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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.

A new method for in vitro chemosensitivity testing of human lymphoma and leukemia patients has been developed. The method is based on the use of ornithine decarboxylase (ODC), a universal marker of proliferation, which is expressed early during the cell cycle and has a short half-life. This marker was detected by a quantitative immunohistochemical analysis, using an ODC antibody and a FITC-linked second antibody. The in vitro chemosensitivity of lymphocytes from four normal individuals was tested by the immunohistochemical method. Lymphocytes from 25 cancer patients were also examined. In drug-sensitive cells, the intensity of the marker declined in the presence of the drug, whereas resistance to the drug was demonstrated by the presence of the marker. A good correlation was found between the predicted chemosensitivity and the outcome of the therapy. It has been suggested that this approach could be used for in vitro chemosensitivity testing of hematological cancers and most likely also for other malignancies.

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.

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).

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.

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.

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 effect of morphine on neuroblastoma x glioma hybrid cells is not limited to the inhibition of adenylate cyclase activity. It is demonstrated that in morphine-treated cells there is a marked reduction in the activity of both cyclic AMP-dependent protein kinase and ornithine decarboxylase (ODC) in response to stimulation by PGE 1 . The effect of morphine is to block a cascade of events which may be crucial for the normal biochemical processes in these cells.

Internal protein precursors (IP) are formed in Escherichia coli B cells, soon after infection with bacteriophage T4. These precursors are cleaved to internal protein products (IP∗) in E. coli B infected with a wild type of T4, but not after infection with an amber mutant defective in gene 21. It has been shown that T4 internal protein products (IP∗), formed either in vitro or in vivo , interact with T4 DNA, but not with the DNA of E. coli B, Clostridium perfringens , bacteriophage T2, lambda, or ∅X174. The specific binding of T4 internal proteins to T4 DNA is not due to glucose residues on phage DNA, since DNA preparations from a glucosyl transferase mutant (T4 am αgt am βgt ) or T4 DNA after digestion with β-glucosidase, retain their ability to bind T4 internal proteins. Internal protein precursors (IP) do not form detectable complexes with phage DNA. The proteins bound to T4 DNA in vitro , have been identified as internal proteins by polyacrylamide gel electrophoresis and have the respective molecular weights of 7600, 8600 and 18,000 d.

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.

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.

Infecting NIH 3T3 cells with different species of mycoplasmas resulted only in a slight decrease in ornithine decarboxylase (ODC) activity and in the appearance of cadaverine in the infected cells. Similarly, the presence of mycoplasma in NIH 3T3 cells infected with a temperature-sensitive mutant of Rous Sarcoma virus did not bring about any significant changes either in the pattern of ODC activity or in putrescine levels, when transferred to the permissive temperature. This indicates that mycoplasmal contamination of cultures may not significantly change the putrescine metabolism in host cells. On the other hand, the presence of cadaverine in cultured cells may be attributed to contamination by mycoplasma.

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. 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.

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.

Growth rates of different clones, all derived from NIH 3T3 mouse fibroblasts, were determined. Four different types of cells were studied: (1) Normal NIH 3T3 fibroblasts; (2) a fast-growing NIH 3T3 clone obtained by repeated passages; (3) transformed clones (obtained by transfecting NIH 3T3 with the oncogene c-Ha- ras ); (4) a slow-growing revertant obtained by repeated passages of the transformed line. Growth rates were determined by the following markers of proliferation: thymidine incorporation, protein accumulation and cell number. In parallel experiments growth rates were determined by a new approach based on measuring ornithine decarboxylase (ODC) activity. Transformed cells, which were characterized by phase-contrast microscopy and by electron microscopy grew rapidly and showed high ODC activity. Similarly, a high-passage NIH 3T3 variant, which grew rapidly, also possesed high ODC activity. On the other hand, high-passage of a transformed clone revealed phenotypic changes confirmed by electron microscopy. These cells exhibited reduced growth rates and their ODC activities were similar to those of the normal NIH 3T3 cells. A confident correlation was found among each of the three conventional parameters of growth and between them and ODC activity. However, in all the cases studied ODC activity appeared early in the cell cycle before the expression of the other markers of proliferation. It has been suggested that ODC is a reliable early marker of cell proliferation and might also serve as an important tool for determining the arrest of growth.

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

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.

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.

dl -α-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum . Asynchronous cultures were pretreated with sorbitol and incubated for 28–30 hr. Then, when cultures consisted of mainly schizont stage parasites, dl -α-difluoromethylornithine was added to the growth medium for another 38–47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4–5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.

