Supercoiled Plasmid Research Articles

Synthesis, DNA binding, topoisomerase I inhibition and antiproliferation activities of three new binuclear terpyridine platinum(II) complexes

Three binuclear platinum(II) complexes, 1 ([Pt2(L)Cl2]Cl2), 2 ([Pt2(L)Br2]Br2) and 3 ([Pt2(L)I2]I2) (where L = 1,4-bi-[4′-(4-Hydroxyphenyl)-2,2′,6′,2″-terpyridine] butane) were synthesized and characterized by nuclear magnetic resonance, elemental analysis and mass spectrometry. The crystal structure of the new ligand L was determined by single crystal X-ray crystallography, and π–π stacking interactions were revealed in the geometry. Interactions of complexes with calf thymus DNA have been investigated by UV–Vis spectroscopy and fluorescence spectroscopy. Due to the structural differences, binuclear complex 3 exhibited higher binding affinity for calf thymus DNA with a DNA-binding constant Kb of 8.72 × 106 M−1. The competitive study indicated that the ethidium bromide (EB) can displaced by complexes 1–3 from the DNA-EB conjugation. The concentration-dependence unwinding of supercoiled circular plasmid pBR322 DNA by complexes 1–3 was observed via agarose gel electrophoresis. Meanwhile, complexes displayed obvious activities for inhibition of topoisomerase I. Herein the halogen leaving groups Cl−/Br−/I− presented in 1–3 showed significant effect on DNA binding and topoisomerase I inhibition activities. An evaluation of in vitro cytotoxicity for three complexes was performed by MTT assay in two cancer cell lines and normal cells as the control, respectively.

Copper(II) complexes of bidentate mixed ligands as artificial nucleases: Synthesis, crystal structure, characterization and evaluation of biological properties

Mixed ligand transition metal complexes play a significant role in biological processes by interacting with small molecules and activating the enzymes for nuclease activity. Four mixed ligand copper(II) complexes [Cu(5-nsal)(bipy)(ClO4)] (1), [Cu(2-hnap)(bipy) (ClO4)] (2), [Cu(5-nsal)(biim)(ClO4)] (3) and [Cu(2-hnap)(biim)(ClO4)] (4) (bipy = 2,2′-bipyridyl, biim = 2,2′-bi-1H-imidazole, 5-nsal = 5-nitrosalicylaldehyde and 2-hnap = 2-hydroxynaphthaldehyde) were successfully synthesized and physico-chemically characterized. The X-ray quality single crystals of copper(II) complexes 1 and 2 showed that the copper atom was penta-coordinated with square-pyramidal geometry. The physicochemical characterizations of the copper(II) complexes also confirmed the formation of anticipated structures. The redox potentials of the complexes assessed by cyclic voltammetry showed a quasi-reversible redox behavior consistent with Cu(II)/Cu(I) reduction in the cathodic region. The results of ctDNA binding studies using absorption, fluorescence and circular dichroism spectra exposed that the synthesized complexes interact via intercalation mode. The molecular docking studies (in silico) showed that complexes interact via major grooves which supported the results obtained from in vitro DNA binding studies. The nuclease activity of the complexes showed a moderate cleave of the supercoiled plasmid pBR322 DNA in presence of reducing agent (MPA) via hydroxy radical formation. All the copper (II) complexes exhibited a significant antimicrobial activity against two gram-positive, two gram-negative bacterial stains and two fungal species.

Noncanonical DNA-binding properties of potential components of an antitumor composition (cytokine AIMP1/p43, lectin SNA, izatizon), which are able to affect different targets

Aim. To search the DNA-binding properties of various drugs promising for the creation of the antitumor composition (AIMP1/p43, SNA-I, izatizon), as well as their ability to influence on key matrix processes – DNA transcription and replication, which are a sensitive target for the action of many pharmacological drugs. Methods. The recombinant protein AIMP1/p43 was obtained from supernatant of E.coli lysed cells by metallic chelating chromatography on a Ni‑NTA‑agarose column. Also there were used the elderberry bark lectin SNA-I (LECTINOTEST, Ukraine), and the preparation of own production izatizon (IMBG NAS of Ukraine). The DNA-binding ability of the drugs was investigated by EMSA. As a transcription test system, an enzyme complex of DNA-dependent RNA polymerase of T7 bacteriophage and plasmid DNA pTZ19R * were used. PCR with Taq DNA polymerase and the plasmid pTZ19R * was used as the model replicative system. Results. Experimental conditions, in which the specificity of each drug action appeared, were found: the cytokine AIMP1/p43 and the SNA-I demonstrated the ability to bind to DNA by the EMSA test; izatizon induced structural changes in DNA, blocked the amplification of DNA during the PCR process, and also in vitro transcription with the participation of DNA-dependent RNA polymerase of the bacteriophage T7. Conclusions. Each of the selected drugs, which according to the literature data have antitumor potential, has demonstrated the ability to interact with the model supercoiled plasmid DNA pTZ19R *. The different ways of the selected drugs influence on DNA allows us to expect their effective combined action and synergism in case of their application as a common antitumor composition.
 Keywords: cytokine AIMP1/p43, SNA-I, izatizon, EMSA method, transcription inhibiting, amplification blocking.

Characteristics of AccSTIP1 in Apis cerana cerana and its role during oxidative stress responses.

Various environmental stresses, such as heat shock, heavy metals, ultraviolet (UV) radiation and different pesticides, induce a cellular oxidative stress response. The cellular oxidative stress response is usually regulated by heat shock proteins (Hsps) acting as molecular chaperones. Stress-induced phosphoprotein 1 (STIP1), one of the most widely studied co-chaperones, functions as an adaptor that directs Hsp90 to Hsp70-client protein complexes. However, the biological functions of STIP1 remain poorly understood in honeybee (Apis cerana cerana). In this study, AccSTIP1 was identified in Apis cerana cerana. AccSTIP1 transcription was found to be induced by heat (42°C), HgCl2, H2O2 and different pesticides (emamectin benzoate, thiamethoxam, hexythiazox and paraquat) and inhibited by CdCl2, UV and kresoxim-methyl. Moreover, western blot analysis indicated that the expression profiles of AccSTIP1 were consistent with its transcriptional expression levels. The disc diffusion assay showed that chemically competent transetta (DE3) bacteria expressing a recombinant AccSTIP1 protein displayed the smaller death zones than did control bacteria after exposure to paraquat and HgCl2. The DNA nicking assay suggested that recombinant purified AccSTIP1 protected supercoiled pUC19 plasmid DNA from damage caused by a thiol-dependent mixed-function oxidation (MFO) system. After knocking down AccSTIP1 gene expression via RNA interference (RNAi), the transcript levels of antioxidation-related genes were obviously lower in dsAccSTIP1 honeybees compared with those in the uninjected honeybees. Collectively, these results demonstrated that AccSTIP1 plays an important role in counteracting oxidative stress. This study lays a foundation for revealing the mechanism of AccSTIP1 in the Apis cerana cerana antioxidant system.

Synthesis, characterization, DNA binding, cytotoxicity and molecular docking properties of Cu (II) and Mn (II) complexes with 1,4‐bis (pyrazol‐1‐yl) terephthalic acid

Two novel complexes, [Cu (L)(H2O)]⋅H2O (1) and [Mn (H2O)6] ⋅L ⋅H2O (2) (L = 1,4‐bis (pyrazol‐1‐yl) terephthalic acid), were synthesized under hydrothermal conditions. They were characterized using elemental analysis, infrared spectroscopy and single‐crystal X‐ray diffraction. Intramolecular weak interactions, such as hydrogen bonds, and intermolecular interactions play important roles in the construction of the complexes. The interaction of these complexes with fish sperm DNA (FS‐DNA) was monitored and binding constants were determined using UV–visible spectroscopy, which revealed their ability to bind to FS‐DNA, with binding constants for the two complexes of 1.88 × 104 M−1 (1) and 1.06 × 104 M−1 (2). Viscosity experiments further demonstrated the binding of the complexes to DNA. The complexes were further studied using gel electrophoresis assay with supercoiled plasmid pBR322 DNA. In addition, anticancer activities of the metal complexes investigated through MTT assays in vitro indicated good cytotoxic activity against cancer cell lines. Flow cytometry and apoptosis experiments showed that these complexes induced apoptosis of two different cancer cell lines (HeLa and KB cells), demonstrating a significant cancer cell inhibitory rate. Finally, a further molecular docking technique was employed to confirm the binding of the complexes towards the molecular target DNA.

