Membrane-bound ATPase in chloroplasts of Euglena gracilis

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca²⁺- and Mg²⁺-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. K_m values of Ca²⁺ and Mg²⁺ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca²⁺- or Mg²⁺-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca²⁺ or Mg²⁺ or from two different enzymes are discussed.     

Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

Catabolism of 6-aminolaevulinic acid in etiolated Euglena gracilis

[4-¹⁴C]-δ-Aminolaevulinic acid (δ-ALA) was employed to study the specificity of δ-ALA incorporation into chlorophyll in etiolated Euglena gracilis. Degradation of chlorophyll a to acetate and propionate, obtained from ring B, indicated that, although some of the radioactivity was incorporated, it did not result from incorporation of intact δ-ALA.     

The bacterial-like lactate shuttle components from heterotrophic Euglena gracilis

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD⁺-independent d-lactate dehydrogenase (d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD⁺-dependent LDH specific for the generation of d- and l-lactate (d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K⁺ or NH₄⁺. Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.     

The effect of submergence,ethylene and gibberellin on polyamines and their biosynthetic enzymes in deepwater-rice internodes

Submergence and treatment with ethylene or gibberellic acid (GA₃) stimulates rapid growth in internodes of deepwater rice (Oryza sativa L. cv. Habiganj Aman II). This growth is based on greatly enhanced rate of cell-division activity in the intercalary meristem (IM) and on increased cell elongation. We chose polyamine biosynthesis as a biochemical marker for cell-division activity in the IM of rice stems. Upon submergence of the plant, the activity of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) in the IM increased six- to tenfold within 8 h; thereafter, SAMDC activity declined. Arginine decarboxylase (ADC; EC 4.1.1.19) showed a similar but less pronounced increase in activity. The activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in the IM was not affected by submergence. The levels of putrescine and spermidine also rose in the IM of submerged, whole plants while the concentration of spermine remained low. The increase in SAMDC activity was localized in the IM while the activity of ADC rose both in the node and the IM above it. The node also contained low levels of ODC activity which increased slightly following submergence. Increased activities of polyamine-synthesizing enzymes in the nodal region of submerged plants probably resulted from the promotion of adventitious root formation in the node. Treatment of excised rice-stem sections with ethylene or GA₃ enhanced the activities of SAMDC and ADC in the IM and inhibited the decline in the levels of putrescine and spermidine. We conclude that SAMDC and perhaps also ADC may serve as biochemical markers for the enhancement of cell-division activity in the IM of deepwater rice.Abbreviations ADC arginine decarboxylase - GA gibberellin - IM intercalary meristem - ODC ornithine decarboxylase - SAM S-adenosylmethionine - SAMDC SAM decarboxylase     

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.     

Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by α-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.     

Properties of purified chloroplastic and cytoplasmicvalyl- and leucyl-tRNA synthetases from Euglena gracilis

The catalytic properties of purified chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetasesfrom Euglena gracilis were studied and compare     

Importance of polyamines in cell cycle kinetics as studied in a transgenic system

Polyamines are organic cations, which are considered essential for normal cell cycle progression. This view is based on results from numerous studies using a variety of enzyme inhibitors or polyamine analogues interfering with either the metabolism or the physiological functions of the polyamines. However, the presence of non-specific effects may be hard to rule out in such studies. In the present study, we have for the first time used a transgenic cell system to analyze the importance of polyamines in cell growth. We have earlier shown that expression of trypanosomal ODC in an ODC-deficient variant of CHO cells (C55.7) supported growth of these otherwise polyamine auxotrophic cells. However, one of the transgenic cell lines grew much slower than the others. As shown in the present study, the level of ODC activity was much lower in these cells, and that was reflected in a reduction of cellular polyamine levels. Analysis of cell cycle kinetics revealed that reduction of growth was correlated to prolongation of the G₁, S, and G₂ + M phases in the cells. Providing exogenous putrescine to the cells resulted in a normalization of polyamine levels as well as cell cycle kinetics indicating a causal relationship.     

Thymidylate synthetase during synchronous nuclear division cycle and differentiation of Physarum polycephalum

Thymidylate synthetase and thymidine kinase activities in wild type strain M₃b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M₃b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism.     

Analysis of a polymorphic region of the Euglena gracilis chloroplast genome

The circular chloroplast genome of Euglena gracilis is known to carry a single region of variable length (polymorphic region) located on BglII·Z fragments (6.1–6.9 kbp). We now have cloned a BglII·Z fragment (6.35 kbp) using as vector a modified pBR322. The cloned BglIl·Z fragment is split by HindIII into fragments of 1.0, 2.6 and 2.75 kbp and by HaeIII into fragments of 0.05, 1.35 and 4.95 kbp, respectively. The zone of variable length on this BglIl·Z fragment can be confined to a BglII-HindIII fragment of 2.6 kbp which is next to the previously mapped BglIl·G₁ (4.7 kbp) and about 4.5 kbp away from the 5′ end of the ‘extra’ 16 S rRNA gene. Two HindIII fragments from the polymorphic region of 5.9 and 6.1 kbp, respectively, were cloned and used in electron microscopic studies. Heteroduplexes formed between the two cloned HindIII fragments show the expected size difference of about 200 bp. Single strand reannealing experiments allow us to place the polymorphic region between two previously mapped short inverted repeats and partial DNA melting experiments indicate that the polymorphic region is very rich in dA-dT.     

The occurrence of 1-O-methyl-2-demethyl-phytylplastoquinol in Euglena gracilis

The isolation and characterization of 1-O-methyl-2-demethylphytylplastoquinol from photoheterotrophic cells of Euglena gracilis is described.     

Rapid loss of ornithine decarboxylase activity during encystation of Entamoeba invadens

Abstract Stimulation of encystation of Entamoeba invadens by incubation of trophozoites under glucose-limiting conditions brought about a dramatic fall of ornithine decarboxylase activity, a key enzyme in polyamine biosynthesis. Levels of enzyme specific activity after 24 and 48 h of encystation represented only 11% and 1.3%, respectively, of those detected at the start of incubation. Induction of encystation in the presence of exogenously added polyamines resulted in a marked reduction in cyst formation. Thus, after 72 h of incubation, 1.0 mM putrescine, 1.0 mM spermidine or 0.5 mM spermine reduced encystation by 48 to 56%. Inhibition was enhanced to 70–73% in response to a two-fold increase in the concentration of either putrescine or spermine. Our results indicate that polyamine biosynthesis from ornithine is rapidly turned off at the onset of encystation.     

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines

Summary Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [³H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.     

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro-morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.