Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase

Phenylketonuria (PKU; OMIM 261600), the most common disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches. Enzyme replacement therapy with hPAH could be regarded as a potential form of PKU treatment if the reported in vitro hPAH instability could be overcome. In this study, we investigated the effect of different polyol compounds, e.g. glycerol, mannitol and PEG-6000 on the in vitro stability of purified hPAH produced in a heterologous prokaryotic expression system. The recombinant human enzyme was stored in the presence of the studied stabilizing agents at different temperatures (4 and −20 °C) during a 1-month period. Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50% glycerol or 10% mannitol, at −20 °C, protected the enzyme from loss of its enzymatic activity. The determined ΔG ⁰ and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.     

Structural elucidation and NMR assignments of a new pyrrolizidine alkaloid from Crotalaria vitellina Ker Gawl

A new pyrrolizidine alkaloid, named crotavitelin, was isolated from fruits of Crotalaria vitellina, Fabaceae (Papilionoideae). The structure was established by spectroscopic techniques such as one‐dimensional and two‐dimensional NMR, IR, and MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Optimization of photo-polymerized sol–gel monolithic stationary phases prepared in polyacrylate-coated fused-silica capillaries for capillary electrochromatography

The preparation of photo-polymerized sol–gel monolithic stationary phases (MSP) within 100 μm internal diameter polyacrylate-coated fused-silica capillaries for use in capillary electrochromatography (CEC) was optimized. Eight mixtures containing different amounts of methacryloxypropyltrimethoxysilane (MPTMS) as the polymeric precursor, hydrochloric acid solution as the catalyst, toluene as the porogen and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (Irgacure 819) as the photo-initiator were irradiated at 370 nm inside the capillaries in order to complete the MSP polymerization, according to a fractional factorial experimental design 2_IV^4-1. All the preparation procedure, from capillary pretreatment until the MSP is ready to use in CEC, were made in less than four hours in mild conditions. A high pressurization injection device (HPID) useful for micro-volume syringes was built in order to achieve practical, controlled and precise injections of sols, solvents and electrolytes in the capillaries. The eight MSP were equally washed, conditioned and submitted to CEC procedures via short-end injection, which showed higher efficiency and peak height taking shorter analysis time. Electrochromatographic behaviors of the MSP were corroborated with morphological characterizations by scanning electron microscopy. The optimum condition, which allowed the separation of standard mixture containing thiourea (marker compound), naphthalene, acenaphthene, fluorene, phenanthrene and anthracene in twelve minutes without external pressure assistance, showed efficiencies up to 51,460 N/m, relative standard deviation from 0.05 to 3.3% for migration/retention time and from 0.14 to 1.6% for relative area (considering thiourea as an internal standard) and also showed no statistical evidence that three MSP prepared at the same condition are different within 95% confidence interval.     

Development of hybrid nanocomposites based on PLLA and low-field NMR characterization

Hybrid nanocomposite based on poly(L-lactic acid) (PLLA) employing silica (A200) and organophilic silica (R972), natural clay (NT-25) and two types of organoclay (S4 and S7) were prepared by the solution casting method. Each nanoparticle type was incorporated at two ratios (3 and 5%) relative to the polymer matrix. The nanocomposites formed were evaluated by X-ray, thermal resistance and molecular dynamic analysis, using proton spin-lattice relaxation time. The results showed that each nanoparticle type acted independently of the polymer/nanoparticle ratio in the new material. Both silica types promoted an increase in rigidity, due to the strong intermolecular forces. The natural clay did not cause any significant change in the molecular mobility of the nanocomposites, probably due to the non-affinity of the chemical structure. Therefore, both organoclays were better dispersed in the polymeric matrix, allowing good intermolecular interaction and, consequently, exfoliated and intercalated nanocomposites were formed.     

High-pressure phase diagram of the drug mitotane in compressed and/or supercritical CO2

This work provides experimental phase diagram of mitotane, a drug used in the chemotherapy treatment of adrenocortical carcinoma, in compressed and/or supercritical CO₂. The synthetic-static method in a high-pressure variable-volume view cell coupled with a transmitted-light intensity probe was used to measure the solid-fluid (SF) equilibrium data. The phase equilibrium experiments were determined in temperature ranging from (298.2 to 333.1) K and pressure up to 22 MPa. Peng–Robinson equation of state (PR-EoS) with classical mixing rule was used to correlate the experimental data. Excellent agreement was found between experimental and calculated values.     

The use of the fractal model to complex resistivity in the interpretation of induced polarization data

Several researchers demonstrated that spectral parameters in induced polarization can be applied to discriminate different IP sources. In this paper it was applied an inversion procedure using the Gauss–Newton method to recover the spectral parameters of fractal model to complex resistivity. The finite element method was applied to carry out the forward modeling. The procedure was applied in synthetic data and simulations were carried out in five different frequencies. The inversion of the data were carried out in each frequency, further the inversion was applied also to each cell of the finite element mesh to recover the fractal parameter in order to analyze the possibility of using the fractal model parameters in the interpretation of the induced polarization response to this geological geometry. The results showed that the anomalies are well detected by the image of the fractal model parameters.     

