Derivados de lignina de DNA como agentes modificadores no desenvolvimento de sensores voltamétricos / Lignin derivatives and DNA as modificating agents in the development of voltammetric sensors

Neste trabalho, 2 diferentes procedimentosforam utilizados para a extração de lignina a partir do licor negro, caracterizando-se quimicamente as amostras de lignina obtidas em relação a umalignina comercial, visando a aplicação no desenvolvimento de sensores voltamétricos. A análise elementar, espectroscopia na região do infravermelho, análise térmica e caracterização eletroquímica do material mostraram que, tanto a origem da lignina quanto a metodologia de obtenção da mesma, a partir do licor negro, podem fornecer materiais com propriedades químicas distintas, embora possuam comportamento eletroquímico similar. Observou-se, também, que a lignina só pode ser armazenada na forma sólida,devido à oxidação pelo oxigênio dissolvidodurante o tempo em que a solução mãe é armazenada.No entanto, a oxidação dalignina é necessária quando se tem por objetivo o desenvolvimento de sensores voltamétricos, devido a predominância de carbonos sp2na estrutura químicaoxidada, condição em que se obtém maior condutividade. Constatou-se também a necessidade de utilizar um transdutor metálico para o desenvolvimento de eletrodos qumicamente modificados com este material, visto que os eletrodos de carbono modificados com lignina oxidada ou não oxidada não apresentaram atividade eletroquímica. Devido à pequena porcentagem de enxofre existente na estrutura química, a lignina oxidada tende a se organizar pelos grupamentos SH quando na presença de ouro, expondo os grupamentos quinônicos eletroativos. A lignina oxidada ainda foi utilizada no preparo de eletrodo de pasta de carbono com nanopartículas de ouro, na qual a lignina oxidada impregnada no grafite atua como redutor "in-situ" do ouro, permitindo o preparo de um sensor voltamétrico versátil, capaz de realizar a determinação de ácido ascórbico, dopamina, nitrito e iodato. No que tange ao comportamento eletroquímico de fármacos e estudos de interação fármaco-DNA, eletrodos de carbono foram modificados com DNAdupla fita com a finalidade de monitorar a interação DNA-Gemcitabina.O fármaco não apresentou atividade eletroquímica tanto na região positiva quanto na região negativa de potencial. A interação do mesmo com o DNA promove a condensação/agregação das duplas fitas do DNA em uma primeiraetapa, seguida da clivagem do nucleosídeo guanosina, formando guanina livre. O comportamento eletroquímico de leflunomida e sulfasalazina, dois fármacos aplicados ao tratamento da artrite reumatóide, foi estudado e mecanismos de oxidação foram propostos para cada fármaco

The chemical properties of samples of lignin, which were precipitated from black liquor using two different methodologies (precipitation with CO2 and H2SO4), were studied and the results compared to those obtained from a commercial lignin sample in order to prepare voltammetric sensors. The elementary analysis, infrared spectroscopy, thermal analysis and electrochemical characterization of the material demonstrated that both, the source of lignin and the precipitation method from the black liquor, can provide lignin samples with different chemical properties, although the electrochemical behavior of all samples has been the same. Lignin could only be stored in solid form as lignin in the black liquor is slowly and quantitatively oxidized by dissolved oxygen, preventing the extraction procedures. However, the lignin as extracted from black liquor cannot be used to modify solid electrodes due its high resistivity. The previous oxidation of the all material was necessary when the aim was its application on the sensors development. The electrical conductivity in the oxidized lignin was achieved, probably due to the predominance of sp2 hybridized carbon atoms, which improved orbital overlapping in the material. In addition, it was necessary to use a metallic transducer to produce electrodes modified with films of lignin with good electrochemical activity. The films drying time was also important parameter, which suggested a specific organization of lignin macromolecules over the electrode surface. Due to the small percentage of sulfur in the material, the oxidized lignin tended to be organized by the SH groups in the presence of metallic substrates, exposing its electroactive quinone groups. The oxidized lignin was further used to prepare carbon paste electrodes modified with gold nanoparticles, in which the impregnated oxidized lignin on graphite acted as an "in situ" reducing agent towards HAuCl4.The resulting composite allowed the preparation of a versatile voltammetric sensor, capable of detecting ascorbic acid, dopamine, nitrite and iodate. Regarding the electrochemical behavior and drug interaction studies DNA-molecule, carbon electrodes were modified with double strand DNA with the purpose of monitoring Gemcitabine-DNA interaction. The drug showed no electrochemical activity both, in the positive and the negative potential. The Gemcitabine-DNA interaction promoted condensation / aggregation of the double strand DNA in a first step, followed by cleavage of the nucleoside guanosine in the form of free guanine. In addition, the electrochemical behavior of sulfasalazine and leflunomide, two pharmacological compounds applied to the treatment of rheumathoid arthritis, were studied and their oxidation mechanisms were proposed

Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII

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.

The molecular physiological and genetic mechanisms underlying the superb efficacy of quinolones / 药学学报

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.

Synthesis of diethylenetriamine polydentate ligands and their DNA-cleaving activity / 药学学报

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.

Cryotop no desenvolvimento de oócitos bovinos imaturos vitrificados / Cryotop and development of vitrified immature bovine oocytes

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10 percent DMSO + 10 percent ethylene glycol (EG) for 30-45sec and 2: 20 percent DMSO + 20 percent EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58 percent respectively. Vitrified oocytes using OPS and EMG showed 26 and 32 percent; and 35 and 46 percent of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes.

Avaliou-se a eficácia de diferentes dispositivos de congelamento (envasamento em palhetas (EP), microscopia eletrônica de grade (MEG) e Cryotop) para vitrificação de ovócitos imaturos de bovinos. Para tal, foram determinados o corpo polar, a metáfase II (MII), a viabilidade e as subsequentes taxas de desenvolvimento. Foram utilizados somente ovócitos com quatro ou cinco camadas de células do cumulus. Os ovócitos foram equilibrados em duas soluções de vitrificação - 1: DMSO (10 por cento) + etilenoglicol (EG; 10 por cento) por 30 a 45 segundos e 2: DMSO (20 por cento) + EG (20 por cento) + sacarose (0,5M) por 25 segundos -, transferidos para os dispositivos de congelamento e mantidos, por 10 dias, em nitrogênio líquido. Imediatamente após serem retirados do nitrogênio, os ovócitos foram removidos dos dispositivos e processados para maturação, fertilização e cultivo in vitro. Os ovócitos vitrificados com o Cryotop apresentaram as maiores taxas de extrusão do corpo polar (CP) e de maturidade nuclear (MII), 41 e 58 por cento, respectivamente. Para os ovócitos vitrificados com EP e MEG, as taxas de CP e as de MII foram, respectivamente, de 26 e 32 por cento e de 35 e 46 por cento. As taxas de viabilidade não diferiram entre os grupos Cryotop e EMG. Os ovócitos vitrificados com Cryotop apresentaram as maiores taxas de clivagem e de blastocisto. Para todas as variáveis estudadas, as taxas para os ovócitos vitrificados foram significativamente menores do que as do grupo-controle (P<0,05). Os resultados deste estudo mostraram a superioridade do dispositivo Cryotop para vitrificação de ovócitos imaturos de bovinos.

Role of prosurvival molecules in the action of lidamycin toward human tumor cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Lidamycin, 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.</p><p><b>METHODS</b>Cleavage 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.</p><p><b>RESULTS</b>SIRT1 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.</p><p><b>CONCLUSIONS</b>Cellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.</p>

Electrochemical, Antifungal, Antibacterial and DNA Cleavage Studies of Some Co(II), Ni(II), Cu(II) and Zn(II)-Copolymer Complexes

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.

Apoptosis Induction of Stomach Cancer Cell by TNF alpha and TGFbeta / 대한암학회지

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.