Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135

Abstract Background The gene coding for the uncharacterized protein PAB1135 in the archaeon Pyrococcus abyssi is in the same operon as the ribonuclease P (RNase P) subunit Rpp30. Findings Here we report the expression, purification and structural analysis of PAB1135. We analyzed the interaction of PAB1135 with RNA and show that it binds efficiently double-stranded RNAs in a non-sequence specific manner. We also performed molecular modeling of the PAB1135 structure using the crystal structure of the protein Af2318 from Archaeoglobus fulgidus (2OGK) as the template. Conclusions Comparison of this model has lead to the identification of a region in PAB1135 that could be involved in recognizing double-stranded RNA.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 degrees C, and R-g is 108 angstrom, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 degrees C and 60 degrees C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 degrees C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of R-g, D-max and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet-> oxy- > met-HbGp. (C) 2012 Elsevier B.V. All rights reserved.

Cone photopigment variations in Cebus apella monkeys evidenced by electroretinogram measurements and genetic analysis

We investigated the color vision pattern in Cebus apella monkeys by means of electroretinogram measurements (ERG) and genetic analysis. Based on ERG we could discriminate among three types of dichromatic males. Among females, this classification is more complex and requires additional genetic analysis. We found five among 10 possible different phenotypes, two trichromats and three dichromats. We also found that Cebus present a new allele with spectral peak near 552 nm, with the amino acid combination SFT at positions 180, 277 and 285 of the opsin gene, in addition to the previously described SYT, AFT and AFA alleles. (C) 2009 Elsevier Ltd. All rights reserved.

Water-in-Oil Micro-Emulsion Enhances the Secondary Structure of a Protein by Confinement

A scheme is presented in which an organic solvent environment in combination with surfactants is used to confine a natively unfolded protein inside an inverse microemulsion droplet. This type of confinement allows a study that provides unique insight into the dynamic structure of an unfolded, flexible protein which is still solvated and thus under near-physiological conditions. In a model system, the protein osteopontin (OPN) is used. It is a highly phosphorylated glycoprotein that is expressed in a wide range of cells and tissues for which limited structural analysis exists due to the high degree of flexibility and large number of post-translational modifications. OPN is implicated in tissue functions, such as inflammation and mineralisation. It also has a key function in tumour metastasis and progression. Circular dichroism measurements show that confinement enhances the secondary structural features of the protein. Small-angle X-ray scattering and dynamic light scattering show that OPN changes from being a flexible protein in aqueous solution to adopting a less flexible and more compact structure inside the microemulsion droplets. This novel approach for confining proteins while they are still hydrated may aid in studying the structure of a wide range of natively unfolded proteins.

Effect of Temperature on the Degree of Conversion and Working Time of Dual-Cured Resin Cements Exposed to Different Curing Conditions

Objectives: This study evaluated the degree of conversion (DC) and working time (WT) of two commercial, dual-cured resin cements polymerized at varying temperatures and under different curing-light accessible conditions, using Fourier transformed infrared analysis (FTIR). Materials and Methods: Calibra (Cal; Dentsply Caulk) and Variolink II (Ivoclar Vivadent) were tested at 25 degrees C or preheated to 37 degrees C or 50 degrees C and applied to a similar-temperature surface of a horizontal attenuated-total-reflectance unit (ATR) attached to an infrared spectrometer. The products were polymerized using one of four conditions: direct light exposure only (600 mW/cm(2)) through a glass slide or through a 1.5- or 3.0-mm-thick ceramic disc (A2 shade, IPS e.max, Ivoclar Vivadent) or allowed to self-cure in the absence of light curing. FTIR spectra were recorded for 20 min (1 spectrum/s, 16 scans/spectrum, resolution 4 cm(-1)) immediately after application to the ATR. DC was calculated using standard techniques of observing changes in aliphatic-to-aromatic peak ratios precuring and 20-min postcuring as well as during each 1-second interval. Time-based monomer conversion analysis was used to determine WT at each temperature. DC and WT data (n=6) were analyzed by two-way analysis of variance and Tukey post hoc test (p=0.05). Results: Higher temperatures increased DC regardless of curing mode and product. For Calibra, only the 3-mm-thick ceramic group showed lower DC than the other groups at 25 degrees C (p=0.01830), while no significant difference was observed among groups at 37 degrees C and 50 degrees C. For Variolink, the 3-mm-thick ceramic group showed lower DC than the 1-mm-thick group only at 25 degrees C, while the self-cure group showed lower DC than the others at all temperatures (p=0.00001). WT decreased with increasing temperature: at 37 degrees C near 70% reduction and at 50 degrees C near 90% for both products, with WT reduction reaching clinically inappropriate times in some cases (p=0.00001). Conclusion: Elevated temperature during polymerization of dual-cured cements increased DC. WT was reduced with elevated temperature, but the extent of reduction might not be clinically acceptable.

