The role of MAPK signal transduction pathways in the response to oxidative stress in the fungal pathogen Candida albicans: implications in virulence

In recent years, Mitogen-Activated Protein Kinase (MAPK) pathways have emerged as major regulators of cellular physiology. In the fungal pathogen Candida albicans, three different MAPK pathways have been characterized in the last years. The HOG pathway is mainly a stress response pathway that is activated in response to osmotic and oxidative stress and also participates regulating other pathways. The SVG pathway (or mediated by the Cek1 MAPK) is involved in cell wall formation under vegetative and filamentous growth, while the Mkc1-mediated pathway is involved in cell wall integrity. Oxidative stress is one of the types of stress that every fungal cell has to face during colonization of the host, where the cell encounters both hypoxia niches (i.e. gut) and high concentrations of reactive oxygen species (upon challenge with immune cells). Two pathways have been shown to be activated in response to oxidative stress: the HOG pathway and the MKC1-mediated pathway while the third, the Cek1 pathway is deactivated. The timing, kinetics, stimuli and functional responses generated upon oxidative stress differ among them; however, they have essential functional consequences that severely influence pathogenesis. MAPK pathways are, therefore, valuable targets to be explored in antifungal research.     

Evaluation of antioxidant capacity and synergistic associations of quinonemethide triterpenes and phenolic substances from Maytenus ilicifolia (Celastraceae)

This work describes the isolation of the secondary metabolites identified as the quinonemethides maytenin (1) and pristimerin (2) from Maytenus ilicifolia extracts obtained from root barks of adult plants and roots of seedlings and their quantification by high performance liquid chromatography coupled to a diode array detector. The electrochemical profiles obtained from cyclic voltammetry and a coulometric detector coupled to high-performance liquid chromatography contributed to the evaluation of their antioxidant capacity. The antioxidant properties of individual components and the crude extracts of the root barks of Maytenus ilicifolia were compared and the possible synergistic associations of quinonemethide triterpenes and phenolic substances were investigated by using rutin as a model phenolic compound.     

Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata

The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm⁻² UVA, 0.07 and 0.9 J cm⁻² UVB, 20 n m β-PDH (pigment dispersing hormone) or 10 μ m SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo . Cultured cells were exposed to 250 μ m L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo . SIN-1 did not induce pigment dispersion in the cell cultures. l-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.     

Steric factors override thermodynamic driving force in regioselectivity of proline hydroxylation by prolyl-4-hydroxylase enzymes

Prolyl-4-hydroxylase is an important nonheme iron-containing dioxygenase in humans involved in the regioselective hydroxylation of a proline residue in a peptide chain on the C(4) position. In biosystems this process is important to create collagen cross-linking and cellular responses to hypoxia. We have performed a series of density functional theory (DFT) studies into the origin of the regioselectivity of proline hydroxylation by P4H enzymes using a minimal active site model (where substrate is unhindered in the binding site) and a larger active site model that incorporates steric hindrance of the substrate by several secondary sphere aromatic residues. Our studies show that thermodynamically the most favorable hydrogen atom abstraction position of proline is from the C(5) position; hence, the small model gives a low reaction barrier and large exothermicity for this process. However, stereochemical repulsions of the substrate with aromatic residues of Tyr(140) and Trp(243) in the second coordination sphere prevent C(5) hydroxylation and make C(4) hydroxylation the dominant mechanism, despite a lesser driving force for the reaction. These studies explain the remarkable regioselectivity of proline hydroxylation by P4H enzymes and show that the regioselectivity is kinetically controlled but not thermodynamically. In addition, we calculated spectroscopic parameters and found good agreement with experimental data.     

Capillary electrophoresis-mass spectrometry of intact basic proteins using Polybrene-dextran sulfate-Polybrene-coated capillaries: System optimization and performance

A capillary electrophoresis-mass spectrometry (CE-MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol-water-acetic acid (75:25:0.1, v/v/v) at 2 μL min⁻¹ resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE-MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE-MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE-MS system, determination coefficients (R²) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM.     