Synthesis, DNA interaction, topoisomerase I, II inhibitory and cytotoxic effects of water soluble silicon (IV) phthalocyanine and napthalocyanines bearing 1-acetylpiperazine units

In this study, axially 1-acetylpiperazine substituted silicon (IV) phthalocyanine, naphthalocyanine 2, 3 and their water soluble derivatives 2a, 3a were synthesized for the first time and their DNA binding modes (absorption spectral, thermal denaturation and electrophoresis studies), DNA cleavage activities, topoisomerases inhibitory and cytotoxicity effects were investigated. Compounds 2a and 3a showed good binding propensity to CT-DNA with Kb values of 1.25 ± (0.01) × 104 and 1.13 ± (0.03) × 104 M−1. In DNA cleavage studies revealed that the compounds cleaved to supercoiled pBR322 plasmid DNA via the oxidative pathway at irradiation. The compounds inhibited topoisomerase I enzyme in a concentration-dependent manner whilst they had low inhibitory effects against topoisomerase II compared to doxorubicin as a positive control. In vitro studies of the cytotoxicity of the compounds on five carcinoma cell lines indicated that compound 3a had the potential to act as an anticancer drug with CC50 values of 7.83, 3.36, 3.18 and 3.36 μM toward BT-20, SNU-398, DU-145 and SK-MEL 128 compared to cis-platin as a positive control.

Efficient copper-based DNA cleavers from carboxylate benzimidazole ligands.

Four copper(II) coordination compounds from 2-benzimidazole propionic acid (Hbzpr) and 4-(benzimidazol-2-yl)-3-thiobutanoic acid (Hbztb) were synthesized and fully characterized by elemental analyses, electronic spectroscopy, FT-IR and mass spectrometry. The molecular structure for the four complexes was confirmed by single-crystal X-ray crystallography. The DNA-interacting properties of the two trinuclear and two mononuclear compounds were investigated using different spectroscopic techniques including absorption titration experiments, fluorescence spectroscopy and circular dichroism spectroscopy. Trinuclear [Cu3(bzpr)4(H2O)2](NO3)2·3H2O·CH3OH (2) and [Cu3(bzpr)4Cl2]·3H2O (3) bind to DNA through non-intercalative interactions, while for mononuclear [Cu(bzpr)2(H2O)]·2H2O (1) and [Cu(bztb)2]·2H2O (4), at minor concentrations in relation to the DNA, a groove binding interaction is favored, while at higher concentrations an intercalative mode is preferred. The nuclease properties of all complexes were studied by gel electrophoresis, which showed that they were able to cleave supercoiled plasmid DNA (form I) to the nicked form (form II). Compound 4 is even capable of generating linear form III (resulting from double-strand cleavage). The proposed mechanism of action involves an oxidative pathway (Fenton-type reaction), which produces harmful reactive species, like hydroxyl radicals.

Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis.

The spread of multidrug-resistant isolates of Mycobacterium tuberculosis requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine (SCN) and N-methyl-seconeolitsine (N-SCN), against M. tuberculosis. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of Streptococcus pneumoniae. Both SCN and N-SCN inhibited M. tuberculosis growth at 1.95–15.6 μM, depending on the strain. In M. smegmatis this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of Escherichia coli. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. In vitro inhibition of MtbTopoI activity by SCN and N-SCN was tested using a plasmid relaxation assay. Both SCN and N-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 μM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated in vivo in plasmid-containing cultures of M. tuberculosis. Plasmid supercoiling densities were −0.060 in cells untreated or treated with boldine, and −0.072 in 1 × MIC N-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of N-SCN. Altogether, these results demonstrate that the M. tuberculosis topoisomerase I enzyme is an attractive drug target, and that SCN and N-SCN are promising lead compounds for drug development.

