Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Filmes ultrafinos de ésteres de celulose: preparo, caracterização e imobilização de proteínas

In this study cellulose acetate butyrate (CAB) and carboxymehtylcellulose acetate butyrate (CMCAB) films adsorbed onto silicon wafers were characterized by means of ellipsometry, atomic force microscopy (AFM), sum frequency generation spectroscopy (SFG) and contact angle measurements. The adsorption behavior of lysozyme (LIS) or bovine serum albumin (BSA) onto CAB and CMCAB films was investigated. The amounts of adsorbed LIS or BSA onto CMCAB films were more pronounced than those onto CAB films due to the presence of carboxymethyl group in the CMCAB structure. Besides, the adsorption of BSA molecules on CMCAB films was more favored than that of LIS molecules. Antimicrobial effect of LIS bound to CAB or CMCAB layers was evaluated using Micrococcus luteus as substrate.

Extraction and concentration of biogenic calcium oxalate from plant leaves

The objective of this study was to extract and concentrate calcium oxalate (CaOx) crystals from plant leaves that form the above mentioned crystals. The chemical and physical studies of CaOx from plant to be performed depend on an adequate amount of the crystals. The plant used in this study was croton (Codiaeum variegatum). The leaves were ground in a heavy duty blender and sieved through a 0.20 mm sieve. The suspension obtained was suspended in distilled water. The crystals were concentrated at the bottom of a test tube. The supernatant must be washed until it is free of plant pigments and other organic substances. Biogenic CaOx crystals have well-defined and sharp peaks, indicating very high crystallinity. Moreover, the CaOx crystals were not damaged during the extraction procedure, as can be seen on the scanning electron microscope images. The porposed method can be considered efficient to extract and concentrate biogenic calcium oxalate.

Influence of Annealing Heat Treatment and Cr, Mg, and Ti Alloying on the Mechanical Properties of High-Silicon Cast Iron

The influence of annealing on the mechanical properties of high-silicon cast iron for three alloys with distinct chromium levels was investigated. Each alloy was melted either with or without the addition of Ti and Mg. These changes in the chemical composition and heat treatment aimed to improve the material's mechanical properties by inhibiting the formation of large columnar crystals, netlike laminae, precipitation of coarse packs of graphite, changing the length and morphology of graphite, and rounding the extremities of the flakes to minimize the stress concentration. For alloys with 0.07 wt.% Cr, the annealing reduced the impact resistance and tensile strength due to an enhanced precipitation of refined carbides and the formation of interdendritic complex nets. Annealing the alloys containing Ti and Mg led to a decrease in the mechanical strength and an increase in the toughness. Alloys containing approximately 2 wt.% Cr achieved better mechanical properties as compared to the original alloy. However, with the addition of Ti and Mg to alloys containing 2% Cr, the chromium carbide formation was inhibited, impairing the mechanical properties. In the third alloy, with 3.5 wt.% of Cr additions, the mechanical strength improved. The annealing promoted a decrease in both hardness and amount of iron and silicon complex carbides. However, it led to a chromium carbide formation, which influenced the mechanical characteristics of the matrix of the studied material.

Phase transformation and residual stress probed by Raman spectroscopy in diamond-turned single crystal silicon

Single-point diamond turning of monocrystalline semiconductors is an important field of research within brittle materials machining. Monocrystalline silicon samples with a (100) orientation have been diamond turned under different cutting conditions (feed rate and depth of cut). Micro-Raman spectroscopy and atomic force microscopy have been used to assess structural alterations and surface finish of the samples diamond turned under ductile and brittle modes. It was found that silicon undergoes a phase transformation when machined in the ductile mode. This phase transformation is evidenced by the creation of an amorphous surface layer after machining which has been probed by Raman scattering. Compressive residual stresses are estimated for the machined surface and it is observed that they decrease with an increase in the feed rate and depth of cut. This behaviour has been attributed to the formation of subsurface cracks when the feed rate is higher than or equal to 2.5 mu m/rev. The surface roughness was observed to vary with the feed rate and the depth of cut. An increase in the surface roughness was influenced by microcrack formation when the feed rate reached 5.0 mu m/rev. Furthermore, a high-pressure phase transformation induced by the tool/material interaction and responsible for the ductile response of this typical brittle material is discussed based upon the presented Raman spectra. The application of this machining technology finds use for a wide range of high quality components, for example the creation of a micrometre-range channel for microfluidic devices as well as microlenses used in the infrared spectrum range.

Using the ultrasound and instrumented indentation techniques to measure the elastic modulus of engineering materials

Currently, the acoustic and nanoindentation techniques are two of the most used techniques for material elastic modulus measurement. In this article fundamental principles and limitations of both techniques are shown and discussed. Last advances in nanoindentation technique are also reviewed. An experimental study in ceramic, metallic, composite and single crystals was also done. Results shown that ultrasonic technique is capable to provide results in agreement with those reported in literature. However, ultrasonic technique does not allow measuring the elastic modulus of some small samples and single crystals. On the other hand, the nanoindentation technique estimates the elastic modulus values in reasonable agreement with those measured by acoustic methods, particularly in amorphous materials, while in some policristaline materials some deviation from expected values was obtained.

Quasiharmonic elastic constants corrected for deviatoric thermal stresses

The quasiharmonic approximation (QHA), in its simplest form also called the statically constrained (SC) QHA, has been shown to be a straightforward method to compute thermoelastic properties of crystals. Recently we showed that for noncubic solids SC-QHA calculations develop deviatoric thermal stresses at high temperatures. Relaxation of these stresses leads to a series of corrections to the free energy that may be taken to any desired order, up to self-consistency. Here we show how to correct the elastic constants obtained using the SC-QHA. We exemplify the procedure by correcting to first order the elastic constants of MgSiO(3) perovskite and MgSiO(3) postperovskite, the major phases of the Earth's lower mantle. We show that this first-order correction is quite satisfactory for obtaining the aggregated elastic averages of these minerals and their velocities in the lower mantle. This type of correction is also shown to be applicable to experimental measurements of elastic constants in situations where deviatoric stresses can develop, such as in diamond-anvil cells.

