Targeting of photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene using oligonucleotide-conjugates containing [Ru(phen)3](2+)-like photosensitiser groups

Photooxidative damage was induced predominantly at a single guanine base in a target DNA by irradiation (lambda > 330 nm) in the presence of complementary oligodeoxynucleotide conjugates (ODN-5'-linker-[Ru(phen)3]2+) (phen = 1,10-phenanthroline). The target DNA represents the b2a2 variant of the chimeric bcr-abl gene implicated in the pathogenesis of chronic myeloid leukaemia, and the sequence of the 17mer ODN component of the conjugate (3' G G T A G T T A T T C C T T C T T 5') was complementary to the junction region of the sense strand sequence of this oncogene. Two different conjugates were prepared, both of them by reaction of the appropriate succinimide ester with 5'-hexylamino-derivatised 17mer ODN. In Ru-ODN-1 (7) the linker was -(CH2)6-NHCO-bpyMe (-bpyMe = 4'-[4-methyl-2,2'-bipyridyl]), whereas in Ru-ODN-2 (13) it was -(CH2)6-NHCO-(CH2)3-CONH-phen. Photoexcitation of either of the conjugates when hybridised with the 32P-5'-end-labelled target 34mer 5'T G A C C A T C A A T A A G G A A G A A G21 C C C T T C A G C G G C C 3' (ODN binding site underlined) led to an alkali-labile site predominantly (> 90%) at the G21 base, which is at the junction of double-stranded and single-stranded regions of the hybrid. Greater yields were found with Ru-ODN-1 (7) than with Ru ODN-2 (13). In contrast to this specific cleavage with Ru-ODN-1 (7) or Ru-ODN-2 (13), alkali-labile sites were generated at all guanines when the 34mer was photolysed in the presence of the free sensitiser [Ru(phen)3]2+. Since [Ru(phen)3]2+ was shown to react with 2'-deoxyguanosine to form the diastereomers of a spiroiminodihydantoin derivative (the product from 1O2 reaction), 1O2 might also be an oxidizing species in the case of Ru-ODN-1 (7) and Ru-ODN-2 (13). Therefore to determine the range of reaction, a series of 'variant' targets was prepared, in which G21 was replaced with a cytosine and a guanine substituted for a base further towards the 3'-end (e.g. Variant 3; 5'T G A C C A T C A A T A A G G A A G A A C C G23 C T T C A G C G G32 C C3'). While it was noted that efficient reaction took place at distances apparently remote from the photosensitiser (e.g at G32, but not G23 for Variant 3), this effect could be attributed to hairpinning of the single-stranded region of the target. These results are therefore consistent with the photooxidative damage being induced by a reaction close to the photosensitiser rather than by a diffusible species such as 1O2.

Phenotypic consequences of genome mistranslation in Candida albicans

Várias espécies do género Candida traduzem o codão CUG de leucine como serina. Em C. albicans este codão é traduzido pelo tRNACAG Ser de serina que é reconhecido por leucil- e seril-tRNA sintetases (LeuRS e SerRS), permitindo a incorporação de leucina ou serina em posições com CUG. Em condições padrão de crescimento os codões CUG é incorporam 3% de leucina e 97% de serina, no entanto estes valores são flexíveis uma vez que a incorporação de serina pode variar entre 0.6% e 5% em resposta a condições de stress. Estudos anteriores realizados in vivo em Escherichia coli sugeriram que a ambiguidade em codões CUG é regulada pela SerRS. De facto, o gene da SerRS de C. albicans tem um codão CUG na posição 197 (Ser197) cuja descodificação ambígua resulta na produção de duas isoformas de SerRS. A isoforma SerRS_Leu197 é mais ativa, apesar de menos estável, que a isoforma SerRS_Ser197, suportando a ideia da existência de um feedback loop negativo, envolvendo estas duas isoformas de SerRS, a enzima LeuRS e o tRNACAG Ser, que mantem os níveis de incorporação de leucina no codões CUG baixos. Nesta tese demonstramos que tal mecanismo não é operacional nas células de C. albicans. De facto, os níveis de incorporação de leucina em codões CUG flutuam drasticamente em resposta a alterações ambientais. Por exemplo, a incorporação de leucina pode chegar a níveis de 49.33% na presença de macrófagos e anfotericina B, mostrando a notória tolerância de C. albicans à ambiguidade. Para compreender a relevância biológica da ambiguidade do código genético em C. albicans construímos estirpes que incorporam serina em vários codões. Apesar da taxa crescimento ter sido negativamente afetada em condições padrão de crescimento, as estirpes construídas crescem favoravelmente em várias condições de stresse, sugerindo que a ambiguidade desempenha um papel importante na adaptação a novos nichos ecológicos. O transcriptoma das estirpes construídas de C. albicans e Saccharomyces. cerevisiae mostram que as leveduras respondem à ambiguidade dos codões de modo distinto. A ambiguidade induziu uma desregulação moderada da expressão génica de C. albicans, mas ativou uma resposta comum ao stresse em S. cerevisiae. O único processo celular que foi induzido na maioria das estirpes foi a oxidação redução. De salientar, que enriquecimento em elementos cis de fatores de transcrição que regulam a resposta à ambiguidade em ambas as leveduras foi distinta, sugerindo que ambas respondem ao stresse de modo diferente. Na globalidade, o nosso estudo aprofunda o conhecimento da elevada tolerância à ambiguidade de codões em C. albicans. Os resultados sugerem que este fungo usa a ambiguidade do codão CUG durante infeção, possivelmente para modular a sua interação com o hospedeiro e a resposta a drogas antifúngicas.

