Dimerization of the AtoC response regulator and modelling of its binding to DNA.

Bacterial signal transduction systems can be viewed as an entity of multi-sensory and output domains, whereas the functions of response regulators play a pivotal role in the complex network interactions. One crucial property among response regulators functions is their oligomerization and subsequent binding to DNA. The AtoS-AtoC two component system, functionally modulated by various agents, influences fundamental cellular processes such as short-chain fatty acid catabolism and poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli. Among the already reported characteristic properties, AtoC binds to a specific site, a palindromic repeat of 20 nucleotides within the atoDAEB promoter. Since experimental structures of AtoC or its complex with DNA are not yet available, an almost complete homology model of AtoC and of its putative entity as a dimer is constructed for this study, as well as a model of its binding to its target DNA sequence. The latter is associated with large conformational changes, as shown by molecular dynamics simulations. Subsequent biochemical study, including cross-linking via chemical agents, revealed the ability of AtoC to form oligomers in vitro.

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.

Effect of histamine on the signal transduction of the AtoS-AtoC two component system and involvement in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli.

AtoS-AtoC two-component system acts directly on the atoDAEB operon transcription to regulate the biosynthesis of short-chain poly-(R)-3-hydroxybutyrate. This study sought to investigate the effect of histamine and compound 48/80 on the regulation of AtoS-AtoC two-component system in Escherichia coli K-12 MA255 (speC(-), speB(-)) and the isogenic E. coli strains BW25113 (atoSC(+)) and BW28878 (DeltaatoSC) transformed with plasmids carrying related genes. Histamine or compound 48/80 induced or tended to reduce atoC transcription, respectively, while neither compound showed any effect on atoDAEB operon transcription. Moreover, histamine down-regulated poly-(R)-3-hydroxybutyrate biosynthesis, whereas compound 48/80 up-regulated its biosynthesis, maximal induction being obtained in the presence of multiple copies of AtoS-AtoC. Interestingly, co-administration of histamine counteracted this inductive effect of compound 48/80. The reported data provide the first evidence for a differential modulator role of histamine and compound 48/80 on the AtoS-AtoC two-component system signaling in potentially pathogenic bacteria, leading to a new perspective on their symbiotic behavior.

A DING phosphatase in Thermus thermophilus.

Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 degrees C. The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3' phosphodiesterase (3'-exonuclease) activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism.

Molecular modeling and functional analysis of the AtoS-AtoC two-component signal transduction system of Escherichia coli.

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.

The unexpected formation of biologically active Cu(II) Schiff mono-base complexes with 2-thiophene-carboxaldehyde and dipropylenetriamine: crystal and molecular structure of CudptaSCl2.

Two novel mononuclear Cu(II) coordination compounds of the type [Cu(dptaS)Cl(2)] and [Cu(dptaS)Br(2)] (dptaS=1,3-propanediamine, N(1)-[3-aminopropyl]-N(3)-[2-thienylmethylidene] or Schiff mono-base of dipropylenetriamine with 2-thiophene-carboxaldehyde) were prepared by the hydrolysis of the di-bases, [Cu(dptaSS)Cl(2)] and [Cu(dptaSS)Br(2)] (dptaSS=1,3-propanediamine, N(1)-[2-thienylmethylidene]-N(3)-[[2-thienylmethylidene]aminopropyl] or Schiff di-base of dipropylenetriamine with 2-thiophenecarboxaldehyde) to mono-bases with the release of one aldehyde molecule and freeing of the -NH(2) group of the coordinated dpta ligand. The X-ray determined structure of the compound [Cu(dptaS)Cl(2)] was confirmed by spectroscopic methods, magnetic and molar conductivity measurements. The Cu(II) atom is a five-coordinated CuN(3)Cl(2) chromophore with three nitrogen atoms coming up from the (dptaS) ligand and two chlorine atoms completing the square pyramidal geometry. Antiproliferative activity of both novel compounds was examined against a panel of different normal and cancer cell lines (MRC5, HeLa, MCF7, HT-29 and T47D) and showed that the Cu(II) Schiff mono-bases exhibit increased activity as compared to the starting materials. In vitro studies of plasmid DNA (pDNA) and double stranded DNA (dsDNA) interaction with the compounds under study support this difference. Some of the important factors contributing to the antiproliferative activity of the compounds under study, such as ionic character and dipole moment were also discussed in terms of the density functional theory calculated electronic structures of the ligands and their Cu(II) compounds.

Copper(II) Schiff base coordination compounds of dien with heterocyclic aldehydes and 2-amino-5-methyl-thiazole: synthesis, characterization, antiproliferative and antibacterial studies. Crystal structure of CudienOOCl2.

A new series of coordination compounds of the starting materials [Cu(dienX(2)Y(2))] and their adducts [Cu(dienXXY(2))(2a-5mt)] (where dien=diethylenetriamine, dienXX=Schiff bases of diethylenetriamine with 2-furaldehyde or 2-thiophene-carboxaldehyde, X=O, S, Y=Cl, Br, NO(3) and 2a-5mt=2-amino-5-methylthiazole) were synthesized by stepwise reactions and their structures were established by C, H, N, Cu analysis, spectroscopic, magnetic and molar conductivity measurements. The isolated compounds are monomers, paramagnetic and electrolytic compounds of the type 1:1. In all cases, the pentadentate Schiff base (dienXX) is bonded in a tridentate fashion through the 3 N atoms. In the CudienXXY(2) compounds the coordination sphere is completed by two Cl or Br or NO(3) groups in a square pyramidal arrangement. The proposed structure for this type of compound was further supported by X-ray diffraction analysis of the compound [Cu(dienOO)Cl(2)]. Its basal plane consists of three Cu-N contacts [2.017(2), 2.025(2) and 2.012(2) A] from dienOO, and the Cl(1) atom, while the Cl(2) atom possesses the apical position, the relevant distances being 2.2732(7) A for Cu-Cl(1) and 2.6051(7) A Cu-Cl(2). In the CudienX(2)Y(2).2a-5mt adducts the coordination sphere of copper is further completed by the nitrogen ring atom of the 2a-5mt, forming an octahedral configuration. The study of the biological activity of the compounds synthesized against a panel of different normal and cancer cell lines (MRC5, HeLa, MCF7, HT-29, OAW42, T47D) and bacteria (E. coli, B. cereus, B. subtilis) showed that the adducts of the type [Cu(dienXXY(2))(2a-5mt)] exhibit increased activity both in cancer cells and in bacteria, compared to the starting material of type [Cu(dienXXY(2))].

The effect of fused 12-membered nickel metallacrowns on DNA and their antibacterial activity.

The synthesis, characterization and the biological study of a series of Ni(ll)(2)(carboxylato)(2) [12- MC(Ni(II)N(shi)2(pko)2)-4][12-MC(Ni(ii)N(sh03(pko))-4] (CH(3)OH)(3)(H(3)O) fused 12-membered metallacrowns with 10 metal ions and commercial available herbicides or anti-inflammatory drugs as carboxylato ligands are reported. All the compounds have a mixed ligand composition with salicylhydroxamic acid and di-2-pyridylketonoxime as chelate agents. The compounds construct metallacrown cores {[12-MC(Ni(n)N(sj02(pko)2)-4][12-MC(Ni(ll)N(shO3(pko))-4]}(2+) following the pattern [-Ni-O-N-](4). The neutral decanuclear [Ni(II)(A)](2)[12-MC(Ni(II)N(shi)2(pko)2)-4][12-MC(Ni(II)N(pko)3(pko))-4] fused metallacrown, consists of two [12-MC(M(ox)N(ligand))-4] units the {Ni(ll)(A)[12-MC(Ni(II)N(shi)2(pko)2)-4]} and {Ni(II)(A)[12-MC(Ni(II)N(shi)3(pko))-4]} with 1(+) and 1(-) charge, respectively. Each metallacrown unit has four ring Ni(II) ions and one additional encapsulated Ni(II) ion in planar arrangement. The anionic unit is bonded with cationic one creating binuclear moieties. The herbicide or antiiflammatory carboxylato ligands are bridging the central octahedral nickel atom with a ring metal ion in a bindetate fashion. The effect on DNA and their antibacterial activity was examined. The changes in the mobility can be attributed to the altered structures of the pDNA treated with Ni(II) complexes. Evaluating the data of the antibacterial activity of the compounds tested, we can conclude that nickel complexes present strong antibacterial activity.

A kinetic model describing cell growth and production of highly active, recombinant ice nucleation protein in Escherichia coli.

