Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO(2) concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana: a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. (C) 2010 Elsevier GmbH. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 angstrom. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. (C) 2009 Elsevier Inc. All rights reserved.

Effects of Micelles and Vesicles on the Oximolysis of p-Nitrophenyl Diphenyl Phosphate: A Model System for Surfactant-Based Skin-defensive Formulations against Organophosphates

The rates of oximolysis of p-nitrophenyl diphenyl phosphate (PNPDPP) by Acetophenoxime; 10-phenyl-10-hydi-oxyiminodecanoic acid; 4-(9-carboxynonanyl)-1-(9-carboxy-1-hydroyiminononanyl) benzene; 1-dodecyl-2-[(hydroxyimino)methyl]-pyridinium chloride (IV) and N-methylpyridinium-2-aldoxime chloride were determined in micelles of N-hexadecyl-N,N,N-trimethylammonium chloride (CTAC), N-hexadecyl-N,N-dimethylammonium propanesulfonate and dioctadecyldimethylammonium chloride (DODAC) vesicles. The effects of CTAC micelles and DODAC vesicles on the rates of oxymolysis of O,O-Diethyl O-(4-nitrophenyl) phosphate (paraoxon) by oxime IV were also determined. Analysis of micellar and vesicular effects on oximolysis of PNPDPP, using pseudophase or pseudophase with explicit consideration of ion exchange models, required the determination of the aggregate`s effects on the pK(a), of oximes and on the rates of PNPDPP hydrolysis. All aggregates increased the rate of oximolysis of PNPDPP and the results were analyzed quantitatively. In particular, DODAC vesicles catalyzed the reaction and increased the rate of oximolysis of PNPDPP by IV several million fold at pH`s compatible with pharmaceutical formulations. The rate increase produced by DODAC vesicles on the rate of oximolysis paraoxon by IV demonstrates the pharmaceutical potential of this system, since the substrate is used as an agricultural defensive agent and the surfactant is extensively employed in cosmetic formulations. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1040-1052, 2009

Purification and partial characterization of a midgut membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera: Cicadidae)

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major alpha-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of alpha-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified alpha-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl alpha-D-glucoside and M(r) of 61,000 (SDS-PAGE). Taking into account V(Max)/K(M) ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q gigas alpha-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed. (C) 2009 Elsevier Inc. All rights reserved.

Rapid method for the determination. of organic acids in wine by capillary electrophoresis with indirect UV detection

A capillary electrophoresis method for organic acids in wine was developed and validated. The optimal electrolyte consisted of 10 mmol/L 3,5-dinitrobenzoic acid (DNB) at pH 3.6 containing 0.2 mmol/L cetyltrimethylammonium bromide as flow reverser. DNB was chosen because it has an effective mobility similar to the organic acids under investigation, good buffering capacity at pH 3.6, and good chromophoric characteristics for indirect UV-absorbance detection at 254 nm. Sample preparation involved dilution and filtration. The method showed good performance characteristics: Linearity at 6 to 285 mg/L (r > 0.99); detection and quantification limits of 0.64 to 1.55 and 2.12 to 5.15 mg/L, respectively; separation time of less than 5.5 min. Coefficients of variation for ten injections were less than 5% and recoveries varied from 95% to 102%. Application to 23 samples of Brazilian wine confirmed good repeatability and demonstrated wide variation in the organic acid concentrations. (C) 2008 Elsevier Ltd. All rights reserved.

Silver Nanoparticles Obtained in PAH/PAA-Based Multilayers by Photochemical Reaction

Carboxylic acid groups in PAH/PAA-based multilayers bind silver cations by ion exchange with the acid protons. The aggregation and spatial distribution of the nanoparticles proved to be dependent oil the process used to reduce the silver acetate aqueous solution. The reducing method with ambient light formed larger nanoparticles with diameters ranging from 4-50 nm in comparison with the reduction method using UV light, which gave particles with diameters of 2-4 nm The high toughness of samples reduced by ambient light is a result of two population distributions of particle sizes caused by different mechanisms when compared with the UV light process. According to these phenomena, a judicious choice of the spectral source call be used as a way to control the type and size of silver nanoparticles formed on PEMs. Depending on the energy of the light source, the Ag nanoparticles present cubic and/or hexagonal crystallographic structures, as confirmed by XRD. Beyond the kinetically controlled process of UV photoinduced cluster formation, the annealing produced by UV light allowed a second mechanism to modify the growth rates, spatial distribution, and phases.

