Supercoiled Form Research Articles

Binding interaction of food preservatives with glycosylated DNA: In vitro assessment and computational studies

This work aims to evaluate the interaction between sodium benzoate (SB), potassium sorbate (PS), and sodium dihydrogen citrate (CIT) as food preservatives, individually, as well as in combinations of two and three, with DNA and glycosylation DNA to determine the allowable amount of food additive consumption to mitigate potential harm to human health. Various techniques including UV-Vis absorption, fluorescence spectroscopy, DNA amadori product formation, and agarose gel electrophoresis, were employed to observe these interactions. Furthermore, pioneering molecular docking and molecular dynamics simulations were performed to gain deeper insights into the interactions between these preservative compounds and DNA. It is also noteworthy that there have been no documented biological or computational studies concerning citrate. The results revealed that all three food preservatives could interact with DNA, leading to conformational changes as indicated by increased absorption and fluorescence intensity. Both SB and PS were found to damage the DNA structure and promote the formation of DNA Amadori products. Moreover, all treatment groups exhibited a decrease in migration speed on the agarose gel, suggesting binding to DNA and alterations in the supercoiled form of DNA structure. Interestingly, the groups treated with citrate showed a smeared appearance, DNA strand fragmentation, and cleavage. Molecular docking complemented the in vitro achievement that SB, PS, and CIT could interact with DNA. DFT analysis showed that CIT exhibited the best thermodynamic and kinetic stability compared to other food additives.

Synthesis, calf-thymus DNA interaction with trimethyl-/triphenyl-tin(IV) derivatives of ɑ-lipoic acid and their mixed ligand complexes with N-donor (1,10-phenanthroline/2,2′-bipyridyl/benzimidazole)

The mixed ligand complexes of trimethyl-/triphenyl-tin(IV) derivatives of ɑ-lipoic acid (HLA), viz. R3Sn(LA) {R = Me (1), and Ph (2)}with 1,10-phenanthroline (phen), viz. [Me3Sn(LA).phen] (3), 2,2′-bipyridyl, viz. [Ph3Sn(LA).by] (4) and benzimidazole (bz), viz. [R3Sn(LA).bz] {R = Me (5), and Ph (6)} were synthesised and satisfactorily characterized by analytical and spectroscopic techniques, especially FT-IR, NMR (1H, 13C, and 119Sn NMR) spectroscopy and ESI-MS spectrometry. The ∠C-Sn-C bond angle calculated by optimization of their gas-phase geometry revealed that complexes (1) and (2) have distorted tetrahedral geometry and complexes (3) and (4) exhibited distorted octahedral geometry while complexes (5) and (6) exhibited trigonal bipyramidal environment around the tin, which agreed with the data obtained from NMR. Spectroscopic titrations (UV–visible absorption, fluorescence titration, and circular dichroism) were performed to ascertain the binding mode of the complex with calf thymus DNA (CT-DNA). The obtained values of Kb in the order of 105 M−1 suggested partial intercalative interactions of complexes with DNA grooves/base pairs. The complexes efficiently cleaved plasmid DNA (pBR322) into supercoiled (Form I), nicked/circular (From II), and linear structure (Form III). The fascinating biophysical data suggests the potential of these complexes as anticancer agents.

Diorganotin(IV) derivatives of ONO tridentate Schiff bases: Synthesis, spectroscopic characterization, DFT studies, CT-DNA binding, and plasmid-DNA cleavage studies