A structured kinetic model, which describes the production of the recombinant ice nucleation protein in different conditions, was applied. The model parameters were estimated based on the variation of the specific growth rate and the intracellular product concentration during cultivation. The equations employed relate the cellular plasmid content or plasmid copy number with the cloned-gene expression; these correlations were successfully tested on the experimental data. The optimal nutrient conditions for the growth of Escherichia coli expressing the inaZ gene of Pseudomonas syringae were determined for the production of active ice nucleation protein. The kinetics of the cultures expressing the inaZ gene were studied in a bioreactor at different growth temperatures and nutrient conditions.

Antiproliferative activity of mixed-ligand dien-Cu(II) complexes with thiazole, thiazoline and imidazole derivatives.

The reaction of [Cu(dien)NO(3)]NO(3) with 2-amino-5-methylthiazole (2A5MT), 2-amino-2-thiazoline (2A-2Tzn), imidazole (im), N,N'-thiocarbonyldiimidazole (Tcdim), 2-aminothiazole (2AT) and 2-ethylimidazole (2Etim), gave a new series of mixed-ligand compounds of the general formula [Cu(dien)(B)NO(3))]NO(3); (dien, diethylenetriamine; B, 2A5MT, 2A-2Tzn, im, Tcdim, 2AT and 2Etim). The complexes have been characterised by elemental analysis, molar conductivity and magnetic measurements, as well as by electronic and IR spectral studies. According to the above measurements the possible structure of the compounds is the square pyramidal in the solid state and the square planar in aqueous solution. We tested all complexes for antiproliferative (cytostatic and cytotoxic) activity against a panel of cell lines (HeLa, L929, HT-29 and T47D). All [(dien)Cu(B)NO(3))](NO(3)) complexes had an activity against colon cancer cells (HT-29), inducing G2/M cell cycle arrest, an effect that for most of the complexes could be attributed to p34cdc2 inhibition by tyrosine-phosphorylation and/or to induction of (cyclin-dependent kinase inhibitor) p21(WAF1). Other cell lines were resistant to the majority of the complexes, except [Cu(dien)(2A5MT)NO(3))](NO(3)), that had showed the highest anti-proliferative activity against HT-29 cells also. The predilection for colon cancer cells and the relatively low toxicity against normal (L929) cells justify further investigation of this group of compounds.

L-asparaginase of Thermus thermophilus: purification, properties and identification 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 degrees C. Chemical modification experiments implied the existence ofhistidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus.

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60 degrees C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.

Xanthan gum production by Xanthomonas campestris w.t. fermentation from chestnut extract.

Xanthomonas campestris w.t. was used for production of xanthan gum in fermentations with chestnut flour for the first time. Fermentations were carried out with either chestnut flour or its soluble sugars (33.5%) and starch (53.6%), respectively, at 28 degrees C and 200 rpm at initial pH 7.0 in flasks. The effect of agitation rate (at 200, 400, and 600 rpm) on xanthan gum production was also studied in a 2-L batch reactor. It was found that xanthan production reaches a maximum value of 3.3 g/100 mL at 600 rpm and 28 degrees C at 45 h.

Anti-inflammatory drugs interacting with Zn(II), Cd(II) and Pt(II) metal ions.

Complexes of Zn(II), Cd(II) and Pt(II) metal ions with the anti-inflammatory drugs, 1-methyl-5-(p-toluoyl)-1H-pyrrole-2-acetic acid (Tolmetin), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (Ibuprofen), 6-methoxy-alpha-methylnaphthalene-2-acetic acid (Naproxen) and 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid (indomethacin) have been synthesized and characterized. In the structurally characterized Cd(naproxen)2 complex the anti-inflammatory drugs acts as bidentate chelate ligand coordinatively bound to metal ions through the deprotonated carboxylate group. Crystal data for 1: [C32H26O8Cd], orthorhombic, space group P22(1)2(1), a = 5.693(2) (A), b = 8.760(3) (A), c = 30.74(1) (A), V = 1533(1) A3, Z = 2. Antibacterial and growth inhibitory activity is higher than that of the parent ligands or the platinum(II) diamine compounds.

Bis(acetato)bis(1-methyl-4,5-diphenylimidazole)copper(II): preparation, characterization, crystal structure, DNA strand breakage and cytogenetic effect.

The preparation, characterization and antitumour properties of the complex [Cu(O2CMe)2L2] (1), where L = 1-methyl-4,5-diphenylimidazole, are described. The crystal structure of 1 (triclinic, space group P1, a = 6.743(1), b = 8.006(1), c = 15.898(1) A, alpha = 102.87(1), beta = 101.10(1), gamma = 76.76(1) degree, Z = 1) has been determined (R = 0.0254, Rw = 0.0275). In the centrosymmetric complex the copper ion is in an essentially square planar environment consisting of two pyridine-type imidazole nitrogen atoms and an oxygen atom from each acetate ligand; the second oxygen atoms of the carboxylate functionalities are involved in weak interactions with the metal completing the coordination to a very distorted tetragonal bipyramid. Complex 1 has been also characterized by elemental analyses, thermal methods, variable-temperature magnetic susceptibility and spectroscopic (IR and far-IR, FT-Raman, UV/VIS, EPR) techniques. The effect of the complex on the in vitro DNA strand breakage was examined. It was found that 1 causes degradation on the linearized pKS DNA, ds and ss DNA. High concentrations of this Cu(II) complex cause scissions on the relaxed and the supercoiled DNA. Furthermore, the in vivo cytogenic effect of 1 was examined on human lymphocyte cells. This study presents indications that 1 could have some relevance in the treatment of tumour cell lines. An orbital interpretation of the interaction of 1 with the DNA bases is proposed.

Phospholipid analysis and fractional reconstitution of the ice nucleation protein activity purified from Escherichia coli overexpressing the inaZ gene of Pseudomonas syringae.

Ice nucleation protein was partially purified from the membrane fraction of E. coli carrying inaZ from Pseudomonas syringae. The ice nucleation protein was totally localized in the bacterial envelope and was extracted by either salt (0.25 M NH4Cl) or the nonionic detergent Tween 20. The extracted protein was partially purified by sequential passage through DEAE-52 cellulose and Sephacryl-S400 columns. The activity of the purified protein was lost after treatment with phospholipase C, and its activity was subsequently restored by addition of the naturally occurring lipid phosphatidylethanolamine. These results suggest that ice nucleation proteins have a requirement for lipids that reconstitute a physiological hydrophobic environment similar to the one existing in vivo, to attain and maintain a structure that enables ice catalysis.

In vitro combined effect of a new series of copper (II) complexes with cisplatin or epirubicin on human breast and cervical cancer cell lines.

The in vitro cytotoxic effects of some recently synthesized copper (II) complexes were investigated in combination with two clinically important anticancer agents, cisplatin (CDDP) and epirubicin (EPR), on three human breast cancer cell lines (BT20, MCF7 and T47D), a human cervical carcinoma cell line (HeLa) and two non-tumour cell lines (BHK-21 and L929). The in vitro antiproliferative effects were measured by means of XTT assay and drug potency was expressed in terms of IC50 values. Synergistic effects were observed for EPR in combination with a Cu(II) malonate derivative in BT20, MCF7 and HeLa cells. Antagonistic effects were apparent when CDDP combined with a pivalate derivative in HeLa cells. For other drug combinations, synergism, adjuvance or antagonism was demonstrable depending on the cell line applied or the varied administration schedules. The results suggest that these novel complexes may enhance the cytotoxic activity of CDDP or EPR against human tumour cell lines when administered in combination with them.

Antiproliferative activity of synthetic tetrapeptides, analogs of AS-I phytotoxin, towards cancer cell lines.

The in vitro chemosensitivity of three cancer cell lines [HT29 (colon), HeLa (cervical) and T47D (breast)] to eight synthetic tetrapeptides, analogs of AS-I toxin, with phytotoxic effect on a series of plants was studied. Mouse fibroblast L929 cell line was also tested for chemosensitivity to these peptides. All cell lines were especially sensitive to Cys-Val-Gly-Glu tetrapeptide with IC50 values of 0.18, 0.3 and 0.63 mM for HT29, HeLa and T47D cells, respectively, whereas the IC50 value for the L929 cells was higher than 1 mM. Antiproliferative activity was also observed with peptides Tyr-Val-Gly-Glu and His-Val-Gly-Glu with IC50 values higher than those obtained for Cys-Val-Gly-Glu. For the rest of the peptides tested the IC50 values were found close to or higher than 3 mM.