Electroanalytical study of the pesticide ethiofencarb

A detailed study of voltammetric behavior of ethiofencarb (ETF) is reported using glassy carbon electrode (GCE) and hanging mercury drop electrode (HMDE). With GCE, it is possible to verify that the oxidative mechanism is irreversible, independent of pH, and the maximum intensity current was observed at +1.20 V vs. AgCl/Ag at pH 1.9. A linear calibration line was obtained from 1.0x10-4 to 8.0x10-4 mol L-1 with SWV method. To complete the electrochemical knowledge of ETF pesticide, the reduction was also explored with HMDE. A well-defined peak was observed at –1.00V vs. AgCl/Ag in a large range of pH with higher signal at pH 7.0. Linearity was obtained in 4.2x10-6 and 9.4x10-6 mol L-1 ETF concentration range. An immediate alkaline hydrolysis of ETF was executed, producing a phenolic compound (2-ethylthiomethylphenol) (EMP), and the electrochemical activity of the product was examined. It was deduced that it is oxidized on GCE at +0.75V vs. AgCl/Ag with a maximum peak intensity current at pH 3.2, but the compound had no reduction activity on HMDE. Using the decrease of potential peak, a flow injection analysis (FIA) system was developed connected to an amperometric detector, enabling the determination of EMP over concentration range of 1.0x10-7 and 1.0x10-5 mol L-1 at a sampling rate of 60 h-1. The results provided by FIA methodology were performed by comparison with results from high-performance liquid chromatography (HPLC) technique and demonstrated good agreement with relative deviations lower than 4%. Recovery trials were performed and the obtained values were between 98 and 104%.

Metabolic and respiratory effects of infused sodium acetate in healthy human subjects.

The metabolic and respiratory effects of intravenous 0.5 M sodium acetate (at a rate of 2.5 mmol/min during 120 min) were studied in nine normal human subjects. O2 consumption (VO2) and CO2 production (VCO2) were measured continuously by open-circuit indirect calorimetry. VO2 increased from 251 +/- 9 to 281 +/- 9 ml/min (P < 0.001), energy expenditure increased from 4.95 +/- 0.17 kJ/min baseline to 5.58 +/- 0.16 kJ/min (P < 0.001), and VCO2 decreased nonsignificantly (211 +/- 7 ml/min vs. 202 +/- 7 ml/min, NS). The extrapulmonary CO2 loss (i.e., bicarbonate generation and excretion) was estimated at 48 +/- 5 ml/min. This observation is consistent with 1 mol of bicarbonate generated from 1 mol of acetate metabolized. Alveolar ventilation decreased from 3.5 +/- 0.2 l/min basal to 3.1 +/- 0.2 l/min (P < 0.001). The minute ventilation (VE) to VO2 ratio decreased from 22.9 +/- 1.3 to 17.6 +/- 0.9 l/l (P < 0.005), arterial PO2 decreased from 93.2 +/- 1.9 to 78.7 +/- 1.6 mmHg (P < 0.0001), arterial PCO2 increased from 39.2 +/- 0.7 to 42.1 +/- 1.1 mmHg (P < 0.0001), pH from 7.40 +/- 0.005 to 7.50 +/- 0.007 (P < 0.005), and arterial bicarbonate concentration from 24.2 +/- 0.7 to 32.9 +/- 1.1 (P < 0.0001). These observations indicate that sodium acetate infusion results in substantial extrapulmonary CO2 loss, which leads to a relative decrease of total and alveolar ventilation.