Two Schiff bases 2-amino-N'-(5‑bromo-2-hydroxybenzylidene)-3-(4-hydroxyphenyl)propanehydrazide (H2L1) and 2-amino-N'-(5‑chloro-2-hydroxybenzylidene)-3-(4-hydroxyphenyl)propanehydrazide (H2L2) and their six diorganotin(IV) complexes (R = C4H9 (1, 4), CH3 (2, 5), and C8H17 (3, 6)) have been synthesized. Schiff bases (H2L1–2) and their R2SnL1–2 complexes (1–6) have been characterized by CHNS analysis, IR spectroscopy, 1H, 13C, 119Sn NMR, and ES-MS. Schiff bases (H2L1–2) act as a dianionic tridentate ligand coordinated to Sn through phenolic and enolic oxygen and azomethine nitrogen in R2SnL1–2 complexes (1–6). A density functional theory (DFT)-based quantum chemical calculation validated the structures of R2SnL1 (1–3) and R2SnL2 (4–6) at the B3LYP/6‐311G(df,pd)/Def2‐SVP(Sn) and B3LYP/6‐31G(d,p)/Def2‐SVP(Sn) levels of theory, respectively. At these levels of theories, comprehensive electronic structure calculations determined atomic charges at selected atoms. Additionally, a molecular electrostatic potential (MEP) map identified electrostatic potential-varying sites on the molecule's surface, and MEP maps used to identify regions where other molecules might interact or react with the molecules on them. Furthermore, a conceptual-DFT-based global reactivity descriptor determined the stability and reactivity behaviour of R2SnL1–2 complexes (1–6). H2L1–2 and R2SnL1–2 complexes (1–6) have binding constants in the range ∼105 M-1 with CT-DNA, as revealed by UV–visible and fluorescence spectral titrations. UV-visible, fluorescence spectroscopy, and CD studies suggested that H2L1–2 and their R2SnL1–2 complexes (1–6) interact with CT-DNA electrostatically or groove-bound. DNA cleavage studies reveal that H2L1–2 and their R2SnL1–2 complexes (1–6) have ability to cleave the plasmid-DNA from its supercoiled form (SC, I) to nicked form (NC, II). However, n-Bu2SnL2 (4) and n-Oct2SnL2 complex (6) have higher cleavage activity than Schiff bases and other R2SnL1–2 complexes (1–6).

The ecological hazards of profenofos revealed by soil beneficial-bacteria, plant seedlings, and plasmid nicking assays: A short-term toxicity investigation

Excessive and indiscriminate use of pesticides may adversely affect the growth and activity of both crop plants and soil microbial populations. The reported study was conducted to evaluate the toxicity of profenofos (PF; an organophosphate insecticide) using bacterial (Pseudomonas fluorescens PSB-3 and Enterobacter cloacae ZSB-8) and plant (Coriandrum sativum and Lactuca sativa L.) bioassays. PF was applied at rates of (0–100 µg mL−1) in vitro. Both bacterial strains were sensitive to PF but showed variable tolerance. Following PF exposure, cellular growth, morphology, survival, and inner membrane permeability of bacterial strains were significantly (p < 0.05) altered. Decreased population size coincided with decline in cellular respiration. The 100 µgmL−1 PF dosage imparted maximum impact on ZSB-8, inhibiting populations by 87%. PF also interfered with bacterial surface adherence (i.e., biofilm formation) in a concentration-dependent manner. Alterations in bacterial biomarker enzymatic activity and oxidative stress were also noted. PGP traits of bacterial strains were negatively and significantly (p ≤ 0.05) affected by insecticide. Under PF stress, reduction in indole-3-acetic and siderophore production followed the order: ZSB-8 > PSB-3. PF-induced phytotoxicity was confirmed via reduction in germination, seedling parameters, survival, tolerance, and vigor indices in both plant species. Additionally, PF caused distortion in morphology of root tips and root surfaces. Under CLSM, PF-exposed C. sativum and L. sativa roots exhibited increased oxidative stress. Cellular death in insecticide-treated roots was observed following staining with Evans blue dye. Insecticide concentration-dependent increase in stress markers (proline and MDA content), and antioxidant enzymatic activities in plant seedlings were observed. A dose-dependent conversion of super-coiled form of DNA to open circular in pBR-322 plasmid revealed the genotoxic potential of PF. These findings provide an understanding of toxic effects of profenofos on beneficial microbes and leafy edible vegetables, including their morphological, and cellular effects. Indeed, insecticidal applications deserve special attention due to their potential environmental hazards.

Analytical and Functional Characterization of Plasmid DNA Topological Forms and Multimers.

Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (∼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.

DNA binding/cleavage study of mixed ligand complexes of di-/triorganotin 4-fluoroanthranilates with 1,10-phenanthroline/benzimidazole: Synthesis, characterization, X-ray structures of [Bu2Sn(FA)2.phen], [Me3Sn(FA).bz], and DFT calculations