Comparison of two extraction methods for evaluation of volatile constituents patterns in commercial whiskeys: Elucidation of the main odour-active compounds

An analytical procedure based on manual dynamic headspace solid-phase microextraction (HS-SPME) method and the conventional extraction method by liquid–liquid extraction (LLE), were compared for their effectiveness in the extraction and quantification of volatile compounds from commercial whiskey samples. Seven extraction solvents covering a wide range of polarities and two SPME fibres coatings, has been evaluated. The highest amounts extracted, were achieved using dichloromethane (CH2Cl2) by LLE method (LLECH2Cl2)(LLECH2Cl2) and using a CAR/PDMS fibre (SPMECAR/PDMS) in HS-SPME. Each method was used to determine the responses of 25 analytes from whiskeys and calibration standards, in order to provide sensitivity comparisons between the two methods. Calibration curves were established in a synthetic whiskey and linear correlation coefficient (r ) were greater than 0.9929 for LLECH2Cl2LLECH2Cl2 and 0.9935 for SPMECAR/PDMS, for all target compounds. Recoveries greater than 80% were achieved. For most compounds, precision (expressed by relative standard deviation, R.S.D.) are very good, with R.S.D. values lower than 14.78% for HS-SPME method and than 19.42% for LLE method. The detection limits ranged from 0.13 to 19.03 μg L−1 for SPME procedure and from 0.50 to 12.48 μg L−1 for LLE. A tentative study to estimate the contribution of a specific compound to the aroma of a whiskey, on the basis of their odour activity values (OAV) was made. Ethyl octanoate followed by isoamyl acetate and isobutyl alcohol, were found the most potent odour-active compounds.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.

Organic acids and/or compound with defined microorganisms to control Salmonella enterica serovar Enteritidis experimental infection in chickens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impact of atmosphere, organic acids, and calcium on quality of fresh-cut 'Kensington' mango

Fresh-cut slices from ripe 'Kensington' mango (Mangifera indica L.) were prepared aseptically and stored under various treatments at 3 degrees C. Treatments included reduced oxygen (2.5%), enhanced carbon dioxide (5-40%), organic acid application, calcium chloride application, and combinations of the above. Symptoms limiting shelf-life were characterised by tissue darkening, development of a 'glassy' appearance, surface desiccation, and loss of firmness. Reduced oxygen (2.5%) was effective at controlling tissue darkening and the development of a 'glassy' appearance, while calcium application (3%) was partly effective at controlling darkening. Calcium chloride however significantly slowed (but did not stop) loss of tissue firmness. Carbon dioxide (5-40%) and citric acid had little positive effect on shelf-life, with both treatments appearing to promote tissue softening. A combination of low oxygen and calcium allowed 'Kensington' slices to be held for at least 15 days at 3 degrees C. (C) 2006 Elsevier B.V All rights reserved.

Supercritical fluid extraction from Lippia alba: global yields, kinetic data, and extract chemical composition

In this work, experimental data for the system Lippia alba + CO2 is presented. The major constituents of the L. alba volatile oil are limonene and carvone. Thus, literature data for the systems limonene + CO2 and carvone + CO2, and the Peng-Robinson equation of state (PR-EOS) were used to select the operating temperature and pressure, which maximize the global yield in L. alba extract. Global yields were determined at 80, 100, and 120 bar and 40, 45, and 50 degrees C. L. alba extracts were also obtained by conventional processes (hydrodistillation, low-pressure ethanol extraction and Soxhlet ethanol). The chemical compositions of the extracts were determined by gas and thin layer chromatography (TLC). The secretor structures of L. alba were observed by scanning electron microscopy (SEM) before and after supercritical extraction. The largest yield (similar to 7%, mass of extract/mass of dry solid) of the CO2-extract was obtained at 318 K and 100 bar. The chemical compositions of the CO2-extracts were different from those of the extracts obtained by Soxhlet and low-pressure solvent extraction (LPSE) because of the co-extraction of heavy substances by ethanol. The operating conditions that maximized the carvone and limomene yields were 80 bar and 323 K (80 mass%) and 120 bar and 323 K (17 mass%), respectively. (c) 2004 Elsevier B.V All rights reserved.

Obtenção e caracterização de extrato microencapsulado de resíduo agroindustrial de acerola