Spectroscopic characterization and crystal structure of cis-Bis(N-(2-benzoyl)-N ',N '-diphenylthioureato-k O-2,S)nickel(II)

The title compound [Ni(C20H15N2OS)(2)] is prepared by the reaction of metal acetate with the corresponding acylthiourea derivative. The complex is characterized by elemental analysis, IR, H-1 and C-13 NMR, and its structure is determined by single crystal X-ray diffraction. The Ni(II) ion is coordinated by the S and O atoms of two N-benzoyl-N',N'-diphenylthiourea ligands in a slightly distorted square-planar coordination geometry. The two O and two S atoms are mutually cis to each other. The substance crystallizes triclinic (P-1 space group) with cell dimensions a = 10.7262(9) , b = 12.938(3) , c = 14.2085(12) , alpha = 74.650(4)A degrees, beta = 78.398(4)A degrees, gamma = 68.200(5)A degrees, and two formula units in the unit cell. The structure is very close to the related N-(2-furoyl) Ni complex reported previously.

Expression, purification and molecular analysis of the human ZNF706 protein

Background: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). Findings: ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. Conclusions: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis.

Persistent luminescence behavior of materials doped with Eu2+ and Tb3+

In this work, the persistent luminescence mechanisms of Tb3+ (in CdSiO3) and Eu2+ (in BaAl2O4) based on solid experimental data are compared. The photoluminescence spectroscopy shows the different nature of the inter- and intraconfigurational transitions for Eu2+ and Tb3+, respectively. The electron is the charge carrier in both mechanisms, implying the presence of electron acceptor defects. The preliminary structural analysis shows a free space in CdSiO3 able to accommodate interstitial oxide ions needed by charge compensation during the initial preparation. The subsequent annealing removes this oxide leaving behind an electron trap. Despite the low band gap energy for CdSiO3, determined with synchrotron radiation UV-VUV excitation spectroscopy of Tb3+, the persistent luminescence from Tb3+ is observed only with UV irradiation. The need of high excitation energy is due to the position of F-7(6) level deep below the bottom of the conduction band, as determined with the 4f(8)-> 4f(7)5d(1) and the ligand-to-metal charge-transfer transitions. Finally, the persistent luminescence mechanisms are constructed and, despite the differences, the mechanisms for Tb3+ and Eu2+ proved to be rather similar. This similarity confirms the solidity of the interpretation of experimental data for the Eu2+ doped persistent luminescence materials and encourages the use of similar models for other persistent luminescence materials. (C) 2012 Optical Society of America

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages. Copyright (c) 2012 Society of Chemical Industry

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Cellular biomarkers to elucidate global warming effects on Antarctic sea urchin Sterechinus neumayeri