The mixed ligand complexes of di-/tri-organotin 4-fluoroanthranilates with 1,10-phenanthroline (phen), viz. [R2Sn(FA)2.phen] {R=Bu (1), Oct (2), Me (3)} and benzimidazole (bz), viz. [R3Sn(FA).bz] {R=Me (4), Ph (5)} have been synthesized and satisfactorily characterized by analytical and spectroscopic techniques, especially FT-IR, NMR (1H, 13C, and 119Sn NMR) spectroscopy and ESI-MS spectrometry. Single crystal-Xray diffraction measurements (Sc-XRD) confirmed a distorted pentagonal-bipyramidal geometry for complex (1), where one carboxylate group is monodentate, and the second (–COO) group behaves as a bidentate, while complex (4) affirms trigonal–bipyramidal geometry around the tin centre. The bond angles, especially ∠C–Sn–C obtained by optimizing complexes’ gaseous phase geometry showed significant conformity with the data obtained from Sc-XRD and NMR. The Kb (binding constant) and Ksv (Stern-Volmer quenching constant) values, ranging from 103 to 107, have been obtained through UV–visible absorption and fluorescence titrations with CT-DNA. These results strongly indicate partial minor groove binding/electrostatic interactions between the complex and CT-DNA, which has also been evidenced by circular dichroism studies. The studied complexes cleaved efficiently pBR322 plasmid DNA in NC (nicked circular form), SC (supercoiled form), and linear form.

De novo design and preparation, structural details, and cytotoxic response of a new water soluble (2,2′–bipy)–(phenylalaninato)–μ–chlorido–copper(II) drug candidate against resistant cancer cells

A new water soluble (2,2′–bipyridine)–(phenylalaninato)–μ–chlorido–copper(II) complex was synthesized using ancillary ligands, l-phenylalanine, and 2,2-bipyridine as a chemotherapeutic anticancer agent. The complex was characterized by employing various spectroscopic (UV–visible, FT-IR, EPR, elemental analysis) and single crystal X-ray diffraction technique which revealed the structure of crystal as monoclinic with P21 space group. The interaction studies of the complex with therapeutic drug target, ct-DNA were caried out using various complimentary biophysical techniques, and the corroborative results of these experiments revealed that complex binds avidly through non-covalent electrostatic binding mode in the groove region of DNA. The binding propensity was quantified by calculating the intrinsic binding constant values (Kb and Ksv) which were found to be 3.1 (±0.95) × 105M−1 and 0.4 (±0.05) × 103M−1, respectively. The cleavage studies of copper(II) based drug candidate 1 with pBR322 plasmid DNA were performed in concentration dependent manner and it was demonstrated that complex was capable of conversion of supercoiled form SC (Form I) into nicked form NC (Form II) upon incubation of 3 μM concentration, implicating efficient single strand cleavage of DNA helix. The mechanistic cleavage studies were also performed in the presence various activators viz., DMSO, NaN3, SOD, and H2O2 and the strong inhibition of cleavage process was observed in presence of NaN3 and SOD that suggested an oxidative pathway (Fenton's mechanism) for the Cu(II) complex. The cytotoxicity of the complex was evaluated by SRB assay against the resistant cancer cell lines, viz., Hop-62, MCF-7, AW13516, MDA-MB-231, and SiHa. The cytotoxicity activity demonstrated good therapeutic potency against all the tested cell lines with GI50 value of <10 μM.

Studying the Functional Potential of Ground Ivy (Glechoma hederacea L.) Extract Using an In Vitro Methodology

Glechoma hederacea L., known as ground ivy, has a long history of use in folk medicine. The main bioactive compounds in ground ivy are polyphenolic compounds known for their potent antioxidant and antimicrobial activities and thus have high potential as functional ingredients against bacterial infections and the occurrence of chronic diseases associated with oxidative stress in the human body. The aim of the present study was to determine the biological activity of ground ivy extract on selected human cell lines, including hepatic (HepG2), tongue (CAL 27), gastric (AGS) and colon (Caco-2) cancer cell lines by evaluating cytotoxicity, formation of reactive oxygen species and genotoxicity. The antioxidant capacity of the extract was additionally evaluated using cellular model macromolecules of protein and DNA, bovine serum album and plasmid phiX174 RF1 DNA. The effect of ground ivy extract on representatives of human microflora, including L. plantarum, E. coli and S. aureus, was also studied. The cytotoxicity of the extract depended on the type of cells treated, and the pro-oxidant effect generally decreased with increasing exposure time. The most pronounced genoprotective effect against hydroxyl radical damage was monitored in model plasmid DNA and occurred at the highest tested concentration (0.25 mg mL−1), with 95.89% preservation of the supercoiled form of the plasmid. This concentration also had the most significant antioxidant activity on the model protein—14.01% more than the positive control prepared using Trolox. The ground ivy extract showed high antimicrobial potential against the pathogenic bacteria E. coli and S. aureus.