Enteral versus parenteral nutrition: comparison of energy metabolism in healthy subjects.

Continuous respiratory exchange measurements were performed on 10 healthy young women for 1 h before, 3 h during, and 3 h after either parenteral (iv) or intragastric (ig) administration of a nutrient mixture (52% glucose, 18% amino acid, and 30% lipid energy) infused at twice the postabsorptive resting energy expenditure (REE). REE rose from 0.98 +/- 0.02 (iv) and 0.99 +/- 0.02 kcal/min (ig) postabsorptively to 1.13 +/- 0.03 (iv) and 1.13 +/- 0.02 kcal/min (ig), resulting in nutrient-induced thermogenesis of 10 +/- 0.6 and 9.3 +/- 0.9%, respectively, when related to the metabolizable energy. The respiratory quotient rose from preinfusion values of 0.81 +/- 0.02 (iv) and 0.80 +/- 0.01 (ig) to 0.86 +/- 0.01 (iv) and 0.85 +/- 0.01 (ig). After nutrient administration the respiratory quotient fell significantly to below the preinfusion values. Plasma glucose and insulin concentrations rose during nutrient administration but were higher during the intravenous route. It is concluded that, although the response time to intragastric administration was delayed, the thermic effects and overall substrate oxidations were comparable during intravenous or intragastric administration, albeit, at lower plasma glucose and insulin concentrations via the intragastric route.

Measurement of 13CO2 in expired air as an index of compliance to a high carbohydrate diet naturally enriched in 13C.

The aim of this study was to determine whether breath 13CO2 measurements could be used to assess the compliance to a diet containing carbohydrates naturally enriched in 13C. The study was divided into two periods: Period 1 (baseline of 4 days) with low 13C/12C ratio carbohydrates. Period 2 (5 days) isocaloric diet with a high 13C/12C ratio (corn, cane sugar, pineapple, millet) carbohydrates. Measurements were made of respiratory gas exchange by indirect calorimetry, urinary nitrogen excretion and breath 13CO2 every morning in post-absorptive conditions, both in resting state and during a 45-min low intensity exercise (walking on a treadmill). The subjects were 10 healthy lean women (BMI 20.4 +/- 1.7 kg/m2, % body fat 24.4 +/- 1.3%), the 13C enrichment of oxidized carbohydrate and breath 13CO2 were compared to the enrichment of exogenous dietary carbohydrates. At rest the enrichment of oxidized carbohydrate increased significantly after one day of 13C carbohydrate enriched diet and reached a steady value (103 +/- 16%) similar to the enrichment of exogenous carbohydrates. During exercise, the 13C enrichment of oxidized carbohydrate remained significantly lower (68 +/- 17%) than that of dietary carbohydrates. The compliance to a diet with a high content of carbohydrates naturally enriched in 13C may be assessed from the measurement of breath 13CO2 enrichment combined with respiratory gas exchange in resting, postabsorptive conditions.

Effect of citrate on the redox properties of cytochrome C and its kinetic and binding behaviour with cytochrome oxidase /

Increasing citrate concentration, at constant ionic strength (30 mM) decreases the rate of cytochrome ~ reduction by ascorbate. This effect is also seen at both high (600 mM) and low (19 mM) ionic strengths, and the Kapp for citrate increases with increasing ionic strength. Citrate binds d both ferri -and ferrocytochrome ~, but with a lower affinity for the latter form (Kox . .red d = 2 mM, Kd = 8 mM) as shown by an equilibrium assay with N,N,N',N', Tetramethyl E- phenylenediamine. The reaction of ferricytochrome ~with cyanide is also altered in the presence of citrate: citrate increases the K~PP for cyanide. Column chromatography of cytochrome ~-cytochrome oxidase mixtures shows citrate increases the dissociation constant of the complex. These results are confirmed in kinetic assays for the "loose"site (Km = 20 pM) only. The effect of increasing citrate observable at the "tight" site (Km = 0.25 pM) is on the turnover number and not on the K . These results suggest a mechanism m where anion binding to cytochrome £ at the tight site affects the equilibrium between two forms of cytochrome c bound cytochrome oxidase: an active and an inactive one.

Fabrication of Electrochemical Sensors for the Determination of Pharmaceuticals

Voltammetric sensors are an important class of electrochemical sensors in which the analytical information is obtained from the measurement of current obtained as a result of electrochemical oxidation/reduction.This current is proportional to the concentration of the analyte.Chemically modified electrodes(CMEs) have great significance as important analytical tools for the electrochemical determination of pharmaceuticals.The modification of electrode results in efficient determination of electro-active biomolecules at very lower potential without its major interferences.The operation mechanism of CMEs depends on the properties of the modifier materials that are used to promote selectivity towards the target analytes.Modified electrodes can be prepared by deposition of various compounds such as organic compounds ,conducting polymers,metal oxides,etc. on the various electrode surfaces.The thesis presents the development ,electrochemical characterization and analytical application studies of eight voltammetric sensors developed for six drugs viz.,Ambroxol,Sulfamethoxazole,PAM Chloride, Lamivudine,Metronidazole and Nimesulide.The modification techniques adopted as part of the present work include Multiwalled Carbon Nanotube(MWCNT) based modification.Electropolymerisation and Gold Nanoparticle (AuNP) based modifications.

Synthesis and reactions of flavazoles

The study deals with the production of l-phenylflavazoles with chloro, amino, hydroxy, chloromethyl, carboxamido, trichloromethyl, N-pyrrolidyl and N-pyrrolidylmethyl groups substituted at position 3. The interconversions of 3-amino, 3-hydroxy and 3-chlorol- phenylflavazoles were also investigated. Further, an unusual phenylation reaction was found to take place if stored or air-oxidised phenylhydrazine was used as the condensing agent for the formation of flavazoles from quinoxaline-2-carboxaldehyde phenylhydrazones. By this phenylation reaction 1,3-diphenyl, l-p-tolyl-3-phenyl, l-p-chlorophenyl-3-phenyl, l-p-bromophenyl- 3-phenyl and l-phenyl-3-p-tolylflavazoles were prepared. In addition to establishing the structure of the phenylation products, the reaction was shown to take place by a free radical mechanism involving phenyl radicals formed from oxidised phenylhydrazine. Also the oxidation, reduction and bromination reactions of l-phenylflavazole were investigated. The product obtained when a mixture of l-phenylflavazole and sodium borohydride in isopropanol was heated under reflux was shown to be 2-anilinoquinoxaline-3-carboxamide which when refluxed with concentrated hydrochloric acid gave the known 2-anilinoquinoxaline. New procedures were worked out for the oxidative cyclisation reactions of quinoxaline-2carboxaldehyde phenylhydrazones to l-phenylflavazoles in excellent yields using azobenzene as a dehydrogenating agent. These cyclisations were also shown to take place, though in low Yield, if the quinoxaline2- carboxaldehyde phenylhydrazones were heated above their melting points in an atmosphere containing oxygen.

Macrobenthos And Its Relation To Ecosystem Dynamics In The Cochin Estuary

Cochin estuary is a shallow brackish water body situated on the south west coast of India. It is a tropical positive estuary extending between 90 40’ and 100 12’ N and 760 10’and 760 30’ E with its northern boundary at Azhikode and southern boundary at Thannermukkom bund.The abundance of benthic fauna in an ecosystem shows the close relationship to its environment and reflects the characteristics of an ecological niche. Seasonal and monthly variations in the distribution of macrobenthos in relation to sediment characteristics were conducted in Cochin estuary from 2009-10 periods. Oxidation-reduction potential showed reducing trends that affected the distribution and diversity of fauna. Seasonal variations in water quality and river discharge pattern affected the faunal composition in the different stations. Sewage mixing was the principal source of organic pollution in the Cochin estuary. The sediment pH was generally on the alkaline side ranging from 4.99 at St.9 and 8.33 at St.1.The Eh ranged from -11mV at St.3 to -625mV at St.2.The temperature varied from 260C to 320C in the estuary. The moisture content ranged from 1.63 to 12.155%, that of organic carbon from 0 09 at St. 6 to 4.29% at St.9 and that of organic matter from 0.16 to 7.39%. Seasonally, the average of Eh was highest during the monsoon (156.22 mV) and in the pre monsoon (140.94 mV). The average pH for the 9 study stations was 7.68 during monsoon period and 7.08 during post monsoon. Based on group wise seasonal analysis, the average mean abundance was maximum for polychaetes (43.47) followed by nematodes (33.62), crustaceans (21.62), molluscs (11.94) and Pisces (0.05) in the estuary. Monsoon season was most favourable for benthic faunal abundance followed by the post monsoon period in the study. The series of human interventions like dredging, discharge of industrial effluents, urbanisation and related aspects had a strong influence on the distribution, abundance of benthic macrofauna in the wetland.

DISTRIBUTION OF Cr, Pb, Cu, Cd AND Zn IN SEDIMENTS OF THE DOBCZYCE DAM RESERVOIR (SOUTHERN POLAND)

Sediments play a fundamental role in the behaviour of contaminants in aquatic systems. Various processes in sediments, eg adsorption-desorption, oxidation-reduction, ion exchange or biological activities, can cause accumulation or release of metals and anions from the bottom of reservoirs, and have been recently studied in Polish waters [1-3]. Sediment samples from layer A: (1 divided by 6 cm depth in direct contact with bottom water); layer B: (7 divided by 12 cm depth moderate contact); and layer C: (12+ cm depth, in theory an inactive layer) were collected in September 2007 from six sites representing different types of hydrological conditions along the Dobczyce Reservoir (Fig. l). Water depths at the sampling points varied from 3.5 to 21 m. We have focused on studying the distribution and accumulation of several heavy metals (Cr, Pb, Cd, Cu and Zn) in the sediments. The surface, bottom and pore water (extracted from sediments by centrifugation) samples were also collected. Possible relationships between the heavy-metal distribution in sediments and the sediment characteristics (mineralogy, organic matter) as well as the Fe, Mn and Ca content of sediments, have been studied. The 02 concentrations in water samples were also measured. The heavy metals in sediments ranged from 19.0 to 226.3 mg/kg of dry mass (ppm). The results show considerable variations in heavy-metal concentrations between the 6 stations, but not in the individual layers (A, B, C). These variations are related to the mineralogy and chemical composition of the sediments and their pore waters.

Comparison of SERS performances of Co and Ni ultrathin films over silver to electrochemically activated Co and Ni electrodes

In this work, the surface-enhanced Raman scattering (SERS) spectra of pyridine (py) on thin films of Co and Ni electrodeposited on an Ag electrode activated by oxidation-reduction cycles (ORC) are presented. The SERS spectra from the thin films were compared to those of py on activated bare transition metal electrodes. It was verified that the SERS spectra of py on 3 monolayers (ML)-thick films of Ni and Co presented only bands assignable to the py adsorbed on transition metal surfaces. It was also observed that even for 50 ML-thick transition metal films, the py SERS intensity was ca. 40% of the intensity from the 3 ML-thick films. The relative intensities of the SERS bands depended on the thickness of the films, and for films thicker than 7 ML for Co and 9 ML for Ni they were very similar to those of the bare transition metal electrodes. The transition metal thin films over Ag activated electrodes presented SERS intensities 3 orders of magnitude higher than the ones from bare transition metal electrodes. These films are more suitable to study the adsorption of low Raman cross-section molecules than are ORC-activated transition metal electrodes.

A New Insight on the Preparation of Stabilized Alpha-Nickel Hydroxide Nanoparticles

Nickel hydroxide can provide an outstanding cathode material in alkaline secondary batteries, however the progressive decrease of the charge capacity as a function of the number of oxidation/reduction cycles is a challenging problem to be solved. New improvements on the electrochemical properties of electrode materials can be achieved by exploiting the much better performance of alpha-nickel hydroxide. Such materials were obtained in a stable form by sol-gel method and characterized by thermogravimetric analyses, UV-Vis spectroscopy, X-ray diffractometry, scanning and transmission electron microscopy, cyclic voltammetry and electrochemical quartz crystal microbalance techniques. The results revealed not only the formation of the alpha-Ni(OH)(2) phase, but also a much better electrochemical reversibility and stability as compared with similar materials obtained by electrochemical precipitation method.

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bio-electrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 mu W cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 mu W cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxiclase bioanodes and/or biofuel cells. (C) 2011 Elsevier Inc. All rights reserved.

A water-soluble antimicrobial acrylamide copolymer containing sulfitobetaine for enhanced oil recovery

Herein, we report a novel acrylamide copolymer with antimicrobial property as an enhanced oil recovery chemical. The copolymer was synthesized from acrylamide (AM), acrylic acid (AA) and 2-((2-(acryloyloxy)ethyl)dimethylammonio)ethyl sulfite (ADMES) using oxidation-reduction initiation system. Subsequently, the copolymer AM/AA/ADMES was evaluated and characterized on several aspects such as IR, ¹H NMR, intrinsic viscosity, and dissolubility. The AM/AA/ADMES solution exerted remarkable thickening ability, salt tolerance ability and viscoelasticity. In addition, the rheological properties, temperature resistance ability and long-term stability of AM/AA/ADMES were investigated systematically in the presence of sulfate-reducing bacteria and relatively low viscosity loss could be obtained compared to partially hydrolyzed polyacrylamide. On the basis of core flooding experiments, AM/AA/ADMES was found to be a valuable prospect with 10.5 resistance factor, 4.6 residual resistance factor and up to 11.0% enhanced oil recovery.