Dimitrios A. Kyriakidis

The regulation and function of ornithine decarboxylase and of the polyamines.

Publisher Summary This chapter highlights the regulation and function of ornithine decarboxylase (ODC) and of the polyamines. ODC catalyzes the synthesis of putrescine. It is necessary to distinguish among several possible functions of polyamines, to define the state of the cell at the time of induction, and to differentiate among various modes of induction of ODC activity. The chapter discusses a few new aspects of the regulation and function of ODC and of the polyamines and some of the uncertainties in the field. It also presents areas that are closely related to the regulation and the interrelationships of the polyamines in a variety of physiological and pathological states. The chapter also explores the possible functions of the polyamines.

Optimization of xanthan gum production by Xanthomonas campestris grown in molasses

Xanthan gum production by Xanthomonas campestris ATCC 1395 using sugar beet molasses as carbon source was studied. The pre-treatment of sugar beet molasses and the supplementation of the medium were investigated in order to improve xanthan gum production. Addition of K2HPO4 to the medium had a significant positive effect on both xanthan gum and biomass production. The medium was subsequently optimized with regard to molasses, K2HPO4 concentration and initial pH. Maximum xanthan gum production (53 g/l) was observed after 24 h at 175 g/l molasses, 4 g/l K2HPO4 and at neutral initial pH. Results indicate that K2HPO4 serves as a buffering agent as well as a nutrient for the growth of X. campestris. Sugar beet molasses appears to be a suitable industrial substrate for xanthan gum fermentations.

Xanthan production by Xanthomonas campestris in batch cultures

The kinetics of growth and xanthan production by Xanthomonas campestris ATCC 1395 in batch culture were studied in a laboratory fermenter without pH control. Fermentations were carried out over a range of stirrer speeds (100–600 rpm) and the pyruvate content, as well as the molecular weight of the product were estimated. Increased agitation levels resulted in higher production rates and biomass levels, while product formation in this fermentation appeared to be partly growth associated. The chemical structure of xanthan was influenced by agitation, as the pyruvate content increased with increasing stirrer speeds. However, no significant effect was observed on xanthan molecular weight as the stirrer speed increased from 100 to 600 rpm.

L-asparaginase of Thermus thermophilus: Purification, properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.

Interactions of the antizyme AtoC with regulatory elements of the Escherichia coli atoDAEB operon

AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here, we show that the same cis elements control both promoter inducibility and AtoC binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at -146 to -107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the sigma54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth: Review

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each others biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S20/L26 and L34, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.

Superinduction of cytosolic and chromatin-bound ornithine decarboxylase activities of germinating barley seeds by actinomycin D

Putrescine in plant cells is formed either from Larginine by L-arginine decarboxylase (EC 4.11.19, ADC) or directly from L-ornithine by L-ornithine decarboxylase (EC 4.11.17, ODC) [1-5]. The contribution of ODC to the formation of polyamines in plants was claimed to be insignificant, since ODC activity in most plant tissues was found to be much lower than that of ADC [6]. The only welldocumented work on ODC activity in plant cells is that in [4] on rapidly proliferating plant cells. Investigations to now on plant ODC were performed in the 10000 × g supernatant of plant tissue homogenates based on the assumption that ODC is a cytosolic enzyme. Here, evidence is presented that in barley seeds germinated for > 90 h, ODC activity is located mainly in the nucleus, tightly bound to chromatin, although the cytosol also possesses considerable activity. Both activities are superinduced when seed germination takes place in the presence of gibberellic acid and actinomycin D.

Structure-Activity Relationships for Some Diamine, Triamine and Schiff Base Derivatives and Their Copper(II) Complexes.

