Increase nitric oxide and oxidative stress in dogs experimentally infected by Ehrlichia canis: Effect on the pathogenesis of the disease

The aim of this study was to evaluate nitric oxide levels, lipid peroxidation, protein oxidation and glutathione reductase activity in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups were used: negative control (n=5) and infected by E. canis (n=5). The concentration of nitrite/nitrate (NOx), lipid peroxidation (TBARS), advanced oxidation protein products (AOPP), and glutathione reductase (GR) activity in sera were evaluated. Samples were collected on days 0, 3, 6, 18 and 30 post-infection (PI). NOx and TBARS levels were significantly (P<0.05) higher in the infected group at 18 and 30 days PI, as well as AOPP levels at 30 days PI when compared to samples from control group. The GR activity was significant (P<0.05) increased in serum of dogs infected by E. canis on days 18 and 30 PI. Based on the increased levels of NOx, TBARS, AOPP and GR activity we concluded that dogs experimentally infected by E. canis develop a state of redox imbalance and that these changes might be involved in the pathophysiology of the disease. © 2013 Elsevier B.V.

Sugarcane bagasse ozonolysis pretreatment: Effect on enzymatic digestibility and inhibitory compound formation

Sugarcane bagasse was pretreated with ozone to increase lignocellulosic material digestibility. Bagasse was ozonated in a fixed bed reactor at room temperature, and the effect of the two major parameters, ozone concentration and sample moisture, was studied. Acid insoluble and total lignin decreased whereas acid soluble lignin increased in all experiments. Pretreatment barely attacked carbohydrates, with cellulose and xylan recovery rates being >92%. Ozonolysis increased fermentable carbohydrate release considerably during enzymatic hydrolysis. Glucose and xylose yields increased from 6.64% and 2.05%, for raw bagasse, to 41.79% and 52.44% under the best experimental conditions. Only xylitol, lactic, formic and acetic acid degradation compounds were found, with neither furfural nor HMF (5-hydroxymethylfurfural) being detected. Washing detoxification provided inhibitor removal percentages above 85%, increasing glucose hydrolysis, but decreasing xylose yield by xylan solubilization. SEM analysis showed structural changes after ozonization and washing. © 2013 Elsevier Ltd.

Characterization of esterase patterns in hepatopancreas of three species of Macrobrachium (Palaemonidae)

The genus Macrobrachium (Bate, 1868) belongs to the Palaemonidae family. These species are commonly found in lakes, floodplains and rivers in tropical and subtropical regions of South America. The Macrobrachium genus encompasses nearly 210 species of ecological and economic importance. In this study, three species of Macrobrachium (M acrobrachium jelskii, M acrobrachium amazonicum and M acrobrachium brasiliense) were studied in order to characterize the esterase patterns in the hepatopancreas, which were still unknown. Esterases are enzymes which catalyze the hydrolysis of esters. In the hepatopancreas, these enzymes play important roles in several metabolic processes involved in some functions of this organ, such as detoxification and digestion. Twelve esterase bands (EST1 to EST12) were detected in these species, and a comparison among them showed no qualitative differences in interspecific bands, or between males and females. Inhibitors were used to classify the esterase bands. The results indicated seven acetylesterases, two carboxylesterases, one arylesterase, and one cholinesterase. The EST11 band was not detected in these procedures because of its lower frequency. Statistical analyses showed no variability among the species, in either interspecific or intraspecific assays. These results support the hypothesis of a high evolutionary conservation of esterases in the hepatopancreas of these crustaceans. The data enabled us to assess the genetic structure of these species through the use of esterasic enzymes. It also contributes to our knowledge about the biology of these poorly studied species. Knowledge on the genetic structure of populations and species are essential when defining priorities for their management and conservation. © 2012 Elsevier Ltd.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Does background colouration affect the behaviour of tadpoles? An experimental approach with an odonate predator

Predation is a primary driver of tadpole assemblages, and the activity rate is a good predictor of the tadpoles' tolerance for predation risk. The conflicting demands between activity and exposure to predation can generate suboptimal behaviours. Because morphological components, such as body colouration, may affect the activity of tadpoles, we predict that environmental features that enhance or match the tadpole colouration should affect their survival or activity rate in the presence of a predator. We tested this prediction experimentally by assessing the mortality rate of tadpoles of Rhinella schneideri and Eupemphix nattereri and the active time on two artificial background types: one bright-coloured and one black-coloured. We found no difference in tadpole mortality due to the background type. However, R. schneideri tadpoles were more active than E. nattereri tadpoles, and the activity of R. schneideri was reduced less in the presence of the predator than that of E. nattereri. Although the background colouration did not affect the tadpole mortality rate, it was a stimulus that elicited behavioural responses in the tadpoles, leading them to adjust their activity rate to the type of background colour. © 2013 Dipartimento di Biologia, Università degli Studi di Firenze, Italia.