<em>In Vitro </em>and <em>in </em><em>Vivo</em> Antitumor Effect of Trachylobane-360, a Diterpene from<em> Xylopia langsdorffiana</em>

Trachylobane-360 (ent-7α-acetoxytrachyloban-18-oic acid) was isolated from Xylopia langsdorffiana. Studies have shown that it has weak cytotoxic activity against tumor and non-tumor cells. This study investigated the in vitro and in vivo antitumor effects of trachylobane-360, as well as its cytotoxicity in mouse erythrocytes. In order to evaluate the in vivo toxicological aspects related to trachylobane-360 administration, hematological, biochemical and histopathological analyses of the treated animals were performed. The compound exhibited a concentration-dependent effect in inducing hemolysis with HC₅₀ of 273.6 μM, and a moderate in vitro concentration-dependent inhibitory effect on the proliferation of sarcoma 180 cells with IC₅₀ values of 150.8 μM and 150.4 μM, evaluated by the trypan blue exclusion test and MTT reduction assay, respectively. The in vivo inhibition rates of sarcoma 180 tumor development were 45.60, 71.99 and 80.06% at doses of 12.5 and 25 mg/kg of trachylobane-360 and 25 mg/kg of 5-FU, respectively. Biochemical parameters were not altered. Leukopenia was observed after 5-FU treatment, but this effect was not seen with trachylobane-360 treatment. The histopathological analysis of liver and kidney showed that both organs were mildly affected by trachylobane-360 treatment. Trachylobane-360 showed no immunosuppressive effect. In conclusion, these data reinforce the anticancer potential of this natural diterpene.     

Magnetoelectric coupling in multiferroic heterostructure of rf-sputtered Ni–Mn–Ga thin film on PMN–PT

In this paper, we report a preparation of multiferroic heterostructure from thin film of Ni–Mn–Ga (NMG) alloy and lead magnesium niobate–lead titanate (PMN–PT) with effective magnetoelectric (ME) coupling between the film as ferromagnetic material and PMN–PT as piezoelectric material. The heterostructure was prepared by relatively low temperature (400 °C) deposition of the film on single crystal of piezoelectric PMN–PT substrate using rf magnetron co-sputtering of Ni₅₀Mn₅₀ and Ni₅₀Ga₅₀ targets. Magnetic measurements by Superconducting Quantum Interference Design (SQIUD) Magnetometer and Vibrating Sample Magnetometer (VSM) on the film revealed that the film is in ferromagnetically ordered martensitic state at room temperature with saturation magnetization of ∼240 emu/cm³ and Curie temperature of ∼337 K. Piezoresponse force microscopy (PFM) measurement done at room temperature on the substrate showed the presence of expected hysteresis loop confirming the stability of the piezoelectric state of the substrate after deposition. Room temperature ME voltage coefficient (α_ME) of the heterostructure was measured as a function of applied bias dc magnetic field in Longitudinal–Transverse (L–T) ME coupling mode by lock-in technique. A maximum ME coefficient α_ME of 3.02 mV/cm Oe was measured for multiferroic NMG/PMN–PT heterostructure which demonstrates that there is ME coupling between the film as ferromagnetic material and PMN–PT as piezoelectric material.     

Perturbed angular correlations investigations on YMnO3 multiferroic manganite

The Perturbed Angular Correlation (PAC) technique was applied to study the yttrium local environment in YMnO₃ multiferroic manganite. The electric field gradients (EFG) at the Y site have been measured as function of temperature, covering both ferroelectric and magnetic transitions. The results were compared with point charge model (PCM) calculations. The experimental results show two different EFG distributions for all temperatures. Only one can be directly attributed to the yttrium crystalline site in the hexagonal structure.     

Hyperfine Fields at the Cd Site in La0.67Cd0.25MnO3 CMR Manganites

Although Cd and Ca ions have the same valence and cation size, their incorporation into vacancy-doped La manganites induce different properties. While the incorporation of Ca leads to high Tc up to 250 K and induces a metallic-like behaviour, the incorporation of Cd severely reduces Tc and promotes insulator-like behaviour. In this work, the Cd hyperfine fields have been measured with the Perturbed Angular Correlations (PAC) technique after implantation and annealing of ^111m Cd in La–Cd–MnO₃ samples. The PAC results are compared with measurements of the resistivity and magnetization performed on the same samples. The mixed La and Mn site Cd occupancy is suggested as a possibility to explain the properties of the La–Cd–MnO₃ system.