Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Influence Of Substrate Steps On The Catalytic Properties Of Pt Layers: The Ethanol Electrooxidation Reaction.

The ethanol oxidation reaction (EOR) is investigated on Pt/Au(hkl) electrodes. The Au(hkl) single crystals used belong to the [n(111)x(110)] family of planes. Pt is deposited following the galvanic exchange of a previously deposited Cu monolayer using a Pt(2+) solution. Deposition is not epitaxial and the defects on the underlying Au(hkl) substrates are partially transferred to the Pt films. Moreover, an additional (100)-step-like defect is formed, probably as a result of the strain resulting from the Pt and Au lattice mismatch. Regarding the EOR, both vicinal Pt/Au(hkl) surfaces exhibit a behavior that differs from that expected for stepped Pt; for instance, the smaller the step density on the underlying Au substrate, the greater the ability to break the CC bond in the ethanol molecule, as determined by in situ Fourier transform infrared spectroscopy measurements. Also, we found that the acetic acid production is favored as the terrace width decreases, thus reflecting the inefficiency of the surface array to cleave the ethanol molecule.

Microtensile Bond Strength Of Methacrylate And Silorane Resins To Enamel And Dentin.

The aim of this study was to evaluate the microtensile bond strength (µTBS) of two substrates (enamel and dentin) considering two study factors: type of composite resin [methacrylate-based (Filtek Supreme) or silorane-based (Filtek LS)] and aging time (24 h or 3 months). Twenty human molars were selected and divided into 2 groups (n=10) considering two dental substrates, enamel or dentin. The enamel and dentin of each tooth was divided into two halves separated by a glass plate. Each tooth was restored using both tested composite resins following the manufacturer's instructions. The samples were sectioned, producing 4 sticks for each composite resin. Half of them were tested after 24 h and half after 3 months. µTBS testing was carried out at 0.05 mm/s. Data were analyzed by three-way ANOVA and Tukey's HSD tests at α=0.05. Significant differences between composite resins and substrates were found (p<0.05), but no statistically significant difference was found for aging time and interactions among study factors. The methacrylate-based resin showed higher µTBS than the silorane-based resin. The µTBS for enamel was significantly higher than for dentin, irrespective of the composite resin and storage time. Three months of storage was not sufficient time to cause degradation of the bonding interaction of either of the composite resins to enamel and dentin.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Comparative In vitro Mechanical Evaluation Of Techniques Using A 2.0 mm Locking Fixation System For Simulated Fractures Of The Mandibular Body.

To perform a comparative evaluation of the mechanical resistance of simulated fractures of the mandibular body which were repaired using different fixation techniques with two different brands of 2.0 mm locking fixation systems. Four aluminum hemimandibles with linear sectioning simulating a mandibular body fracture were used as the substrates and were fixed using the two techniques and two different brands of fixation plate. These were divided into four groups: groups I and II were fixed with one four-hole plate, with four 6 mm screws in the tension zone and one four-hole plate, with four 10 mm screws in the compression zone; and groups III and IV were fixed with one four-hole plate with four 6 mm screws in the neutral zone. Fixation plates manufactured by Tóride were used for groups I and III, and by Traumec for groups II and IV. The hemimandibles were submitted to vertical, linear load testing in an Instron 4411 servohydraulic mechanical testing unit, and the load/displacement (3 mm, 5 mm and 7 mm) and the peak loads were measured. Means and standard deviations were evaluated applying variance analysis with a significance level of 5%. The only significant difference between the brands was seen at displacements of 7 mm. Comparing the techniques, groups I and II showed higher mechanical strength than groups III and IV, as expected. For the treatment of mandibular linear body fracture, two locking plates, one in the tension zone and another in the compression zone, have a greater mechanical strength than a single locking plate in the neutral zone.

Analysis Of Cellular Adhesion On Superhydrophobic And Superhydrophilic Vertically Aligned Carbon Nanotube Scaffolds.

We analyzed GFP cells after 24h cultivated on superhydrophilic vertically aligned carbon nanotube scaffolds. We produced two different densities of VACNT scaffolds on Ti using Ni or Fe catalysts. A simple and fast oxygen plasma treatment promoted the superhydrophilicity of them. We used five different substrates, such as: as-grown VACNT produced using Ni as catalyst (Ni), as-grown VACNT produced using Fe as catalyst (Fe), VACNT-O produced using Ni as catalyst (NiO), VACNT-O produced using Fe as catalyst (FeO) and Ti (control). The 4',6-diamidino-2-phenylindole reagent nuclei stained the adherent cells cultivated on five different analyzed scaffolds. We used fluorescence microscopy for image collect, ImageJ® to count adhered cell and GraphPad Prism 5® for statistical analysis. We demonstrated in crescent order: Fe, Ni, NiO, FeO and Ti scaffolds that had an improved cellular adhesion. Oxygen treatment associated to high VACNT density (group FeO) presented significantly superior cell adhesion up to 24h. However, they do not show significant differences compared with Ti substrates (control). We demonstrated that all the analyzed substrates were nontoxic. Also, we proposed that the density and hydrophilicity influenced the cell adhesion behavior.

Enzymatic Biodiesel Synthesis From Yeast Oil Using Immobilized Recombinant Rhizopus Oryzae Lipase.

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

Towards A Deeper Understanding Of Structural Biomass Recalcitrance Using Phase-contrast Tomography.