Organotin (2-amino-4-fluorobenzoates): Synthesis, characterization, DFT studies, DNA interaction and cleavage studies

Synthesis of organotin(IV) complexes of 2-amino-4-fluorobenzoic acid (AFA), namely C16H16F2N2O4Sn (1), C22H28F2N2O4Sn (2), C10H14FNO2Sn (3), and C25H20FNO2Sn (4), have been accomplished, and all complexes 1–4 have been characterized using various spectroscopic and analytical techniques, viz. FT–IR and NMR spectroscopy (1H, 13C and 119Sn), ESI–mass spectrometry and elemental analysis. DFT calculations revealed that complexes 1 and 2 exhibited bicapped tetrahedral geometry, while 3 and 4 exhibited distorted tetrahedral environments around tin. Intrinsic binding constants (Kb ≈ 105 M−1) for AFA and complexes 1–4 have been determined by performing UV–vis spectral titration with Calf thymus-DNA (CT-DNA). Further, AFA and complexes 1–4 are luminescent with Kb values of the order of 104 M−1. A significant shift is observed in the circular dichroism (CD) spectra of CT-DNA in the presence of AFA and organotin(IV) complexes 1–4. Based on the UV–vis, fluorescence, and CD studies, it is concluded that AFA and it’s all complexes 1–4 may interact with CT-DNA by minor/major groove binding or electrostatic interaction. Interestingly, triorganotin(IV) complexes 3 and 4 more efficiently cleave the supercoiled form (SC, I) into linear form (L, III) along with the nicked form (NC, II) as compared to complexes 1 and 2, and AFA.

Assessing phytotoxicity and cyto-genotoxicity of two insecticides using a battery of in-vitro biological assays.

Intensive use of chemical pesticides in agriculture poses environmental risks and may have negative impacts on agricultural productivity. The potential phytotoxicity of two chemical pesticides, chlorpyrifos (CPS) and fensulfothion (FSN), were evaluated using Cicer arietinum and Allium cepa as model crops. Different concentrations (0-100 μgmL-1) of both CPS and FSN decreased germination and biological attributes of C. arietinum. High pesticide doses significantly (p≤0.05) caused membrane damage by producing thiobarbituric acid reactive substances (TBARS) and increasing proline (Pro) content. Pesticides elevated ROS levels and substantially increased the superoxide anions and H2O2 concentrations, thus aggravating cell injury. Plants exposed to high pesticide dosages displayed significantly higher antioxidant levels to combat pesticide-induced oxidative stress. Ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) increased by 48%, 93%, 71%, 52% and 94%, respectively, in C. arietinum roots exposed to 100 µgFSNmL-1. Under CLSM, pesticide-exposed C. arietinum and 2',7'-dichlorodihydrofluorescein diacetate (2'7'-DCF) and 3,3'-diaminobenzidine stained roots exhibited increased ROS production in a concentration-dependent manner. Additionally, enhanced Rhodamine 123 (Rhd 123) and Evan's blue fluorescence in roots, as well as changes in mitochondrial membrane potential (ΔΨm) and cellular apoptosis, were both associated with high pesticide dose. Allium cepa chromosomal aberration (CAs) assay showed a clear reduction in mitotic index (MI) and numerous chromosomal anomalies in root meristematic cells. Additionally, a-dose-dependent increase in DNA damage in root meristematic cells of A. cepa and conversion of the super-coiled form of DNA to open circular in pBR322 plasmid revealed the genotoxic potential of pesticides. The application of CPS and FSN suggests phytotoxic and cyto-genotoxic effects that emphasize the importance of careful monitoring of current pesticide level in soil before application and addition at optimal levels to soil-plant system. It is appropriate to prepare both target-specific and slow-release agrochemical formulations for crop protection with concurrent safeguarding of agroecosystems.