Acerola (Malpighia emarginata D.C.) is a red fruit widely cultivated in Brazil, especially in the Northeastern region. Its increasing demand is attributed to its high ascorbic acid contents. Besides ascorbic acid, widely known by its health-benefit effects, acerola is rich in anthocyanins, which contribute for the antioxidant power of the fruit. Acerola processing produces a bright-red pomace, usually discarded. The further processing of this pomace, in order to explore its antioxidant compounds, could enhance acerola market value and rentability of its processing. Both ascorbic acid and anthocyanins are highly susceptible to degradation, that can be delayed by microencapsulation, which consists on packing particles (core) in an edible matrix (wall material). This work has been made with the purpose of producing a microencapsulated acerola pomace extract, which could be used by the food industry as a functional ingredient with antioxidant and coloring properties. Antioxidant compounds were recovered by pressing the pomace diluted in a solvent (a citric acid aqueous solution), by using a central composite design, with two variables: citric acid concentration in the solvent (0-2%), and solvent: pomace mass ratio (2:1-6:1). The acerola pomace extract was then microencapsulated by spray drying. A central composite design was adopted, with three variables: inlet temperature of the spray dryer (170o-200oC), wall material: acerola solids mass ratio (2:1-5:1), and degree of maltodextrin replacement by cashew tree gum as wall material (0-100%). The cashew tree gum was used because of its similarity to arabic gum, which is regarded as the wall material by excellence. The following conditions were considered as optimal for extraction of anthocyanins and ascorbic acid: solvent/pomace ratio, 5:1, and no citric acid in the solvent. 82.47% of the anthocyanins were recovered, as well as 83.22% of the ascorbic acid. Anthocyanin and ascorbic acid retentions were favored by lower inlet temperatures, higher wall material: acerola solids mass ratio and higher maltodextrin replacement by cashew tree gum, which was presented as a promising wall material. The more adequate microencapsulation conditions, based not only on retention of antioxidant compounds but also on physical properties of the final powder, were the following: inlet temperature, 185oC; wall material: acerola solids mass ratio, 5:1, and minimum degree of maltodextrin replacement by cashew tree gum, 50%

Extração do óleo essencial de Cymbopogom winterianus J. com CO2 pressurizado

The extraction with pressurized fluids has become an attractive process for the extraction of essential oils, mainly due the specific characteristics of the fluids near the critical region. This work presents results of the extraction process of the essential oil of Cymbopogon winterianus J. with CO2 under high pressures. The effect of the following variables was evaluated: solvent flow rate (from 0.37 to 1.5 g CO2/min), pressure (66.7 and 75 bar) and temperature (8, 10, 15, 20 and 25 ºC) on the extraction kinetics and the total yield of the process, as well as in the solubility and composition of the C. winterianus essential oil. The experimental apparatus consisted of an extractor of fixed bed and the dynamic method was adopted for the calculation of the oil solubility. Extractions were also accomplished by conventional techniques (steam and organic solvent extraction). The determination and identification of extract composition were done by gas chromatography coupled with a mass spectrometer (GC-MS). The extract composition varied in function of the studied operational conditions and also related to the used extraction method. The main components obtained in the CO2 extraction were elemol, geraniol, citronellol and citronellal. For the steam extraction were the citronellal, citronellol and geraniol and for the organic solvent extraction were the azulene and the hexadecane. The most yield values (2.76%) and oil solubility (2.49x10-2 g oil/ g CO2) were obtained through the CO2 extraction in the operational conditions of T = 10°C, P = 66.7 bar and solvent flow rate 0.85 g CO2/min

Preparação e caracterização de um material híbrido, à base de vermiculita modificada com quitosana, para a remoção de íons de chumbo presentes em efluentes sintéticos

Actually, surveys have been developed for obtaining new materials and methodologies that aim to minimize environmental problems due to discharges of industrial effluents contaminated with heavy metals. The adsorption has been used as an alternative technology effectively, economically viable and potentially important for the reduction of metals, especially when using natural adsorbents such as certain types of clay. Chitosan, a polymer of natural origin, present in the shells of crustaceans and insects, has also been used for this purpose. Among the clays, vermiculite is distinguished by its good ion exchange capacity and in its expanded form enhances its properties by greatly increasing its specific surface. This study aimed to evaluate the functionality of the hybrid material obtained through the modification of expanded vermiculite with chitosan in the removal of lead ions (II) in aqueous solution. The material was characterized by infrared spectroscopy (IR) in order to evaluate the efficiency of modification of matrix, the vermiculite, the organic material, chitosan. The thermal stability of the material and the ratio clay / polymer was evaluated by thermogravimetry. To evaluate the surface of the material was used in scanning electron microscopy (SEM) and (BET). The BET analysis revealed a significant increase in surface area of vermiculite that after interaction with chitosan, was obtained a value of 21, 6156 m2 / g. Adsorption tests were performed according to the particle size, concentration and time. The results show that the capacity of removal of ions through the vermiculite was on average 88.4% for lead in concentrations ranging from 20-200 mg / L and 64.2% in the concentration range of 1000 mg / L. Regarding the particle size, there was an increase in adsorption with decreasing particle size. In fuction to the time of contact, was observed adsorption equilibrium in 60 minutes with adsorption capacity. The data of the isotherms were fitted to equation Freundlich. The kinetic study of adsorption showed that the pseudo second- order model best describes the adsorption adsorption, having been found following values K2=0,024 g. mg-1 min-1and Qmax=25,75 mg/g, value very close to the calculated Qe = 26.31 mg / g. From the results we can conclude that the material can be used in wastewater treatment systems as a source of metal ions adsorbent due to its high adsorption capacity