The development of technological routes to convert lignocellulosic biomass to liquid fuels requires an in-depth understanding of the cell wall architecture of substrates. Essential pretreatment processes are conducted to reduce biomass recalcitrance and usually increase the reactive surface area. Quantitative three-dimensional information about both bulk and surface structural features of substrates needs to be obtained to expand our knowledge of substrates. In this work, phase-contrast tomography (PCT) was used to gather information about the structure of a model lignocellulosic biomass (piassava fibers). The three-dimensional cellular organization of piassava fibers was characterized by PCT using synchrotron radiation. This technique enabled important physical features that describe the substrate piassava fibers to be visualized and quantified. The external surface area of a fiber and internal surface area of the pores in a fiber could be determined separately. More than 96% of the overall surface area available to enzymes was in the bulk substrate. The pore surface area and length exhibited a positive linear relationship, where the slope of this relationship depended on the plant tissue. We demonstrated that PCT is a powerful tool for the three-dimensional characterization of the cell wall features related to biomass recalcitrance. Original and relevant quantitative information about the structural features of the analyzed material were obtained. The data obtained by PCT can be used to improve processing routes to efficiently convert biomass feedstock into sugars.

New Interaction Partners For Nek4.1 And Nek4.2 Isoforms: From The Dna Damage Response To Rna Splicing.

Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins. Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins. This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.

Condutividade e difusividade térmica do figo (Ficus carica L.) Roxo de Valinhos

The post harvest cooling and/or freezing processes for horticultural products have been carried out with the objective of removing the heat from these products, allowing them a bigger period of conservation. Therefore, the knowledge of the physical properties that involve heat transference in the fig fruit Roxo de Valinhos is useful for calculating projects and systems of food engineering in general, as well as, for using in equations of thermodynamic mathematical models. The values of conductivity and thermal diffusivity of the whole fig fruit-rami index were determined, and from these values it was determined the value of the specific heat. For these determination it was used the transient method of the Line Heat Source. The results shown that the fig fruit has a thermal conductivity of 0.52 W m-1°C, thermal diffusivity of 1.56 x 10-7 m² s-1, pulp density of 815.6 kg m-3 and specific heat of 4.07 kJ kg-1 °C.

Rigidez do papelão ondulado: comparação entre resultados experimentais e os obtidos por cálculo analítico

Cardboard packing for horticultural products has as main function to protect them. The design of a cardboard packing request the knowledge of the bending stiffens which is depending on the modulus of elasticity. The objective of this work was to calculate the cardboard modulus of elasticity from data obtained in laboratory using physical characterization test, with different methods, and comparing the results with the values obtained experimentally. Ten samples of each cardboard selected for this study were tested in the paper fabrication direction and in its transverse direction. The papers liner and medium resistance to the traction, used to calculate the bending stiffness, was determined in a universal machine test. To obtaining of the bending stiffens the four points test was accomplished. Expressive variations among the methods from which the modulus of elasticity is obtained were observed and that influence the bending stiffness of the structure. The stiffness values obtained experimentally were always greater than the values obtained from analytical method. This difference can be attributed to two factors, the production processes that assurance a larger rigidity than the components separately and the addition of the adhesive layer that is not taken in consideration in the analytic calculations.

Plasticidade neural: relações com o comportamento e abordagens experimentais

Behavioral adaptiveness to different situations as well as behavioral individuality result from the interrelations between environmental sitmuli and the responses of an organism.These kind of interrelationships also shape the neural circuits as well as characterize the plasticity and the neural individuality of the organism. Studies on neural plasticity may analyze changes in neural circuitry after environmental manipulations or changes in behavior after lesions in the nervous system. Issues on neural plasticity and recovery of function refer both to physiology and behavior as well as to the subjacent mechanisms related to morphology, biochemistry and genetics. They may be approached at the systemic, behavioral, cellular and molecular levels. This work intends to characterize these kinds of studies pointing to their relations with the analyis of behavior and learning.The analysis of how the environmental-organismic interrelationships affect the neural substrates of behavior is pointed as a very stimulating area for investigation.

Ambientes de cultivo, recipientes e substratos na produção de biomassa foliar e radicular em mudas de maracujazeiro amarelo em Aquidauana - MS

The objective of the work was to evaluate the effects of environment, recipients, and substrate compositions in passion fruit (Passiflora edulis Sims f. flavicarpa Deg.) seedlings biomass production in Pantanal region from September to November of 2006. Experimental trials were conducted in four protected environments, in two types of containers and three different substrate compositions. The environments were: A1 (greenhouse covered with low-density, 150-microns-thick polyethylene film), A2 (monofilament black screened with mesh for 50% of shade), A3 (aluminized screened with mesh for 50% of shade) and A4 (environment covered with straw of native coconut palm); the recipients were: polyethylene bags (R1) (15 x 25 cm) and polystyrene trays (R2) (with 72 cells). There substrates were: S1 (soil + organic compost + vermiculite, 1:1: 1 v/v), S2 (soil + organic compost + sawdust, 1:1: 1 v/v) and S3 (soil + organic compost + vermiculite + sawdust, 1:1: 1/2: 1/2 v/v). The experimental design was completely randomized statistical analysis in split-split-plot, with fifteen replications. The treatments in the plot were environments, in the subplots were pots, and subsubplots were substrates (4 x 2 x 3 = 24 treatments). Fresh and dry mass of aerial and root system parts were evaluated. Environments with screen showed better results for seedlings of yellow passion fruit biomass in polyethylene bags. Polyethylene bags promoted higher biomasses. The substrate with vermiculite showed better results for both types of containers. The substrate with a higher percentage of sawdust showed the worst result.