In vitro and in silico evaluation of the inhibitory effect of a curcumin-based oxovanadium (IV) complex on alkaline phosphatase activity and bacterial biofilm formation.

The scientific interest in the development of novel metal-based compounds as inhibitors of bacterial biofilm-related infections and alkaline phosphatase (ALP) deregulating effects is continuous and rising. In the current study, a novel crystallographically defined heteroleptic V(IV)-curcumin-bipyridine (V-Cur) complex with proven bio-activity was studied as a potential inhibitor of ALP activity and bacterial biofilm. The inhibitory effect of V-Cur was evaluated on bovine ALP, with two different substrates: para-nitrophenyl phosphate (pNPP) and adenosine triphosphate (ATP). The obtained results suggested that V-Cur inhibited the ALP activity in a dose-dependent manner (IC50 = 26.91 ± 1.61 µM for ATP, IC50 = 2.42 ± 0.12 µM for pNPP) exhibiting a mixed/competitive type of inhibition with both substrates tested. The evaluation of the potential V-Cur inhibitory effect on bacterial biofilm formation was performed on Gram (+) bacteria Staphylococcus aureus (S. aureus) and Gram (-) Escherichia coli (E. coli) cultures, and it positively correlated with inhibition of bacterial ALP activity. In silico study proved the binding of V-Cur at eukaryotic and bacterial ALP, and its interaction with crucial amino acids of the active sites, verifying complex's inhibitory potential. The findings suggested a specific anti-biofilm activity of V-Cur, offering a further dimension in the importance of metal complexes, with naturally derived products as biological ligands, as therapeutic agents against bacterial infections and ALP-associated diseases. KEY POINTS: • V-Cur inhibits bovine and bacterial alkaline phosphatases and bacterial biofilm formation. • Alkaline phosphatase activity correlates with biofilm formation. • In silico studies prove binding of the complex on alkaline phosphatase.

Administration of the extra virgin olive oil (EVOO) in mild cognitive impairment (MCI) patients as a therapy for preventing the progress to AD.

OBJECTIVE: Although Mediterranean diet is connected with longevity and lower rate of many disorders including Alzheimer's disease (AD), the effect of olive oil, which is the principal component of the Mediterranean diet, on fibrinolytic system related to AD and especially on plasminogen activator inhibitor-1 (PAI-1) and a2-antiplasmin in aged participants are not yet examined. This study was performed on 108 aged participants allocated into 5 groups: Mild Cognitive Impairment (MCI) (36) patients subjected to 1-year therapy with extra virgin olive oil (EVOO), MCI without therapy patients (26), MCI without therapy 1-year later patients (11), AD patients (30) and healthy individuals (16). Hypothesis/Purpose: To examine the effect of EVOO therapy on the fibrinolytic factors PAI-1 and a2-antiplasmin, on hallmarks of AD, tau and Aß amyloid fragments and on an oxidative stress biomarker, MDA in the serum of MCI patients aiming to be exploited as a future preventive therapy. RESULTS: Using ELISA method, the levels of both fibrinolytic factors PAI-1 and a2- antiplasmin in the serum of MCI patients were reduced notably in the EVOO treated patients versus the control group and were lower than those of all other groups. For better determination of AD from other pathological conditions the ratio Aß1-42/Aß1-40 was measured in serum of all participants. The more lessened the ratio is, the more cognitive impairment is observed in patients. The MCI group with one-year EVOO therapy displayed a ratio similar to this of healthy individuals. Moreover, patients with EVOO therapy showed decreased tau protein levels in comparison with all the other groups. The levels of the oxidative stress's biomarker, malondialdehyde (MDA) showed a significant decrease in MCI patients subjected to EVOO therapy revealing the involvement of the beneficial antioxidative properties of EVOO in the progression of AD. CONCLUSION: We demonstrated that EVOO therapy may prevent the risk of patients with MCI to progress to AD via decreasing fibrinolytic factors PAI-1 and a2 antiplasmin that reflecting in the diminution of the hallmarks proteins of AD, tau and Aß amyloid as well and in a biomarker of oxidative stress, MDA.

Silver(I) complexes of N-methylbenzothiazole-2-thione: synthesis, structures and antibacterial activity.

Three silver(I) complexes containing N-methylbenzothiazole-2-thione (mbtt) have been prepared and structurally characterized by X-ray single-crystal analysis. Silver(I) nitrate, and silver(I) triflate react with mbtt to give homoleptic complexes of formula [(mbtt)2Ag(µ-mbtt)2Ag(mbtt)2](NO3)2 (1) and [Ag(mbtt)3](CF3SO3) (2) respectively, while silver(I) chloride gives the binuclear halide-bridged [(mbtt)2Ag(µ2-Cl)2Ag(mbtt)2] (3). In the binuclear complex 1 the two metal ions, separated by 3.73 Å from each other, are doubly bridged by the exocyclic S-atoms of two mbtt ligands, with the tetrahedral environment around each silver ion being completed by the S-atoms of two terminally bonded mbtt units. Compound 2 is mononuclear with the metal ion surrounded by the exocyclic S-atoms of three mbtt ligands in a nearly ideal trigonal planar arrangement. The new complexes showed significant in vitro antibacterial activity against certain Gram-positive and Gram-negative bacterial strains.

Copper(I) halide complexes of N-methylbenzothiazole-2-thione: synthesis, structure, luminescence, antibacterial activity and interaction with DNA.

The reactions of copper(I) halides, CuX (X=Cl, Br, I) with N-methylbenzothiazole-2-thione (mbtt), independent of the molar ration chosen (1:2 or 1:3), led to the formation of dinuclear complexes of the formula [CuX(mbtt)2]2, whereas the reactions of CuX and mbtt in the presence of two equivalents of triphenylphosphine (PPh3) afforded mononuclear mixed-ligand complexes of the formula [CuX(PPh3)2(mbtt)]. The molecular structure of a representative compound from each of the two above types of complexes, namely [CuCl(mbtt)2]2 and [CuI(PPh3)2(mbtt)] have been established by single-crystal X-ray diffraction. The new complexes are strongly emissive both in the solid state and in solution. The complexes were also screened for antibacterial activity and their ability to interact with native calf thymus DNA (CT-DNA) in vitro. Both types of complexes showed significant activities against all the bacteria tested as compared to that of standard antibiotic ampicillin, however, the three mixed-ligand complexes including triphenylphosphane as ligand exhibited perceptibly stronger antibacterial activity than the three homoleptic ones possessing only the mbtt ligand. DNA electrophoretic mobility experiments showed that all complexes bind to CT-ds DNA resulting in high molecular weight complexes ending in DNA degradation.

On the Thermus thermophilus HB8 potential pathogenicity triggered from rhamnolipids secretion: morphological alterations and cytotoxicity induced on fibroblastic cell line.

A limited number of bacterial strains usually grown under nutrient limitation secrete rhamnolipids (RLs), which are recorded as virulence factors that are implicated in the pathogenicity of a microorganism. The non-pathogenic T. thermophilus HB8 produces extracellular rhamnolipids (TthRLs) under defined cultivation conditions using sunflower seed oil and sodium gluconate as carbon sources. In particular, the secreted TthRLs have been isolated, purified and identified with ATR-FTIR. Their effects on the cells' viability were examined when they were supplemented in a culture of human skin fibroblasts. Purified TthRLs triggered a sequence of rapid and pronounced morphological alterations characterized by transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation, rounding up, distortion of nuclei and loss of lamellar processes, and finally disruption of membrane. The addition of TthRLs in the cultured fibroblasts caused cytotoxicity, in contrast to that of rhamnose that stimulated viability, as it was assessed by MTT test. These results revealed that among the constituents of RLs that are implicated in the cytotoxicity, it has to be attributed to the lipidic chain variation and not to the carbohydrate part. TthRLs cytotoxicity on fibroblasts is comparable, and provoked similar effects, to that caused by saponin white, a known surfactant. TthRLs secretion might be a crucial point for the transformation of a non-pathogenic bacterium to a pathogenic one under certain environmental conditions favoring their secretion. RLs secretion in the microorganism's world might be a general route for the passage in the pathogenicity to ensure their survival under nutrient limitation conditions.

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.

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.

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.

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.

Synthesis and cytogenetic effects of aminoquinone derivatives with a di- and a tripeptide.

Quinones are of significant interest due to their important role in specific cellular functions. Quinoproteins are a big class of oxyreductive agents occurring in bacteria and other organisms. In this investigation derivatives of 2-amino-1,4-benzoquinone, 2-amino-1,4-naphthoquinone and 2-amino-5,8-dihydroxy-1,4-naphthoquinone with a di- and a tripeptide were prepared for first time. The effect of the synthesized compounds on sister chomatid exchange (SCE) rates and human lymphocyte proliferation kinetics on a molar basis was studied. Among these coupled products the most effective in inducing SCEs and depressing proliferation rate indices is the coupling product of 2-amino-1,4-naphthoquinone with the tripeptide GHK (10). Next in order of magnitude in inducing cytogenetic effects is 2-amino-1,4-naphthoquinone (2) and its coupling products with glycine and serine (4 and 5), while the rest displayed marginal activity.

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.

A combined biochemical and cytogenetic study of thioridazine-induced damage to nucleic acids.

In this work the biochemical effects of thioridazine, a commonly used phenothiazine, have been studied upon native double- and single-stranded DNA and also upon a supercoiled plasmid. The results indicate that thioridazine causes damage and scissions to these nucleic acids but only at concentrations much higher than the one used in our cytogenetic experiments and that the damage seems to depend on the concentrations used. Furthermore, we studied the action of thioridazine alone or in combination with caffeine and/or melphalan upon human lymphocytes in vitro. Thioridazine and caffeine (a well-known inhibitor of cellular repair mechanisms) were shown to act synergistically to potentiate the cytogenetic effect of melphalan on human lymphocytes. It is suggested that thioridazine alone or in combination with caffeine may exert its synergistic effect on melphalan cytotoxicity to cultured human lymphocytes not only indirectly, i.e. as a strong calmodulin inhibitor by facilitating the intracellular retention of melphalan, but also directly by reaction with nucleic acids and by causing scissions in and damage to them. Therefore, thioridazine (as chlorpromazine) has some potential as an adjuvant chemotherapeutic agent for the treatment of human cancer.

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.

Hyperalkaline and thermostable phosphatase in Thermus thermophilus.

The phosphatases existing in the extreme thermophilic bacterium Thermus 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 with p-nitrophenylphosphate, and two with optimum pH between 7.0 and 11.0, with 32P-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 degrees C for 10 min, 75% of their activity remains. The physiological role and function of these phosphatases need further investigation.