Targeted LC–MS derivatization for aldehydes and carboxylic acids with a new derivatization agent 4-APEBA

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 °C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described.     

3-MCPD in food other than soy sauce or hydrolysed vegetable protein (HVP)

This review gives an overview of current knowledge about 3-monochloropropane-1,2-diol (3-MCPD) formation and detection. Although 3-MCPD is often mentioned with regard to soy sauce and acid-hydrolysed vegetable protein (HVP), and much research has been done in that area, the emphasis here is placed on other foods. This contaminant can be found in a great variety of foodstuffs and is difficult to avoid in our daily nutrition. Despite its low concentration in most foods, its carcinogenic properties are of general concern. Its formation is a multivariate problem influenced by factors such as heat, moisture and sugar/lipid content, depending on the type of food and respective processing employed. Understanding the formation of this contaminant in food is fundamental to not only preventing or reducing it, but also developing efficient analytical methods of detecting it. Considering the differences between 3-MCPD-containing foods, and the need to test for the contaminant at different levels of food processing, one would expect a variety of analytical approaches. In this review, an attempt is made to provide an up-to-date list of available analytical methods and to highlight the differences among these techniques. Finally, the emergence of 3-MCPD esters and analytical techniques for them are also discussed here, although they are not the main focus of this review.     

Determination of synthetic pharmaceuticals in phytotherapeutics by capillary zone electrophoresis with contactless conductivity detection (CZE-CD)

In this work, a method for simultaneous determination of amfepramone, fenproporex, sibutramine and fluoxetine was developed by capillary zone electrophoresis with capacitively coupled contactless conductivity detection (C⁴D) using a homemade capillary electrophoretic system. The optimized conditions for the separation of the pharmaceuticals by CZE were as follows: 50 mmol L^− 1 phosphate buffer (pH 5.0) in 50/50 (v/v) mixture of water/acetonitrile as the working electrolyte, 15 kV separation voltage, 25 °C separation temperature, hydrodynamic injection by gravity using 20 cm injection height and 60 s injection time. The detection by C⁴D was carried out by using a homemade detector, which employs a sinusoidal wave generator operating at 600 kHz frequency and 2 V_pp wave amplitude. The optimized and validated CZE-C⁴D method was applied for the determination of the studied pharmaceuticals as adulterants in phytotherapeutic formulations commercialized in Brazil for slimming purposes.     

(Liquid + liquid) equilibrium of {water + phenol + (1-butanol,or 2-butanol,or tert-butanol)} systems

(Liquid + liquid) equilibrium (LLE) and binodal curve data were determined for the systems (water + phenol + tert-butanol) at T = 298.15 K, (water + phenol + 2-butanol) and (water + phenol + 1-butanol) at T = 298.15 K and T = 313.15 K by the combined techniques of densimetry and refractometry. Type I curve (for tert-butanol) and Type II curves (for 1- and 2-butanol) were found. The data were correlated with the NRTL model and the parameters estimated present root mean square deviations below 2% for the system with tert-butanol and lower than 0.8% for the other systems.     

Radiation processing of natural polymers: The IAEA contribution

Radiation processing offers a clean and additive-free method for preparation of value-added novel materials based on renewable, non-toxic, and biodegradable natural polymers. Crosslinked natural polymers can be used as hydrogel wound dressings, face cleaning cosmetic masks, adsorbents of toxins, and non-bedsore mats; while low molecular weight products show antibiotic, antioxidant, and plant-growth promoting properties. Recognizing the potential benefits that radiation technology can offer for processing of natural polymers into useful products, the IAEA implemented a coordinated research project (CRP) on “Development of Radiation-processed products of Natural Polymers for application in Agriculture, Healthcare, Industry and Environment”. This CRP was launched at the end of 2007 with participation of 16 MS to help connecting radiation technology and end-users to derive enhanced benefits from these new value-added products of radiation-processed natural materials. In this paper the results of activities in participating MS related to this work will be presented.