Global warming is a reality and its effects have been widely studied. However, the consequences for marine invertebrates remain poorly understood. Thus, the present study proposed to evaluate the effect of elevated temperature on the innate immune system of Antarctic sea urchin Sterechinus neumayeri. Sea urchins were collected nearby Brazilian Antarctic Station "Comandante Ferraz" and exposed to 0 (control), 2 and 4A degrees C for periods of 48 h, 2, 7 and 14 days. After the experimental periods, coelomic fluid was collected in order to perform the following analyses: coelomocytes differential counting, phagocytic response, adhesion and spreading coelomocytes assay, intranuclear iron crystalloid and ultra structural analysis of coelomocytes. The red sphere cell was considered a biomarker for heat stress, as they increased in acute stress. Besides that, a significant increase in phagocytic indexes was observed at 2A degrees C coinciding with a significant increase of intranuclear iron crystalloid at the same temperature and same time period. Furthermore, significant alterations in cell adhesion and spreading were observed in elevated temperatures. The ultra structural analysis of coelomocytes showed no significant difference across treatments. This was the first time that innate immune response alterations were observed in response to elevated temperature in a Polar echinoid.

Use of a Single L1 GPS Receiver for Monitoring Structures: First Results of the Detection of Millimetric Dynamic Oscillations

This paper presents preliminary results to determine small displacements of a global positioning system (GPS) antenna fastened to a structure using only one L1 GPS receiver. Vibrations, periodic or not, are common in large structures, such as bridges, footbridges, tall buildings, and towers under dynamic loads. The behavior in time and frequency leads to structural analysis studies. The hypothesis of this article is that any large structure that presents vibrations in the centimeter-to-millimeter range can be monitored by phase measurements of a single L1 receiver with a high data rate, as long as the direction of the displacement is pointing to a particular satellite. Within this scenario, the carrier phase will be modulated by antenna displacement. During a period of a few dozen seconds, the relative displacement to the satellite, the satellite clock, and the atmospheric phase delays can be assumed as a polynomial time function. The residuals from a polynomial adjustment contain the phase modulation owing to small displacements, random noise, receiver clock short time instabilities, and multipath. The results showed that it is possible to detect displacements of centimeters in the phase data of a single satellite and millimeters in the difference between the phases of two satellites. After applying a periodic nonsinusoidal displacement of 10 m to the antenna, it is clearly recovered in the difference of the residuals. The time domain spectrum obtained by the fast Fourier transform (FFT) exhibited a defined peak of the third harmonic much more than the random noise using the proposed third-degree polynomial model. DOI: 10.1061/(ASCE)SU.1943-5428.0000070. (C) 2012 American Society of Civil Engineers.

Comparison of two experimental models of pulmonary hypertension

Objective: To compare two models of pulmonary hypertension (monocrotaline and monocrotaline+pneumonectomy) regarding hemodynamic severity, structure of pulmonary arteries, inflammatory markers (IL-1 and PDGF), and 45-day survival. Methods: We used 80 Sprague-Dawley rats in two study protocols: structural analysis; and survival analysis. The rats were divided into four groups: control; monocrotaline (M), pneumonectomy (P), and monocrotaline+pneumonectomy (M+P). In the structural analysis protocol, 40 rats (10/group) were catheterized for the determination of hemodynamic variables, followed by euthanasia for the removal of heart and lung tissue. The right ventricle (RV) was dissected from the interventricular septum (IS), and the ratio between RV weight and the weight of the left ventricle (LV) plus IS (RV/LV+IS) was taken as the index of RV hypertrophy. In lung tissues, we performed histological analyses, as well as using ELISA to determine IL-1 and PDGF levels. In the survival protocol, 40 animals (10/group) were followed for 45 days. Results: The M and M+P rats developed pulmonary hypertension, whereas the control and P rats did not. The RV/LV+IS ratio was significantly higher in M+P rats than in M rats, as well as being significantly higher in M and M+P rats than in control and P rats. There were no significant differences between the M and M+P rats regarding the area of the medial layer of the pulmonary arteries; IL-1 and PDGF levels; or survival. Conclusions: On the basis of our results, we cannot conclude that the monocrotaline+pneumonectomy model is superior to the monocrotaline model.

Interaction of gramicidin with DPPC/DODAB bilayer fragments

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids. (C) 2012 Elsevier B.V. All rights reserved.

Cationic nanoparticles for delivery of amphotericin B: preparation, characterization and activity in vitro

Abstract Background Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans. Results Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings. Conclusion Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.