Ethylenediamine (en), putrescine (pu), diethylenetriamine (dien), dipropylenetriamine (dpta), spermidine (spmd) and their CuII compounds as well as the Schiff bases with 2-furaldehyde (dienOO), 2- thiophenecarboxaldehyde (dienSS) and pyrrole-2-carboxaldehyde (dienNN) of dien and that of dpta with 2- thiophenecarboxaldehyde (dptaSS), were prepared and characterised. They were tested against Bacillus substilis, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Xanthomonas campestris as antibacterial reagents, the highest activity being exhibited by Cu(dptaSS)(NO3)2 complex, which acts as antibiotic. In the antiproliferative tests (vs. T47D,L929 and BHK21/c13 cell lines) the best results were obtained with Cu(dptaSS)2+ and Cu(dienSS)2+. Electronic structure calculations gave for dptaSS and dienSS the higher negative charges on the N atoms. The counter-ions (Br-, NO3- and SO42-) play an important role by modulating the reagents selectivity versus the bacteria [Gram(+) or Gram(-)], but they have no effect on the antiproliferative activity.

DNA interaction studies and evaluation of biological activity of homo- and hetero-trihalide mononuclear Cu(II) Schiff base complexes. Quantitative structure-activity relationships.

A new series of mixed-ligand mono- or hetero-trihalide Cu(II) complexes of the type [Cu(dienXX)Y(YZ(2))], where dienXX=Schiff dibase of diethylenetriamine with 2-thiophene-carboxaldehyde (dienSS), 2-furaldehyde (dienOO) or 2-pyrrole-2-carboxaldehyde (dienNN), Y=Cl, Br and Z=Br, I was synthesized by the reaction of the precursors of the type [Cu(dienXX)Y]Y with iodine or bromine in 1:1 molar ratio. The distorted square pyramidal configuration of the new homo- and hetero-trihalide Cu(II) mononuclear complexes was identified by C, H, N, Cu analysis, spectroscopic methods (IR, UV-visible), molar conductivity and magnetic measurements. The basal plane consists of three nitrogen atoms of the Schiff base and one halogen (terminal) atom while another axially located trihalogen moiety occupies the fifth side of the square pyramid as a YZ(2) entity, adopting an almost linear configuration. The equilibrium geometry of these complexes was further corroborated by theoretical studies at the B3LYP/DGDZVP level. A series of quantum chemical descriptors (e.g. SOMO (singly occupied molecular orbital) LUMO (lowest occupied molecular orbital), SOMO and LUMO energies, SOMO-LUMO gap, dipole moment, polarizability, molar volume, etc.) have been utilized in order to deduce quantitative structure-activity relationships (QSARs). The effect of the new compounds on the single stranded (ss), double stranded (ds) and pDNA led either to the formation of a DNA-complex cationic adduct, or to its degradation, evidenced by DNA electrophoretic mobility and DNA interaction spectroscopic titration studies. Moreover, the antimicrobial activity of Cu(II) complexes against Gram(+) and Gram(-) bacteria can be attributed to the synergistic action of the dissociation species, namely the cationic [Cu(dienXX)Y](+) and anionic [YZ(2)](-) ones. Finally, de Novo linear regression analysis correlating the bioactivity of these complexes with their structural substituents has been carried out, leading to some interesting qualitative observations/conclusions.

Hyperalkaline and thermostable phosphatase in Thermus thermophilus

The phosphatases existing in the extreme thermophilic bacteriumThermus thermophilus have been studied. Utilizing ion exchange, hydrophobic, pseudoaffinity, and affinity chromatography, a number of distinct phosphatase activities were identified. At least four phosphatases, with optimum pH ranging between 5.0 and 11.5, were assayed withp-nitrophenylphosphate, and two with optimum pH between 7.0 and 11.0, with32P-casein as substrate. The authors have focused on the hyperalkaline phosphatase and have tried to purify and characterize it. This hyperalkaline phosphatase reaches a maximal level at the stationary phase of the growth, and is co-purified with alkaline phosphatase with optimum pH of 10.2. The enzymes present a relative mol wt of 65 and 58 kDa, respectively, as judged by SDS-PAGE and Sephadex G-150 column, and possess similar properties, indicating that they are isoforms. These enzymes barely function in the presence of tartrate, and are inhibited by EDTA, pyrophosphate, and molybdate. Among the metals tested, Hg2+ appeared as the strongest inhibitor of the hyperalkaline phosphatase. The two enzymes are thermostable and, upon treatment at 90°C for 10 min, 75% of their activity remains. The physiological role and function of these phosphatases need further investigation.