Absence of RstA results in delayed initiation of DNA replication in Escherichia coli

RstB/RstA is an uncharacterized Escherichia coli two-component system, the regulatory effects of which on the E. coli cell cycle remain unclear. We found that the doubling time and average number of replication origins per cell in an ΔrstB mutant were the same as the wild-type, and the average number of replication origins in an ΔrstA mutant was 18.2% lower than in wild-type cells. The doubling times were 34 min, 35 min, and 40 min for the wild-type, ΔrstB, and ΔrstA strains, respectively. Ectopic expression of RstA from plasmid pACYC-rstA partly reversed the ΔrstA mutant phenotypes. The amount of initiator protein DnaA per cell was reduced by 40% in the ΔrstA mutant compared with the wild-type, but the concentration of DnaA did not change as the total amount of cellular protein was also reduced in these cells. Deletion or overproduction of RstA does not change the temperature sensitivity of dnaA46, dnaB252 and dnaC2. The expression of hupA was decreased by 0.53-fold in ΔrstA. RstA interacted with Topoisomerase I weakly in vivo and increased its activity of relaxing the negative supercoiled plasmid. Our data suggest that deletion of RstA leads to delayed initiation of DNA replication, and RstA may affect initiation of replication by controlling expression of dnaA or hupA. Furthermore, the delayed initiation may by caused by the decreased activity of topoisomerase I in RstA mutant.

Synthesis, characterization, DNA binding, topoisomerase I inhibition, and antiproliferation activities of (di-tert-butylbipyridine) platinum(II) complexes

Two mononuclear Pt(II) complexes, Pt(dbbpy)Cl2 (1) and [Pt(dbbpy)2](PF6)2 (2) (dbbpy = 4,4′-ditertbutyl-2,2′-biyridine) were synthesized and characterized by single-crystal X-ray diffraction analysis, elemental analysis, 1H NMR, and ESI–MS. Their binding affinities for both double-stranded (DS) calf thymus DNA (ct-DNA) and G-quadruplex DNA (HT21 and BCL-2) were investigated. In addition to structural differences, complex 1 displayed higher binding affinity for DS ct-DNA, whereas positively charged complex 2 was selective for binding to G-quadruplex DNA over DS DNA. The time-dependent cleavage of supercoiled circular plasmid pBR322 DNA by 1 was observed using agarose gel electrophoresis, whereas complex 2 hardly cleaved DS DNA. Stabilization of G-quadruplex HT21 DNA by both complexes was assessed by PCR stop assays. Both complexes exhibited moderate activities for inhibition of topoisomerase I as well as modest antiproliferation activities toward cancer cells in CKK-8 assays.

Identification and characterization of DNA endonucleases in Plasmodium falciparum 3D7 clone

BackgroundPlasmodium falciparum is the most virulent parasite of the five Plasmodium species that cause human malaria, and biological analysis of the parasite is critical for the development of novel strategies for disease control. DNA endonucleases are important for maintaining the biological activity, gene stability of the parasite and interaction with host immune systems. In this study, ten sequences of DNA endonucleases were found in the genome of P. falciparum 3D7 clone, seven of them were predicted to contain an endonuclease/exonuclease/phosphatase (IPR005135) domain which plays an important role in DNA catalytic activity. The seven DNA endonucleases of P. falciparum were systematically investigated.MethodsPlasmodium falciparum 3D7 clone was cultured in human O+ RBCs, RNA was extracted at 8, 16, 24, 32, 40, and 48 h post invasion and real-time quantitative PCR was carried out to analyse the transcription of the seven DNA endonuclease genes in asexual stages. Immunofluorescence assay was carried out to confirm the location of the encoded proteins expressed in the erythrocytic stages. Finally, the catalytic activity of the DNA nucleases were tested.ResultsOf the seven proteins analysed, two proteins were not soluble. Fragments derived from the rest five endonuclease sequences were successfully expressed as soluble proteins, and which were used to generate antisera for protein localization. The proteins were all located in the nucleus at ring and trophozoite stages. While at schizont stage, proteins encoded by PF3D7_1238600, PF3D7_0107200 and PF3D7_0319200 were in the punctuated forms in the parasite most likely around nuclei of the merozoites. But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane. The enzymatic activity of the recombinant GST-PF3D7_1238600 was very efficient without divalent iron, while the activity of the rest four enzymes was iron dependent. Further, divalent irons did not show any specific enhancement on the activity of GST-PF3D7_1238600, but the activity of GST-PF3D7_0107200, GST-PF3D7_1363500 and GST-PF3D7_0319200 were Cu2+ dependent. The activity of GST-PF3D7_0305600 was dependent on Mg2+ and Mn2+. Except GST-PF3D7_1363500, four of the GST tagged recombinant proteins hydrolysed the supercoiled circular plasmid DNA with or without divalent metal ions. The GST-PF3D7_1363500 protein only changed the supercoiled circular plasmid DNA into nicked plasmids, even with Cu2+.ConclusionsFragments derived from five of the endonuclease sequences of P. falciparum 3D7 clone were successfully expressed. The proteins displayed diverse cell distribution, biochemical and enzymatic activities, which indicated that they carried different biological function in the development of the parasite in the erythrocytes. The DNA repair and DNA degradation capacity of the DNA endonucleases in the biology of the parasite remained further studied.

Arginine homopeptides for plasmid DNA purification using monolithic supports

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.

Visualizing structure-mediated interactions in supercoiled DNA molecules.

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid’s structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

A Novel and One‐pot Green Synthesis of Vanadium Oxide Nanorods Using a Phytomolecule Isolated from Phyllanthus amarus

AbstractThe nanorods of vanadium oxide (V2O5) is synthesized by environmentally benign synthetic approach using a phytomolecule, 4‐(3‐(3,4‐dimethoxybenzyl)‐4‐methoxy‐2‐(methoxymethyl)butyl)‐3,6‐dimethoxybenzene‐1,2‐diol, isolated from Phyllanthus amarus. The rod shaped V2O5 nanostructures with the widths about 10–60 nm and the lengths reaching up to 1.5 μm was evident from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis. The orthorhombic structure of V2O5 is apparent from X‐ray diffraction (XRD) results. Furthermore, the crystalline nature of V2O5 nanorods was supported by the results of high‐resolution TEM (HRTEM) and selected area electron diffraction (SAED) analysis, which showed that the interplanar distance between two lattice fringes is 0.33 nm corresponding to (001) reflection plane. The X‐ray photoelectron spectroscopic (XPS) studies revealed that the peak at 533.6 eV indicates the presence of metal oxide and also confirms that the oxidation state of vanadium in as‐prepared V2O5 is V5+. The scission of supercoiled plasmid DNA was observed from 2D gel‐image, which clearly indicated that the propensity of plasmid cleavage increases with increase in concentration of V2O5 nanorods. Interestingly, high degree of cell viability with relatively less cytotoxicity was observed for V2O5 nanorods than phytomolecule in comparison with standard drug, Methotrexate against breast cancer cell line MDA MB 231 and MCF 12 A human cell line, respectively.

Combined use of plasmid drug pCMV-VEGFA and autodermoplasty for stimulation of skin defects healing in the experiment

To find effective ways to stimulate chronic skin wounds healing (including deep burns, diabetic and trophic ulcers) is an actual multidisciplinary task. The aim of our study was to assess the potential of using autodermoplasty in combination with plasmid drug pCMV-VEGFA to optimize skin defects repair in the experiment. Autodermoplasty was performed on Wistar rats. The size of the skin flap was 2x2 cm. Immediately after surgery the animals of the test group (n=8) underwent intradermal injection in the periphery of autotransplant with 1 ml solution containing 0.3 mg of supercoiled plasmid DNA pCMV-VEGFA, rats of the control group (n=8) received 1 ml of 0.9 % NaCl. The results were analyzed in 3, 6, 9 12, 18 days using macroscopic evaluation, laser Doppler flowmetry, histological methods. Macroscopically in the test group necrosis of the transplanted skin flap was found at later periods of observation, in one case complete survival of autotransplant was observed. The results of laser Doppler flowmetry in the group with plasmid DNA did not have statistically significant differences with control. The wound defect diameter in the test group at 12 days was 5,52± 4.80 mm, in the control - 12,45±0,82 mm (p=0,03); 2,53±of 2,94 mm and 4,23±3,5 mm (p=0,067) at 18 days, respectively. At 18 days, the average number of vessels under the flap in the central zone were: of 26±2,9 in the test group and 20±8 - in control; it the peripheral zone - 27±3,4 and of 12,1±3,9 (p=0,035), respectively; in the skin muscle - 21,2±of 3,9 and 12,4±3,6 (p=0,04), respectively. Thus, the use of plasmid drug pCMV-VEGFA improved the skin healing after autodermoplasty.

