreated DNA, suggesting interstrand cross-linking.

Spermine, after oxidation by highly purified serum amine oxidase (amine: O2 oxidoreductase (deaminating)), inhibited the multiplication of Ehrlich ascites cells. The oxidation product, oxidized spermine, was separated from the amine oxidase by gel filtration and its toxicity verified. The related triamines, spermidine and diaminodipropylamine, were also oxidized by purified serum amine oxidase, giving rise to cytotoxic products. The polyamines in the absence of amine oxidase did not affect the multiplication of the Ehrlich ascites cells, whereas purified amine oxidase alone exhibited a slight cytotoxic effect. Oxidized [14C]spermine was rapidly taken up by Ehrlich ascites cells on incubation at 37°. The incorporation of radioactive valine, thymidine or uracil, into the corresponding cellular macromolecules, was markedly reduced after treating Ehrlich ascites cells with oxidized spermine for 30 min at 37°.

1. 1. Oxidized spermine, an iminodialdehyde, obtained by the enzymic oxidation of spermine, inhibits the incorporation of labeled thymidine, and uracil into the nucleic acids of growing Escherichia coli cells. The incorporation of labeled valine into bacterial proteins is arrested only after a definite lag period. 2. 2. Oxidized spermine also inhibits nucleic acid synthesis in cell-free systems, using purified DNA-dependent RNA polymerase and DNA polymerase. The inhibition of DNA synthesis by oxidized spermine, is somewhat greater than that of RNA synthesis. 3. 3. The inhibition of nucleic acid synthesis is due to the binding of oxidized spermine to the DNA primer and not to its interaction with the nucleoside triphosphates or the enzymes. 4. 4. Inhibition of DNA replication by oxidized spermine is dependent on the nature of the DNA used and is higher when guanine-cytosine-rich primers are used.

Ornithine decarboxylase (ODC, Lornithine carboxylyase; EC 4.1.17), catalyzes the conversion of ornithine to the diamine, putrescine. This reaction is the ratelimiting step in the synthesis of putrescine and the naturally occurring polyamines, spermidine and spermine which have been linked to many processes crucial for cell growth, division and differentiation [ 1,2] . Recent studies indicated the presence of polyamines in neural tissues [3,4] and in cultures of mouse neuroblastoma cells [5,6]. This paper deals with the induction of ODC in C6-BU-1 giioma and N115 neuroblastoma cells by fresh medium added to confluent cultures. A similar induction of ODC by fresh medium has been reported for other cell lines [ 7121.

1. Introduction The polyamines putrescine, spermidine and spermine, which occur widely in nature [l-5], are involved in cellular growth processes [6]. At physiol- ogical levels, they stimulate DNA [7], RNA [8] and protein ]9,10] synthesis, and reach their peak levels during periods of maximal growth [I]. They are required for optimal growth in bacteria, where their limitation leads to reduced cellular growth [ 111 and their concentration is markedly increased in rapidly growing mammalian cells [l], with this growth being arrested on inhibition of polyamine synthesis [ 121. It was shown that polyamines also occur in various trypanosomatid protozoa, where they are at their maximal levels during the logarithmic phase of growth [13-l 61. We have demonstrated that some antileishmanial drugs and polyamine analogues, i.e., pentamidine isethionate, ethidium bromide and methyglyoxal-bis (guanyl hydrazone) (MGBG), impair leishmanial growth by interfering with polyamine biosynthesis [ 161. In order to demonstrate the causal relationship between leishmanial growth and polyamine biosynthesis, a correlation between growth rate and putrescine to spermidine ratio must be established. In this study we demonstrate such a correlation. 2. Materials and methods The two strains studied were obtained from the 202 culture collection of the World Health Organisation’s International Reference Centre for Leishmaniasis (WHO-LRC), housed in the Department of Protozoology of the Hebrew University-Hadassah Medical School, Jerusalem. Their origins and some of their intrinsic taxonomic characters are given in table 1. These strains were maintained at 28°C by biweekly passage on semisolid Locke-blood-agar [ 191 or NNN diphasic medium [20]. For experimental studies, the strains were grown in a fully-liquid, Panmede (Paines and Byrne Ltd., Greenford, Middlesex, England) based medium, containing 7.5% normal rabbit blood

A proteolytic enzyme has been extracted from Escherichia coli B cells infected with bacteriophage T4. The enzyme has been isolated in a highly purified form. The protein has a molecular weight of 31,000, an isoelectric point of about 9, and a sedimentation constant of 3.12 s .