Fontes de carbono de baixo custo para a produção de xilanase termoestável por aspergillus niger

A strain of the flamentous fungus Aspergillus niger was isolated and shown to possess extracellular xylanolytic activity. These enzymes have biotechnological potential and can be employed in various industries. This fungus produced its highest xylanase activity in a medium made up of 0.1% CaCO3, 0.5% NaCl, 0.1% NH4Cl, 0.5% corn steep liquor and 1% carbon source, at pH 8.0. A low-cost hemicellulose residue (powdered corncob) proved to be an excellent inducer of the A. niger xylanolytic complex. Filtration of the crude culture medium with suspended kaolin was ideal for to clarify the extract and led to partial purifcation of the xylanolytic activity. The apparent molecular mass of the xylanase was about 32.3 kDa. Maximum enzyme activity occurred at pH 5.0 and 55-60oC. Apparent Km was 10.41 ± 0.282 mg/mL and Vmax was 3.32 ± 0.053 U/mg protein, with birchwood xylan as the substrate. Activation energy was 4.55 kcal/mol and half-life of the crude enzyme at 60oC was 30 minutes. Addition of 2% glucose to the culture medium supplemented with xylan repressed xylanase production, but in the presence of xylose the enzyme production was not affected.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

Production, purification, and characterization of a major penicillium glabrum xylanase using brewer's spent grain as substrate

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. © 2013 Adriana Knob et al.

Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human neuronal SH-SY5Y cells by decreasing reactive oxygen species production

Scope. To elucidate the morphological and biochemical in vitro effects exerted by caffeine, taurine, and guarana, alone or in combination, since they are major components in energy drinks (EDs). Methods and Results. On human neuronal SH-SY5Y cells, caffeine (0.125-2 mg/mL), taurine (1-16 mg/mL), and guarana (3.125-50 mg/mL) showed concentration-dependent nonenzymatic antioxidant potential, decreased the basal levels of free radical generation, and reduced both superoxide dismutase (SOD) and catalase (CAT) activities, especially when combined together. However, guarana-treated cells developed signs of neurite degeneration in the form of swellings at various segments in a beaded or pearl chain-like appearance and fragmentation of such neurites at concentrations ranging from 12.5 to 50 mg/mL. Swellings, but not neuritic fragmentation, were detected when cells were treated with 0.5 mg/mL (or higher doses) of caffeine, concentrations that are present in EDs. Cells treated with guarana also showed qualitative signs of apoptosis, including membrane blebbing, cell shrinkage, and cleaved caspase-3 positivity. Flow cytometric analysis confirmed that cells treated with 12.5-50 mg/mL of guarana and its combinations with caffeine and/or taurine underwent apoptosis. Conclusion. Excessive removal of intracellular reactive oxygen species, to nonphysiological levels (or antioxidative stress), could be a cause of in vitro toxicity induced by these drugs. © 2013 Fares Zeidán-Chuliá et al.

Produção de glicose e frutose por invertase de Saccharomyces cerevisiae imobilizada em suporte MANAE-agarose

Invertase from Saccharomyces cerevisiae was immobilized on agarose beads, activated with various groups (glyoxyl, MANAE or glutaraldehyde), and on some commercial epoxy supports (Eupergit and Sepabeads). Very active and stable invertase derivatives were produced by the adsorption of the enzyme on MANAE-agarose, MANAE-agarose treated with glutaraldhyde and glutaraldehyde-agarose supports. At pH 5.0, these derivatives retained full activity after 24h at 40°C and 50 °C. When assayed at 40°C and 50°C, with the pH adjusted to 7.0, the invertase-MANAE-agarose derivative treated with glutaraldehyde retained 80% of the initial activity. Recovered activities of the derivatives produced with MANAE, MANAE treated with glutaraldehyde and glutaraldehyde alone were 73.5%, 44.4% and 36.8%, respectively. These three preparations were successfully employed to produce glucose and fructose in 3 cycles of sucrose hydrolysis.

Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii

The stimulation by Mg2+, Na+, K+, NH 4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na +, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg-1, K 0.5 = 0.10 ± 0.01 mmol L-1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg-1, K 0.5 = 1.30 ± 0.03 mmol L -1), Mg2+ (V M = 115.0 ± 4.6 U mg -1, K 0.5 = 0.96 ± 0.03 mmol L-1), NH4 + (V M = 141.0 ± 5.6 U mg -1, K 0.5 = 1.90 ± 0.04 mmol L-1), and K+ (V M = 120.0 ± 2.4 U mg-1, K M = 2.74 ± 0.08 mmol L-1) followed single saturation curves and, except for K+, exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L-1. Complementary inhibition studies suggest the presence of F0F1-, Na+-, or K +-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. © 2013 Springer Science+Business Media New York.

Gastroprotective mechanisms of the chloroform and ethyl acetate phases of Praxelis clematidea (Griseb.) R.M.King & H.Robinson (Asteraceae)

Flavonoid-rich Praxelis clematidea (Griseb.) R.M.King & H.Robinson (Asteraceae) is a native plant of South America. This study evaluates the gastroprotective activity and possible mechanisms for both the chloroform (CHCl3P) and ethyl acetate phases (AcOEtP) obtained from aerial parts of the plant. The activity was investigated using acute models of gastric ulcer. Gastric secretion biochemical parameters were determined after pylorus ligature. The participation of cytoprotective factors such as mucus, nitric oxide (NO), sulfhydryl (SH) groups, prostaglandin E2 (PGE 2), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), reduction of lipid peroxidation (malondialdehyde level), and polymorphonuclear infiltration (myeloperoxidase activity), was also investigated. CHCl3P (125, 250, and 500 mg/kg) and AcOEtP (62.5, 125, and 250 mg/kg) showed significant gastroprotective activity, reducing the ulcerative index by 75, 83, 88 % and 66, 66, 81 % for ethanol; 67, 67, 56 % and 56, 53, 58 % for a non-steroidal anti-inflammatory drug (NSAID); and 74, 58, 59 % and 64, 65, 61 % for stress-induced gastric ulcer, respectively. CHCl3P (125 mg/kg) and AcOEtP (62.5 mg/kg) significantly reduced the ulcerative area by 78 and 83 %, respectively, for the ischemia-reperfusion model. They also did not alter the biochemical parameters of gastric secretion, the GSH level or the activities of SOD, GPx or GR. They increased the quantity of gastric mucus, not dependent on NO, yet dependent on SH groups, and maintained PGE2 levels. The P. clematidea phases demonstrated gastroprotective activity related to cytoprotective factors. © 2012 The Japanese Society of Pharmacognosy and Springer.

The characterization of a thermostable and cambialistic superoxide dismutase from thermus filiformis

The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0·41, 0·56 and 13·73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis. © 2013 The Society for Applied Microbiology.

Zymographic patterns of MMP-2 and MMP-9 in the CSF and cerebellum of dogs with subacute distemper leukoencephalitis

Distemper leukoencephalitis is a disease caused by the canine distemper virus (CDV) infection. It is a demyelinating disease affecting mainly the white matter of the cerebellum and areas adjacent to the fourth ventricle; the enzymes of the matrix metalloproteinases (MMPs) group, especially MMP-2 and MMP-9 have a key role in the myelin basic protein fragmentation and in demyelination, as well as in leukocyte traffic into the nervous milieu. To evaluate the involvement of MMPs during subacute distemper leukoencephalitis, we measured the levels of MMP-2 and MMP-9 by zymography in the cerebrospinal fluid (CSF) and in the cerebellum of 14 dogs naturally infected with CDV and 10 uninfected dogs. The infected dogs presented high levels of pro-MMP-2 in the CSF and elevated levels of pro-MMP-2 and pro-MMP-9 in the cerebellar tissue. Active MMP-2 was detected in the CSF of some infected dogs. As active MMP-2 and MMP-9 are required for cellular migration across the blood-brain barrier and any interference between MMPs and their inhibitors may result in an amplification of demyelination, this study gives additional support to the involvement of MMPs during subacute distemper leukoencephalitis and suggests that MMP-2 and MMP-9 may take part in the brain inflammatory changes of this disease. © 2013 Elsevier B.V.

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production. © 2013 Alvarez et al.