First Report of Tomato Leaf Curl Bangladesh Virus Associated with Leaf Yellowing Symptoms in Lablab purpureus in Bangladesh

First Report of Tomato Leaf Curl Bangladesh Virus Associated with Leaf Yellowing Symptoms in <i>Lablab purpureus</i> in Bangladesh

First Report of Squash leaf curl China virus Associated with Mosaic and Mild Leaf Curl Disease of Pumpkin in Bangladesh

First Report of <i>Squash leaf curl China virus</i> Associated with Mosaic and Mild Leaf Curl Disease of Pumpkin in Bangladesh

A new device-mediated miniprep method

Small-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use the alkali-SDS-based method in a three-solution format and require spin column-based purification steps. This procedure requires the vortexing or pipetting of pelleted bacteria by centrifugation and manual mixing of the solutions. Here, we describe a centrifuge/mixer-based instrument with the ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that by applying rotor oscillation-driven mixing of solutions added in the lysis and neutralization steps, homogeneous mixing was achieved within 5 s at a rotor oscillation amplitude of 45° and oscillation frequency of 400 ± 30 rpm, yielding the maximal quantity and quality of plasmid DNA. No increase in host chromosome presence purified by this approach occurs for high-copy-number plasmids compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of the chromosomal fraction in low-copy-number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45° does not turn into an open circular (OC) isoform when the plasmid is stored for 1 year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitudes of 270°, 180° and 90°. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs.

Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

Evaluation of Leea rubra Leaf Extract for Oxidative Damage Protection and Antitumor and Antimicrobial Potential.

Background The leaves of Leea rubra contain an abundance of phenolic constituents and have medicinal uses as antipyretic and diaphoretic agents and are also used in the treatment of stomach ache, rheumatism, arthritis etc. In spite of the traditional uses, data on the scientific evaluation of the plant are not sufficient. So, the present study was designed to evaluate the protective role of the extract against oxidative damage to DNA and human erythrocytes as well as antitumor and antibacterial activities against some resistant bacteria. Methods The protective activity of the ethyl acetate fraction (EAF) of the extract was investigated by evaluating the inhibition of oxidative damage of pUC19 plasmid DNA as well as hemolysis and lipid peroxidation damage to human erythrocytes induced by 2,2′-azobis-2-amidinopropane (AAPH). Antitumor activity was assessed by evaluating the percentage inhibition of cell growth, morphological changes of Ehrlich's ascites carcinoma (EAC) cells, and hematological parameters. Antimicrobial activity was determined by the disc diffusion method against different resistant microorganisms. Results EAF effectively inhibited AAPH-induced oxidative damage to DNA because it can inhibit the transformation of the supercoiled form of plasmid DNA to open circular and further linear form. The oxidative hemolysis caused by AAPH in human erythrocytes was inhibited by EAF extract in a time-dependent manner, and the production of malondialdehyde (MDA) was significantly reduced, which indicates the prevention of lipid peroxidation. In antitumor assay, 76% growth of inhibition of EAC was observed compared with the control mice (p < 0.05) at a dose of 100 mg/kg body weight. Antimicrobial activity was evaluated against two pathogenic resistant microorganisms (Escherichia coli and Pseudomonas aeruginosa), and the highest antimicrobial activity was observed against Pseudomonas spp. Conclusion EAF may have great importance in preventing oxidative damage to DNA, erythrocytes, and other cellular components as well as can be a good candidate in cancer chemotherapy and treating infectious diseases caused by antibiotic-resistant bacteria.

Dual Reactive Oxygen Species Generator Independent of Light and Oxygen for Tumor Imaging and Catalytic Therapy

Dual Reactive Oxygen Species Generator Independent of Light and Oxygen for Tumor Imaging and Catalytic Therapy

Synthesis and biological activity of pyrrolidine/piperidine substituted 3-amido-9-ethylcarbazole derivatives

In this study, pyrrolidine/piperidine substituted 3-amido-9-ethylcarbazole derivatives were synthesized and characterized by FT-IR, 1H NMR, 13C NMR spectroscopic and HRMS techniques. Acetylcholinesterase inhibition activity of the compounds were determined. Also, total antioxidant capacity, DPPH radical scavenging and metal chelating activity methods were used to evaluate the antioxidant effects of the compounds. In addition, the antiproliferative effect of the compounds on HT-29 and SH-SY5Y cells were assessed by the MTT assay. In addition, gel electrophoresis was utilized to determine DNA cleavage and topoisomerase II inhibition activities of 2a and 2b. Moreover, the features of the interaction between the compounds and calf thymus DNA (ct-DNA) were evaluated using UV–Vis absorption spectroscopic titration method. The biological activitiy demonstrated an acetylcholinesterase inhibition activity, antioxidant activity, and antiproliferative effect of the compounds on HT-29 and SH-SY5Y cells. The compounds also augmented DNA cleavage from the supercoiled form (SC, Form I) to the nicked circular form (NC, Form II), a process free of simultaneous Form III formation, which indicated the cleavage of single-strand DNA. The compounds prevented topoisomerase II functions at varying concentrations, and are connected to ct-DNA through the intercalation mode. Therefore, the compounds may be offered as anti-cancer drug candidate for use in cancer treatment.

