A series of multinuclear diethylenetriamine ligands were synthesized and used as artificial nuclease enzyme model. Target compounds were characterized by 1H NMR, 13C NMR, IR and ESI-MS. Preliminary studies on the cleavage of pUC19 DNA in the presence of metal complexes have also been performed and the results revealed that these complexes could act as powerful catalysts for the cleavage of pUC19 DNA after 48 h under physiological conditions. The hydrolytic cleavage mechanism of DNA plasmid by title compound was confirmed by T4 DNA ligase experiment.
DNA ; metabolism ; DNA Cleavage ; Ligands ; Magnetic Resonance Spectroscopy ; Polyamines ; chemical synthesis ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Spectrophotometry, Infrared

PURPOSE: Apoptosis is a physiological mechanism for deleting cells from the body for development and homeostasis. Exogenous cytokines such as tumor necrosis factor alpha (TNFalpha) and transforming growth factor beta (TGF beta) are known to modulate apoptosis, thus can provide a new therapeutic modality for various malignancies. We studied whether TNFalpha or TGFbeta can induce apoptosis or exert antiproliferative effect on human gastric cancer cell line (AGS) and which genes are involved in the cytokine-induced apoptotic pathway. MATERIALS AND METHODS: To examine the effect of TNFalpha or TGF beta on AGS cell line (human gastric adenocarcimoma), we performed following tests; MTT test, trypan blue dye exclusion assay and colony forming efficiency. Total DNA was extracted from the TNFalpha-treated AGS cells and DNA ladder was detected as the hallmark of apoptosis, and flow cytometry analysis was performed for another apoptotic index. The effects of TNFalpha on c-myc expression was observed using RT-PCR. RESULTS: TNFalpha suppressed AGS cell growth, in a time- and dose-dependent manner, but TGFbeta had no effect on AGS cell growth. Electrophoretic analysis of total cellular DNA revealed the pattern of internucleosomal DNA cleavage, which is specific for apoptosis and the effect was observed from 24 to 72 hrs after 50 ng/ml TNFalpha treatment. Time-dependent increse of apoptotic cells by TNFalpha was detected by flow cytometry analysis. Morphological changes such as cell to cell contacts and extension of cell processes were observed in TNFalpha-treated AGS cells. RT-PCR using c-myc primers showed thatthe mRNA levels were increased 6 hrs after TNFalpha treatment and persisted for 72 hrs. CONCLUSION: It is suggested that TNFalpha, but not TGF beta, functions as an important inducer of apoptosis in AGS cell line, and c-myc may function as a critical endogenous activator of the pathway leading to cell death of AGS cells.
Apoptosis* ; Cell Death ; Cell Line ; Cytokines ; DNA ; DNA Cleavage ; Flow Cytometry ; Homeostasis ; Humans ; RNA, Messenger ; Stomach Neoplasms* ; Stomach* ; Transforming Growth Factor beta* ; Trypan Blue ; Tumor Necrosis Factor-alpha

Cyclic voltammetric measurements were performed for Co(II), Ni(II), Cu(II) and Zn(II) complexes of 1 : 1 alternating copolymer, poly(3-nitrobenzylidene-1-naphthylamine-co-succinic anhydride) (L) and Ni(II) and Cu(II) complexes of 1 : 1 alternating copolymer, poly(3-nitrobenzylidene-1-naphthylamine-co-methacrylic acid) (L1). The in vitro biological screening effects of the investigated compounds were tested against the fungal species including Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola and Candida albicans and bacterial species including Staphylococcus aureus, Escherichia coli, Klebsiella pneumaniae, Proteus vulgaris and Pseudomonas aeruginosa by well diffusion method. A comparative study of inhibition values of the copolymers and their complexes indicates that the complexes exhibit higher antimicrobial activity. Copper ions are proven to be essential for the growth-inhibitor effect. The extent of inhibition appeared to be strongly dependent on the initial cell density and on the growth medium. The nuclease activity of the above metal complexes were assessed by gel electrophoresis assay and the results show that the copper complexes can cleave pUC18 DNA effectively in presence of hydrogen peroxide compared to other metal complexes. The degradation experiments using Rhodamine B dye indicate that the hydroxyl radical species are involved in the DNA cleavage reactions.
Aspergillus flavus ; Aspergillus niger ; Candida albicans ; Cell Count ; Coordination Complexes ; Copper ; Diffusion ; DNA ; DNA Cleavage ; Electrophoresis ; Escherichia coli ; Hydrogen Peroxide ; Hydroxyl Radical ; Ions ; Klebsiella ; Mass Screening ; Proteus vulgaris ; Pseudomonas aeruginosa ; Rhizoctonia ; Rhizopus ; Rhodamines ; Staphylococcus aureus

OBJECTIVETo analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.

METHODSTwo specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.

RESULTSAfter reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.

CONCLUSIONSCleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.

5' Untranslated Regions ; metabolism ; Base Sequence ; DNA, Catalytic ; genetics ; metabolism ; Hepacivirus ; enzymology ; genetics ; Humans ; Molecular Sequence Data ; RNA Processing, Post-Transcriptional ; RNA, Catalytic ; metabolism ; RNA, Viral ; metabolism ; mRNA Cleavage and Polyadenylation Factors ; genetics ; metabolism

The fluoroquinolones are the most widely used broad-spectrum antibiotics, accounting for 18% of global antibacterial market share. They can kill bacteria rapidly with variety of derivatives available. Different quinolones vary significantly in rate and spectrum of killing, oxygen requirement for metabolism and reliance upon protein synthesis. Further understanding the sophisticated mechanisms of action of this important antibiotic family based on the molecular genetic response of bacteria can facilitate the discovery of better quinolone derivatives. Factors such as SOS response, bacterial toxin-antitoxin system, programmed death, chromosome fragmentation and reactive oxygen have been implicated in the action to some extent. "Two steps characteristic" of quinolones killing is also emphasized, which might inspire future better quinolones modification.
Anti-Bacterial Agents ; pharmacology ; Apoptosis ; drug effects ; Bacteria ; drug effects ; enzymology ; genetics ; Chromosomes, Bacterial ; drug effects ; DNA Cleavage ; drug effects ; DNA Gyrase ; drug effects ; DNA Replication ; drug effects ; DNA Topoisomerases ; drug effects ; Fluoroquinolones ; pharmacology ; Quinolones ; pharmacology ; Reactive Oxygen Species ; SOS Response (Genetics) ; drug effects

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.
Amino Acid Sequence ; Base Sequence ; Calorimetry, Differential Scanning ; Catalytic Domain ; Crystallography, X-Ray ; DNA ; metabolism ; DNA Cleavage ; Deoxyribonucleases, Type II Site-Specific ; chemistry ; genetics ; metabolism ; Escherichia coli ; metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity

OBJECTIVELidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses.

METHODSCleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively.

RESULTSSIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin.

CONCLUSIONSCellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Cell Death ; drug effects ; Cell Line, Tumor ; DNA Cleavage ; Doxorubicin ; pharmacology ; Enediynes ; pharmacology ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Mitogen-Activated Protein Kinase Kinases ; genetics ; metabolism ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; Sirtuin 1 ; Sirtuins ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism