Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Atividade da redutase do nitrato em cana-de-açúcar

Pós-graduação em Agronomia (Ciência do Solo) - FCAV

Effects of Natural Radiation, Photosynthetically Active Radiation and Artificial Ultraviolet Radiation-B on the Chloroplast Organization and Metabolism of Porphyra acanthophora var. brasiliensis (Rhodophyta, Bangiales)

We undertook a study of Porphyra acanthophora var. brasiliensis to determine its responses under ambient conditions, photosynthetically active radiation (PAR), and PAR+UVBR (ultraviolet radiation-B) treatment, focusing on changes in ultrastructure, and cytochemistry. Accordingly, control ambient samples were collected in the field, and two different treatments were performed in the laboratory. Plants were exposed to PAR at 60 mu mol photons m(-2) s(-1) and PAR+UVBR at 0.35 W m(-2) for 3 h per day during 21 days of in vitro cultivation. Confocal laser scanning microscopy analysis of the vegetative cells showed single stellate chloroplast in ambient and PAR samples, but in PAR+UVBR-exposed plants, the chloroplast showed alterations in the number and form of arms. Under PAR+UVBR treatment, the thylakoids of the chloroplasts were disrupted, and an increase in the number of plastoglobuli was observed, in addition to mitochondria, which appeared with irregular, disrupted morphology compared to ambient and PAR samples. After UVBR exposure, the formation of carpospores was also observed. Plants under ambient conditions, as well as those treated with PAR and PAR+UVBR, all showed different concentrations of enzymatic response, including glutathione peroxidase and reductase activity. In summary, the present study demonstrates that P. acanthophora var. brasiliensis shows the activation of distinct mechanisms against natural radiation, PAR and PAR+UVBR.

Enzymatic Logic Gates with Noise-Reducing Sigmoid Response

Biochemical computing is an emerging field of unconventional computing that attempts to process information with biomolecules and biological objects using digital logic. In this work we survey filtering in general, in biochemical computing, and summarize the experimental realization of an and logic gate with sigmoid response in one of the inputs. The logic gate is realized with electrode-immobilized glucose-6-phosphate dehydrogenase enzyme that catalyzes a reaction corresponding to the Boolean and functions. A kinetic model is also developed and used to evaluate the extent to which the performance of the experimentally realized logic gate is close to optimal.

Characterization of glycerol kinase from baker's yeast: Response surface modeling of the enzymatic reaction

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved.

Varied Immune Response to FVIII: Presence of Proteolytic Antibodies Directed to Factor VIII in Different Human Pathologies

The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation of the complement and its activation, and activation of effector cells. In addition to this plethora of functions, antibodies are capable of expressing enzymatic activity. Antibodies with catalytic function are a result of the productive interplay between the highly evolved machinery of the immune system and the chemical framework used to induce them (antigens). Catalytic antibodies are immunoglobulins with an ability to catalyze the reactions involving the antigen for which they are specific. Catalytic immunoglobulins of the IgM and IgG isotypes have been detected in the serum of healthy donors. In addition, catalytic immunoglobulins of the IgA isotype have been detected in the milk of healthy mothers. Conversely, antigen-specific hydrolytic antibodies have been reported in a number of inflammatory, autoimmune, and neoplastic disorders. The pathophysiological occurrence and relevance of catalytic antibodies remains a debated issue. Through the description of the hydrolysis of coagulation factor VIII as model target antigen, we propose that catalytic antibodies directed to the coagulation factor VIII may play a beneficial or a deleterious role depending on the immuno-inflammatory condition under which they occur.

Non-Enzymatic Electroanalysis of Glucose on Electrodeposited Au-PEDOT Dendrites

Electrodeposition of Au on poly (3,4-ethylenedioxythiophene) (PEDOT) coated carbon paper electrode results in the formation of a stable 3-D urchin-like morphology. Au-PEDOT/C electrode exhibits higher surface area, greater catalytic activity, higher sensitivity and lower detection limit for glucose analysis in an alkaline medium than Au/C electrode. Au-PEDOT/C electrode exhibits a linear current response in glucose concentration ranging up to 10 mu M with sensitivity of 515 mu A cm(-2) mu M-1 (on the basis of geometric area) and a low detection limit of 0.03 mu M with signal to noise ratio of 3. Thus, the PEDOT under-layer improves the property of Au for glucose analysis. (c) 2013 The Electrochemical Society.

Non-enzymatic electronic detection of glucose using aminophenylboronic acid functionalized reduced graphene oxide

We report the non-enzymatic electronic detection of glucose using field effect transistor (FET) devices made of aminophenylboronic acid (APBA) functionalized reduced graphene oxide (RGO). Detection of glucose molecules was carried out over a wide dynamic range of concentration varying from 100 pM to 100 mM with a detection limit of similar to 2 nM using both covalently and non-covalently functionalized APBA-RGO complex. The normalized change in electrical conductance data shows that the FET devices made of non-covalently functionalized APBA-RGO complex (nc-APBA-RGO) exhibited a linear response to glucose aqueous solution of concentrations varying from 1 nM to 10 mM and showed 4 times enhanced sensitivity over the devices made of covalently functionalized APBA-RGO complex (c-APBA-RGO). Specificity of APBA-RGO complex to glucose is confirmed from the observation of negligible change in electrical conductance after exposure to 0.1 mM of lactose and other interfering factors. (C) 2015 Elsevier B.V. All rights reserved.