The Concentration Factor Effect Study of Etoposide Drug and Annona squamosa L. Seeds Extracts on Topoisomerase II Inhibition Model: Catalytic Inhibitory and Interfacial Poisons

Background: Topoisomerase II plays an important role in cell proliferation and DNA regulation. It isresponsible for loosening the supercoiled form of DNA and for condensing chromosomes in eukaryotic cells.Custard apple (Annona squamosa L.) (Annonaceae) has been one of the main ingredients of ethnomedicalpreparation for the treatment of tumors in some countries, and has cytotoxic effects on cancerous cell lines.Aim: assessment of Qualitative Inhibition (Catalytic Inhibitor or Interfacial Poisons) and Quantitative(IC50%) of Custard apple Plant Seeds; Crude Extract (CCE) and Alkaloid Extract (CAE) of HumanToposomerase IIA (TOP2A) compared to etoposide (VP-16) as a controlled drug. Methods: TOP2A (0.5unit) and pHOT1 DNA plasmid (62.5ng), DNA plasmid relaxation assay required for inhibition testing.Inhibitors, the seeds plant was extracted by 100% methanol for the preparation of crude with concentrations(240, 160, 100, 80, 40, 20, and 10 µg/ml) and alkaloid (100, 80, 60, 40, 20, 10 and 5 µg/ml), and the VP-16as a positive control drug (150, 100, 50, 25, 12.5, and 3.125 µg/ml). Results and Discussion: The CAEshowed a higher DNA plasmid relaxation inhibition, IC50% (27.46). The higher concentrations of CCEand CAE play as a Catalytic Inhibitory Compounds CICs (as suppressor compound) rather than InterfacialPoisons IFPs (as poisons compounds), quite the opposite at low concentrations. Higher concentrations mayinhibit the formation of the enzyme-DNA complex, resulting in the accumulation of the supercoiled formof the DNA plasmid due to inactivation of the enzyme function. In comparison, at low concentrations ofinhibiting factors the linear structure is accumulated, which means that both strands cannot be rejoined.Conclusions: The CAE had the highest inhibition activity on the TOPO2A compared to the other agents. Incontrast to the VP-16 drug, the CAE and CCE concentrations of this plant affected inhibition models.

Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing.

SummaryAccumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.

An in vitro selective inhibitory effect of silver(i) aminoacidates against bacteria and intestinal cell lines and elucidation of the mechanism of action by means of DNA binding properties, DNA cleavage and cell cycle arrest.

Novel silver(i) aminoacidate complexes {[Ag(HVal)(H2O)(NO3)]}n (AgVal) and {[Ag3(HAsp)2(NO3)]}n·nH2O (AgAsp) were prepared, investigated and fully characterized by vibrational spectroscopy (mid-IR), elemental analysis, thermogravimetric analysis, X-ray crystallography and mass spectrometry. Their stability in D2O and PBS buffer was verified by time-dependent 1H and 13C NMR measurements. Their in vitro antibacterial activity (against pathogenic Staphylococcus aureus CCM4223, Escherichia coli CCM4787) and that against probiotic bacteria Lactobacillus plantarum CCM7102 and Lactobacillus reuteri (L26) were determined and potential dosing concentration was evaluated. The cytotoxicity of both the complexes against intestinal porcine epithelial (IPEC-1) and human epithelial colorectal adenocarcinoma (CaCo-2) cell lines was determined using the colorimetric MTT assay and against human metastatic melanoma (A2058), human pancreatic adenocarcinoma (PaTu 8902), human cervical adenocarcinoma (HeLa), human colorectal carcinoma (HCT116), human leukaemic T cell lymphoma (Jurkat), and human dermal fibroblasts (HDF) using colorimetric MTS assay. The selectivity index (SI) was identified for intestinal cancer (CaCo-2) and healthy (IPEC-1) cells. The mechanism of action of AgVal and AgAsp was further elucidated and discussed by the study of their binding affinity toward the CT DNA, the ability to cleave the supercoiled form of pUC19 DNA and the ability to influence numbers of cells within each cell cycle.