POLYAMINE biosynthesis is one of the earliest events occurring during cellular proliferation1,2, and the rate at which it proceeds3 is limited by ornithine decarboxylase (ODC), an enzyme which catalyses the formation of putrescine from ornithine. ODC levels are low, but stable, in resting cells, and a large increase in activity occurs rapidly after the onset of growth4,5. Elevated ODC levels occur in certain tumour cells, including hepatoma, neuroblastoma and SV40-transformed cells3–7. Fast growing hepatomas have higher enzyme levels than slow growing ones6. During Rous sarcoma virus (RSV) transformation, elevation of ODC levels precedes morphological change8.

Although diamines and polyamines are widespread in biological material [ 1,2] little is known about their physiological role. Numerous studies have established the metabolic relationship of the putrescine (1,4-diaminobutane) and the polyamine spermidine. In various systems, putrescine serves as a precursor for spermidine, which is synthesized by the condensation of the diamine with S-adenosyl-L-methionine [ 1,2] . The biosynthesis of putrescine, on the other hand, is catalyzed by ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) which converts ornithine to putrescine [2]. Coliphages of the T-even series contain considerable amounts of polyamines [3-S] . According to Ames and Dubin [S] ,8 X 10” Escherichia coli B cells, contain 11.7 nmoles putrescine and 1.6 E.tmole spermidine. These cells give rise to 5 X IO’ 3 T4 phages which contain 12.5 fitmoles putrescine and 3.75 pmoles spermidine. It thus appears that phage-infected bacteria should synthesize polyamines at a fast rate, to supply the polycations required for the phages and to make up for the polyamines (up to 50% of the putrescine) which leak from the cells soon after infection [6-81. In the present paper we demonstrate the stimulation of ornithine decarboxylase activity after infecting E. coli with T2 phage. It will be shown that the activity is maximal 6 to 9 min post-infection.

Polyamines are widely distributed in nature, but their precise role in cellular processes is not fully understood. Polyamines are associated with cell proliferation, tissue regeneration and malignancy [l-3]. Most of the information on the biosynthetic pathways of putrescine, spermlne and spermidine, their regulation and possible sites of action has been obtained from studies with micro-organisms or mammalian cells. In mammalian cells, putrescine is synthesized from Lornithine by Lornithine decarboxylase (ODC EC 4.11.17) [2]. In plants, however, it is commonly accepted that putrescine is formed from Larginine by Larginine decarboxylase (ADC EC 4.11.19), via the intermediate agmatine. The activity of ODC in plants is usually found to be much lower than that of ADC [4-71, and ODC has therefore been claimed to be of little significance in the formation of putrescine in plants. We felt that in plant cells such an essential biosynthetic pathway of putrescine should not differ from that in mammalian cells and therefore decided to study ODC activity in plant systems. We chose to search for ODC activity in two plant systems of rapidly proliferating cells the XD cell line of tobacco in suspension culture and tomato ovaries before and after pollination. After subculture of XD cells there is usually a lag period of a few days, after which the culture enters an exponential phase, lasting for several generations [8]. The culture then enters a stationary phase. In tomato ovaries before pollination there is some cell division; after pollination there is very intense cell division, which lasts for

Purified aminoaldehydes produced by polyamine oxidation were toxic to the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes. There was a profound effect on young ring forms, and, during maturation, parasites became more sensitive to the aldehydes. Oxidation of the aldehydes abolished the lethal effect. The plasmodia within glucose-6-phosphate-dehydrogenase (G6PD)-deficient erythrocytes were more sensitive to mono- and di-aldehydes than were parasites in normal erythrocytes. G6PD-deficient erythrocytes were also more sensitive to pretreatment with the dialdehyde produced by the oxidation of spermine. Pretreatment prevented further invasion by the parasites.

Summary Newcastle disease and influenza viruses were purified by centrifuging in sucrose and potassium tartrate gradients and analysed for polyamines by thin-layer chromatography of dansyl derivatives. It has been shown that both viruses contained spermine, spermidine and putrescine in concentrations sufficient to neutralize 30 to 40% of virus RNA phosphates.

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.