Relaxase MobM Induces a Molecular Switch at Its Cognate Origin of Transfer.

The MOBV1 family of relaxases is broadly distributed in plasmids and other mobile genetic elements isolated from staphylococci, enterococci, and streptococci. The prototype of this family is protein MobM encoded by the streptococcal promiscuous plasmid pMV158. MobM cleaves the phosphodiester bond of a specific dinucleotide within the origin of transfer (oriT) to initiate conjugative transfer. Differently from other relaxases, MobM and probably other members of the family, cleaves its target single-stranded DNA through a histidine residue rather than the commonly used tyrosine. The oriT of the MOBV1 family differs from other well-known conjugative systems since it has sequences with three inverted repeats, which were predicted to generate three mutually-exclusive hairpins on supercoiled DNA. In this work, such hypothesis was evaluated through footprinting experiments on supercoiled plasmid DNA. We have found a change in hairpin extrusion mediated by protein MobM. This conformational change involves a shift from the main hairpin generated on “naked” DNA to a different hairpin in which the nick site is positioned in a single-stranded configuration. Our results indicate that the oriTpMV158 acts as a molecular switch in which, depending on the inverted repeat recognized by MobM, pMV158 mobilization could be turned “on” or “off.”

Phosphorus-nitrogen compounds. Part 40. The syntheses of (4-fluorobenzyl) pendant armed cyclotetraphosphazene derivatives: Spectroscopic, crystallographic and stereogenic properties, DNA interactions and antimicrobial activities

The Cl substitution reaction of octachlorocyclotetraphosphazene, N4P4Cl8 (1), with 1.2 equimolar amounts of sodium salt of N/O donor-type bidentate ligand (2) gave four different new (4-fluorobenzyl) pendant armed cyclotetraphosphazene derivatives; namely, mono-(4-fluorobenzyl)-spiro- (spiro; 3), mono-(4-fluorobenzyl)-2-cis-4-dichloro-ansa- (2,4-ansa; 4), bis-(4-fluorobenzyl)-2-trans-6-dispiro (5) and bis-(4-fluorobenzyl)-2-trans-4-dispiro (6) cyclotetraphosphazenes of which 3 was the major product (yield 55%). Compound 3 was treated with mono and difunctional reagents to prepare the fully substituted products (3a-3j) due to its very high yield. However, tetrapyrrolidino-2-cis-4-dichloro-ansa- product (4a) was obtained from the reaction of 2,4-ansa (4) with excess pyrrolidine. The new cyclotetraphosphazenes were characterized by elemental analyses, mass spectrometry (ESI-MS), Fourier transform infrared (FTIR), 1H, 13C, and 31P NMR techniques. The structures of 3 and 3g were determined by X-ray crystallography. 2,4-Ansa compounds (4 and 4a) have two stereogenic P atoms. The stereogenic property of 4 was identified by 31P NMR spectra in the addition of the chiral solvating agent, (R)-(+)-2,2,2-trifluoro-1-(9′-anthryl)-ethanol (CSA). The tetraspiro compounds (3i and 3j) look similar to a propeller, and they may have P and M atropisomers. The antimicrobial activities of the compounds were screened against some G(−)/G(+) bacteria and yeast strains. The interactions of the compounds with supercoiled plasmid pBR322 DNA and their inhibited DNA restriction were examined.