A new sensitive and rapid chemiluminescence-based method for the determination of diamines and polyamines is described. Phosphocellulose paper strips are used for the removal of neutral or negatively charged molecules from polyamine-containing fluid. The procedure is based on the determination of hydrogen peroxide, produced during the oxidation of polyamines, by a fairly specific serum amine oxidase. A plant diamine oxidase is used for the assay of diamines. This method permits the determination of diamines and polyamines in a range of 10 to 100 pmol and may be used for the assay of urinary polyamines.

The activity of transglutaminase (TGase) was measured in cultured C6 glioma cells after their stimulation by either isoproterenol and isobutyl-methylxanthine or by a serum-containing medium. The activity fluctuated in a biphasic manner, with the peaks at 2-3 hr and 7-8 hr poststimulation. The first peak of TGase activity was affected neither by cycloheximide nor by actinomycin D, which inhibited protein synthesis. The second peak, on the other hand, was completely eliminated by cycloheximide and was reduced by actinomycin D. Immunological procedures were employed to find out whether or not the activity of TGase corresponded with the presence of the TGase antigen in the cultured cells. Indirect immunofluorescent staining and radioimmunoblot techniques suggested that unstimulated cells contained an inactive enzyme. This inactive, or cryptic, enzyme had the same molecular weight as its active counterpart. Activation of the enzyme was mediated by cell stimulation, probably by its release from the membrane. This step did not require protein synthesis, unlike the second step, which was dependent on de novo protein synthesis.

Human erythrocytes contain only trace amounts of polyamines and lack active polyamine biosynthetic enzymes. A remarkable increase in polyamine content, and in the activity of ornithine and S-adenosyl-L-methionine decarboxylases, is noted in synchronous cultures of the malarial parasite, Plasmodium falciparum. Polyamine biosynthesis reached peak values during the early trophozoite stage, whereas nucleic acid and protein synthesis occurred later in mature trophozoites. DL-alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, did not interfere with merozoite invasion and with ring-form development, but prevented the transformation of trophozoites to schizonts. Concomitantly, the synthesis of proteins and nucleic acids was significantly inhibited. These inhibitory effects could be readily reversed by the diamine putrescine. Macromolecular synthesis and schizogony were normal when 5-10 mM-DL-alpha-difluoromethylornithine and 0.1 mM-putrescine were added to the cultures simultaneously.

The activity of ornithine decarboxylase (EC 4.1.1.17; L-ornithine carboxy-lyase) of C6-BU-1 glioma and N115 neuroblastoma cells increases significantly when confluent cultures are treated with compounds that increase cellular cAMP levels. These include norepinephrine or isoproterenol, and prostaglandin E1 or adenosine, which stimulate ornithine decarboxylase activity in C6-BU-1 glioma and N115 neuroblastoma cells, respectively. Ornithine decarboxylase activity is also elevated in confluent C6-BU-1 glioma cells treated with dibutyrylcAMP and theophylline, or after the glioma cells are fed with a serum-depleted medium in the presence of catecholamines and inhibitors of cyclic nucleotide phosphodiesterase. The activity of the enzyme increases 500- to 1000-fold, 2-6 hr after stationary-phase N115 neuroblastoma cells are fed with a serum-free medium, supplemented with phosphodiesterase inhibitors, adenosine, or prostaglandin E1. This stimulation is antagonized by carbamoyl choline and is blocked by actinomycin D or cycloheximide. These results suggest that the synthesis of ornithine decarboxylase of C6-BU-1 glioma and N115 neuroblastoma cells is controlled by cAMP.

Friedman, Mischa E. (Hebrew University, Jerusalem, Israel), and Uriel Bachrach . Inhibition of protein synthesis by spermine in growing cells of Staphylococcus aureus . J. Bacteriol. 92: 49–55. 1966.— Staphylococcus aureus SFL 9725 incorporated valine- C 14 into cellular protein. This incorporation was inhibited by spermine when the p H of the culture was adjusted to 7.8, and the inhibition was antagonized partially by Mg ++ . The incorporation of C 14 -labeled leucine, phenylalanine, lysine, arginine, and possibly glutamic acid was inhibited to a much greater extent than that of alanine, glycine, or threonine. The uptake of spermine- C 14 by S. aureus cells was rapid. More than 50% of the radioactivity resided in the soluble extract. The protein fraction of the soluble extract contained 99% of the recovered label, whereas the ribonucleic acid and deoxyribonucleic acid fractions contained little or no spermine- C 14 .