The role of crystalline anisotropy in mechanical property extractions through Berkovich indentation

This work uses crystal plasticity finite element simulations to elucidate the role of elastoplastic anisotropy in instrumented indentation P-h(s) curve measurements in face-centered Cubic (fcc) crystals. It is shown that although the experimental fluctuations in the loading stage of the P-h(s) curves can be attributed to anisotropy, the variability in the unloading stage of the experiments Is much greater than that resulting from anisotropy alone. Moreover, it is found that the conventional procedure used to evaluate the contact variables ruling the unloading P-h(s) curve introduces all uncertainty that approximates to the more fundamental influence of anisotropy. In view of these results, a robust procedure is proposed that uses contact area measurements in addition to the P-h(s) curves to extract homogenized J(2)-Plasticity-equivalent mechanical properties from single crystals.

Polyelectrolyte-protein complexation driven by charge regulation

The interplay between the biocolloidal characteristics (especially size and charge), pH, salt concentration and the thermal energy results in a unique collection of mesoscopic forces of importance to the molecular organization and function in biological systems. By means of Monte Carlo simulations and semi-quantitative analysis in terms of perturbation theory, we describe a general electrostatic mechanism that gives attraction at low electrolyte concentrations. This charge regulation mechanism due to titrating amino acid residues is discussed in a purely electrostatic framework. The complexation data reported here for interaction between a polyelectrolyte chain and the proteins albumin, goat and bovine alpha-lactalbumin, beta-lactoglobulin, insulin, k-casein, lysozyme and pectin methylesterase illustrate the importance of the charge regulation mechanism. Special attention is given to pH congruent to pI where ion-dipole and charge regulation interactions could overcome the repulsive ion-ion interaction. By means of protein mutations, we confirm the importance of the charge regulation mechanism, and quantify when the complexation is dominated either by charge regulation or by the ion-dipole term.

A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 A, beta = 96.03 degrees. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V (M) of 2.12 A3 Da-1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.

Expression, crystallization and preliminary crystallographic analysis of PilZ(XAC1133) from Xanthomonas axonopodis pv. citri

Proteins containing PilZ domains are widespread in Gram-negative bacteria and have recently been shown to be involved in the control of biofilm formation, adherence, aggregation, virulence-factor production and motility. Furthermore, some PilZ domains have recently been shown to bind the second messenger bis(3'-> 5') cyclic diGMP. Here, the cloning, expression, purification and crystallization of PilZ(XAC1133), a protein consisting of a single PilZ domain from Xanthomonas axonopodis pv. citri, is reported. The closest PilZ(XAC1133) homologues in Pseudomonas aeruginosa and Neisseria meningitidis control type IV pilus function. Recombinant PilZ(XAC1133) containing selenomethionine was crystallized in space group P6(1). The unit-cell parameters were a = 62.125, b = 62.125, c = 83.543 angstrom. These crystals diffracted to 1.85 angstrom resolution and a MAD data set was collected at a synchrotron source. The calculated Matthews coefficient suggested the presence of two PilZ(XAC1133) molecules in the asymmetric unit.

Crystallization and preliminary X-ray analysis of LipL32 from Leptospira interrogans serovar Copenhageni

LipL32 is a major surface protein that is expressed during infection by pathogenic Leptospira. Here, the crystallization of recombinant LipL32(21-272), which corresponds to the mature LipL32 protein minus its N-terminal lipid-anchored cysteine residue, is described. Selenomethionine-labelled LipL32(21-272) crystals diffracted to 2.25 angstrom resolution at a synchrotron source. The space group was P3(1)21 or P3(2)21 and the unit-cell parameters were a = b = 126.7, c = 96.0 angstrom.

The influence of adsorption phenomena on the impedance spectroscopy of an electrolytic cell

In this work we investigate the influence of the adsorption of ions on the impedance spectroscopy of an electrolytic cell. We consider that the positive and negative ions present in a dielectric liquid are adsorbed in the electrode surfaces with different adsorption energies. This difference in adsorption energies causes an additional plateaux in the limit of the low-frequency range of the real part of the impedance Z. In the same frequency range, a second minimum in the imaginary part of Z is predicted. The theory is illustrated with measurements of the impedance of an electrolytic solution in the frequency range from 10(-2) Hz to 1 KHz. A comparison between the present model and others from the literature to describe the experimental results is also made.

The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of similar to 35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(l), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some refolding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

Elastic Maier-Saupe-Zwanzig model and some properties of nematic elastomers

We introduce a simple mean-field lattice model to describe the behavior of nematic elastomers. This model combines the Maier-Saupe-Zwanzig approach to liquid crystals and an extension to lattice systems of the Warner-Terentjev theory of elasticity, with the addition of quenched random fields. We use standard techniques of statistical mechanics to obtain analytic solutions for the full range of parameters. Among other results, we show the existence of a stress-strain coexistence curve below a freezing temperature, analogous to the P-V diagram of a simple fluid, with the disorder strength playing the role of temperature. Below a critical value of disorder, the tie lines in this diagram resemble the experimental stress-strain plateau and may be interpreted as signatures of the characteristic polydomain-monodomain transition. Also, in the monodomain case, we show that random fields may soften the first-order transition between nematic and isotropic phases, provided the samples are formed in the nematic state.