Oxovanadium phenanthroimidazole derivatives: synthesis, DNA binding and antitumor activities

Four unsymmetrical oxovanadium phenanthroimidazole complexes, [VO(hntdtsc)(NPIP)] (1), [VO(hntdtsc)(CPIP)] (2), [VO(hntdtsc)(MEPIP)] (3) and [VO(hntdtsc)(HPIP)] (4) (hntdtsc = 2-hydroxy-1-naphthaldehyde thiosemicarbazone, NPIP = 2-(4-nitrophenyl)-imidazo[4,5-f]1,10-phenanthroline, CPIP = 2-(4-chlorphenyl)-imidazo[4,5-f]1,10-phenanthroline), MEPIP = 2-(4-methylphenyl)-imidazo[4,5-f]1,10-phenanthroline), HPIP = 2-(4-hydroxylphenyl)-imidazo[4,5-f] 1,10-phenanthroline), have been synthesized and characterized. Their DNA binding and antitumor activities were determined by biochemical methods. All four oxovanadium complexes can bind with CT-DNA by an intercalation model and can also cleave supercoiled plasmid DNA in the presence of H2O2. The antitumor properties and mechanism of the complexes have been analyzed by MTT assay, cell cycle analysis, apoptosis assay and Western blot analysis. The results showed that the free ligands and their corresponding complexes all possess antiproliferative activities with very low IC50 values against Hela, BIU-87 and SPC-A-1 cell lines. Complex 1, which has a strongly electron-withdrawing nitro group, exhibited the best antiproliferative activities. Complex 1 caused G0/G1 phase arrest of the cell cycle and induced apoptosis in Hela cells. Additionally, complex 1 attenuated the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2).This indicates that inhibition of the ERK1/2 signaling pathway may contribute to the antitumor effects of these complexes.

OXIDATIVE DNA DAMAGE PROTECTIVE AND HEPATOPROTECTIVE ACTIVITY OF TERMI-NALIA BELLERICA (BAHEDA)

Objective: To investigate the ethanolic extracts of Terminalia bellerica for its in vitro DNA damage protective activity against hydroxyl radical (OH•) and hepatoprotective activity against CCl4 induced toxicity in HepG2 liver cells.Methods: The DNA damage assay was performed using supercoiled pBR322 plasmid DNA with Fenton’s reagent. Protection of human liver-derived HepG2 cells against CCl4 induced damage was determined by trypan blue exclusion assay and Tetrazolium bromide salt MTT assay. Liver cells were pre-exposed to 0.5 µmol/ml of CCl4 for 60 min at room temperature and screening assay was carried out in HepG2 cells to evaluate the cytotoxicity of tested extracts with concentration 0.001 to 100 µg/ml.Results: The low dose (0.001-0.1 µg/ml) of leaf and bark has an effect on the percentage viability (101±4.04) and 97±4.04) for leaf and bark extracts respectively) of HepG2 cells. Whereas, the percentage cell viability 99±2.89 and 103±4.04 were found for fruit pulp and seed extracts respectively. The uniform DNA damage protective activity was observed in a dose-dependent manner for leaf, fruit pulp, seed and bark extracts of T. bellerica. Conclusion: Leaf and bark of T. bellerica exhibit 10 fold less toxicity compared to fruit pulp and seed during CCl4 exposure on HepG2 cells suggesting that leaf and bark has more therapeutic potential against hepatotoxicity.

Antioxidant and Cytoprotective Activities of Enzymatic Extracts from Rhizoid of Laminaria japonica.

Rhizoid of Laminaria japonica was hydrolyzed with proteases and carbohydrases to obtain antioxidant materials. Oxygen radical absorbance capacity (ORAC) of the enzymatic extracts was evaluated and the Protamex extract (PE) exhibited the highest ORAC value. PE also potently scavenged 2,2-diphenyl-1-picrylhydrazyl radical, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid cation radical, and hydrogen peroxide (H2O2) and had good reducing power. PE inhibited hydroxyl radical-induced DNA scission by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. The cytoprotective effect of PE against H2O2-induced hepatic cell damage was also investigated. PE showed a dose-dependent cytoprotective effect in cultured hepatocytes by inhibiting intracellular reactive oxygen species scavenging activity. In addition, PE up-regulated the expression of heme oxygenase-1, which is a cytoprotective enzyme, by activating translocation of nuclear factor-erythroid 2-related factor 2. Taken together, the enzymatic extract of rhizoid of L. japonica, particularly PE, may be useful for antioxidant additives.