Biogeochemical study of poly aromatic hydrocarbons (PAHs) and its physiological affection response in mudskipper (Boleophthalmus dussumieri) at coast of Khuzestan

Study on the biomarkers types to assess health status of marine ecosystems in environmental biomonitoring has an important value. Accordingly, accumulation of polycyclic aromatic hydrocarbons(PAHs) in sediment, water and tissues (liver and gill) of mudskipper(i.e. Boleophthalmus dussumieri) and some physiological responses like lysosomal membrane change performed on haemocytes, stability of red blood cell membrane and the Glutathione-S Transferase (GST) activity in the liver were measured in mudskipper. Samples were obtained from five sites along north western coast of the Persian Gulf (Khuzestan coast). Red blood cell membrane changes after different concentration of PAHs at different time was also studied to evaluate impact of PAHs compound on cell membrane. PAHs concentration was measured by HPLC method. The activity of GST enzyme was analysed by spectrophotometric method. Lysosomal membrane change was measured by NRR time method and stability of red blood cell membrane was evaluated by EOF test. Total PAH concentrations in the coastal sea water, the sediments, the liver and the gill tissues ranged between 0.80-18.34 μg/l, 113.50-3384.34 ng g-1 (dry weight), 3.99-46.64 ng g-1 dw and 3.11-17.76 ng g-1 dw, respectively. Highest PAHs pollution was found at Jafari while the lowest was detected at Bahrakan sampling sites. The lowest enzymatic activity was identified at Bahrakan (7.19 ± 1.541 nmol/mg protein/min), while the highest was recorded at Jafari (46.96 ± 7.877 nmol/mg protein/min). Comparative analysis of GST activity in the liver of mudskippers showed significant difference (p < 0.05) between the locations of Jafari and Bahrakan, and with other sites. Moreover, no significant difference was detected between the locations of Arvand, Zangi and Samayeli (p < 0.05). The mean RT was below 90 minutes in all sampling sites. Values of mean RT of the dye ranged from 34 (for the blood samples of mudskipper collected from Jafari site) to 78 minutes (for the blood samples of mudskipper collected from Bahrakan site). Spatial evaluation revealed the longest RT in fish from Bahrakan as compared with those from other sites. Preliminary results showed a significant difference (p < 0.05) among sampling sites except between Arvand and Zangi (p > 0.05). Osmotic fragility curves indicated that erythrocytes collected from mudskippers at Jafari were the most 009 fragile followed by Zangi> Arvand> Samayeli> and Bahrakan. The mean erythrocyte fragility was significantly higher at Jafari site (p < 0.05) when compared to other sites. Significant differences were found between the various sites (p < 0.05).The result indicated no significant differences between the control and treatments of mudskipper RBC exposed to field concentrations of PAHs (P>0.05). The results further indicated significant differences (P<0.05) between the control and treatments of mudskipper RBC exposed to acute. Potency Divisor concentrations. It is clear from the present result that chronic. Potency Divisor concentrations protect red cells against osmotic hemolysis. This study, however, showed that PAH concentrations in this region are not higher than the available standards. The findings showed that Lysosomal membrane destabilization, liver GST activities and fragility of red cell membrane are highly sensitive in the mudskipper, B. dussumieri. Thus, mudskipper perceived to be good sentinel organisms for PAH pollution monitoring. Sediment PAH concentrations were strongly correlated with biomarkers, indicating that PAH type pollutants were biologically available to fish. One of the possible risk assessment implications of this study is that biomarkers can be applied not only to characterize biological effects of pollution exposures, but also to determine the bioavailability of pollution in aquatic systems. The results also indicated that PAHs compound possess anti haemolytic property.

Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M-aeruginosa

The aim of this study was to examine the effects of chemical nonylphenols (NPs) on the antioxidant system of Microcystis aeruginosa strains. The degradation and sorption of NPs by M. aeruginosa were also evaluated. High concentrations of NPs (1 and 2 mg/l) were found to cause increases in superoxidase dismutase (SOD) and glutathione-S-transferase (GST) activities and in glutathione (GSH) levels. These results suggest that toxic stress manifested by elevated SOD and GST levels and GSH contents may be responsible for the toxicity of NPs to M. aeruginosa and that the algal cells could improve their antioxidant and detoxification ability through the enhancement of enzymatic and nonenzymatic prevention substances. The observed elevations in GSH levels and GST activities were relatively higher than those in SOD activities, indicating that GSH and GST contributed more in eliminating toxic effects than SOD. Low concentrations of NPs (0.05-0.2 mg/l) enhanced cell growth and decreased GST activity in algal cells of M. aeruginosa, suggesting that NPs may have acted as a protecting factor, such as an antioxidant. The larger portion of the NPs (> 60%) disappeared after 12 days of incubation, indicating the strong ability of M. aeruginosa to degrade the moderate persistent NP compounds. The sorption ratio of M. aeruginosa after a 12-day exposure to low nominal concentrations of NPs (0.02-0.5 mg/l) was relatively high (> 30%). The fact that M. aeruginosa effectively resisted the toxic effects of NPs and strongly degraded these pollutants indicate that M. aeruginosa cells have a strong ability to adapt to variations in environmental conditions and that low and moderate concentrations of organic compounds may favor its survival. Further studies are needed to provide detailed information on the fate of persistent organic pollutants and the survival of algae and to determine the possible role of organic pollutants in the occurrence of water blooms in eutrophic lakes.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 mug(-1) microcystin-RR. The results showed that the growth of Synechococcus elongatus ( expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Signi. cant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Kinetic Model Study on Enzymatic Hydrolysis of Cellulose Using Artificial Neural Networks

Enzymatic hydrolysis of cellulose was highly complex because of the unclear enzymatic mechanism and many factors that affect the heterogeneous system. Therefore, it is difficult to build a theoretical model to study cellulose hydrolysis by cellulase. Artificial neural network (ANN) was used to simulate and predict this enzymatic reaction and compared with the response surface model (RSM). The independent variables were cellulase amount X-1, substrate concentration X-2, and reaction time X-3, and the response variables were reducing sugar concentration Y-1 and transformation rate of the raw material Y-2. The experimental results showed that ANN was much more suitable for studying the kinetics of the enzymatic hydrolysis than RSM. During the simulation process, relative errors produced by the ANN model were apparently smaller than that by RSM except one and the central experimental points. During the prediction process, values produced by the ANN model were much closer to the experimental values than that produced by RSM. These showed that ANN is a persuasive tool that can be used for studying the kinetics of cellulose hydrolysis catalyzed by cellulase.

Induction of cytogenetic adaptive response in spermatogonia and spermatocytes by pre-exposure of mouse testis to low-dose C-12(6+) ions

To investigate the effects of pre-exposure of mouse testis to low-dose C-12(6+) ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation. the testes of outbred Kun-Ming strain mice were irradiated with 0.05 Gy of C-12(6+) ions as the pre-exposure dose, and then irradiated with 2 Gy as challenging dose at 4 h after per-exposure. Poly(ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4 h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8 h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2 Gy of C-12(6+) ions. However, pre-exposure of mouse testes to a low dose of C-12(6+) ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose C-12(6+) ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations. (C) 2008 Elsevier B.V. All rights reserved.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions).

In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system.

Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR).

Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions. This article is part of a Special Issue entitled: “Farm animal proteomics”.

Effect of high CO2 and ocean acidification on photosynthesis and response to oxidative stress in seagrasses

Climate change scenarios comprise significant modifications of the marine realm, notably ocean acidification and temperature increase, both direct consequences of the rising atmospheric CO2 concentration. These changes are likely to impact marine organisms and ecosystems, namely the valuable seagrass-dominated coastal habitats. The main objective of this thesis was to evaluate the photosynthetic and antioxidant responses of seagrasses to climate change, considering CO2, temperature and light as key drivers of these processes. The methodologies used to determine global antioxidant capacity and antioxidant enzymatic activity in seagrasses were optimized for the species Cymodocea nodosa and Posidonia oceanica, revealing identical defence mechanisms to those found in terrestrial plants. The detailed analysis and identification of photosynthetic pigments in Halophila ovalis, H.stipulacea, Zostera noltii, Z marina, Z. capricorni, Cymodocea nodosa and Posidonia oceanica, sampled across different climatic zones and depths, also revealed a similarity with terrestrial plants, both in carotenoid composition and in the pigment-based photoprotection mechanisms. Cymodocea nodosa plants from Ria Formosa were submitted to the combined effect of potentially stressful light and temperature ranges and showed considerable physiological tolerance, due to the combination of changes in the antioxidant system, activation of the VAZ cycle and accumulation of leaf soluble sugars, thus preventing the onset of oxidative stress. Cymodocea nodosa plants living in a naturally acidified environment near submarine volcanic vents in Vulcano Island (Italy) showed to be under oxidative stress despite the enhancement of the antioxidant capacity, phenolics concentration and carotenoids. Posidonia oceanica leaves loaded with epiphytes showed a significant increase in oxidative stress, despite the increase of antioxidant responses and the allocation of energetic resources to these protection mechanisms. Globally, the results show that seagrasses are physiologically able to deal with potentially stressful conditions from different origins, being plastic enough to avoid stress in many situations and to actively promote ulterior defence and repair mechanisms when under effective oxidative stress.