Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca(2+) homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn(2+) or Ca(2+) with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses. (c) 2008 Elsevier B.V. All rights reserved.

Development of impedimetric and optical calcium biosensor by using modified gold electrode with porcine S100A12 protein

We describe the development of a label free method to analyze the interactions between Ca(2+) and the porcine S100A12 protein immobilized on polyvinyl butyral (PVB). The modified gold electrodes were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and surface plasmon resonance (SPR) techniques. SEM analyses of PVB and PVB-S100A12 showed a heterogeneous distribution of PVB spherules on gold surface. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due the adsorption of PVB-S100A12, and confirm the existence of a positive response of the immobilized S100Al2 to the presence of calcium ions. The biosensor exhibited a wide linear response to Ca(2+) concentrations ranging from 12.5 to 200 mM. The PVB-S100A12 seems to be bound to the gold electrode surface by physical adsorption: we observed an increase of 1184.32 m degrees in the SPR angle after the adsorption of the protein on the PVB surface (in an indication that 9.84 ng of S100A12 are adsorbed per mm(2) of the Au-PVB electrode), followed by a further increase of 581.66 m degrees after attachment of the Ca(2+) ions. In addition, no SPR response is obtained for non-specific ions. These studies might be useful as a platform for the design of new reusable and sensitive biosensing devices that could find use in the clinical applications. (C) 2010 Elsevier B.V. All rights reserved.

Mineralogical and microthermometric studies and structural aspects of imperial topaz mineralization in Antonio Pereira, Ouro Preto District (Minas Gerais) - Brazil

The occurrences of imperial topaz in the Antonio Pereira mine, Ouro Preto, Minas Gerais, are associated with the metamorphic carbonate rocks of the Minas Supergroup. The crystals have densities varying from 3.46 to 3.58. The parameters of the unitary cells obtained were: 4.658 to 4.663 angstrom (ao), 8.823 to 8.832 angstrom (b(o)), 8.382 to 8.389 angstrom (c(o)), and 344.65 to 345.46 angstrom 3 (V). The refraction indices presented the following variations: 1.622 to 1.630 (nX), 1.624 to 1.632 (nY), 1.633 to 1.640 (nZ), and 0.008 to 0.011 (B). These properties are coherent with the low fluorine contents obtained (16,48%/17,05wt%). Infrared spectroscopy and microthermometry showed that the fluid inclusions, which represent the mineralizing fluids, are formed by H(2)O (with Ca(2+), Mg(2+) and Na(+)), and CO(2) +/- CH(4). The minimal trapping T-P conditions of 290/320 degrees C and 2,349/2,497bar were obtained for the primary fluid inclusions. The pseudo-secondary fluid inclusions were trapped at conditions of lower temperatures and variable pressures, during the deformation process under local alternating states of stress. The microthermometric studies, the structural analysis and the fluorine contents suggest that the mineralized veins were formed from hydrothermal fluids originated during the Brasiliano tectono-metamorphic event.

Precambrian geotectonic units of the Rio de La Plata craton

The main Precambrian tectonic units of Uruguay include the Piedra Alta tectonostratigraphic terrane (PATT) and Nico Perez tectonostratigraphic terrane (NPTT), separated by the Sarandi del Yi high-strain zone. Both terranes are well exposed in the Rio de La Plata craton (RPC). Although these tectonic units are geographically small, they record a wide span of geologic time. Therefore improved geological knowledge of this area provides a fuller understanding of the evolution of the core of South America. The PATT is constituted by low-to medium-grade metamorphic belts (ca. 2.1 Ga); its petrotectonic associations such as metavolcanic units, conglomerates, banded iron formations, and turbiditic deposits suggest a back-arc or a trench-basin setting. Also in the PATT, a late to post-orogenic, arc-related layered mafic complex (2.3-1.9 Ga), followed by A-type granites (2.08 Ga), and finally a taphrogenic mafic dike swarm (1.78 Ga) occur. The less thoroughly studied NPTT consists of Palaeoproterozoic high-grade metamorphic sequences (ca. 2.2 Ga), mylonites and postorogenic and rapakivi granites (1.75 Ga). The Brasiliano-Pan African orogeny affected this terrane. Neoproterozoic cover occurs in both tectonostratigraphic terranes, but is more developed in the NPTT. Over the past 15 years, new isotopic studies have improved our recognition of different tectonic events and associated processes, such as reactivation of shear zones and fluids circulation. Transamazonian and Statherian tectonic events were recognized in the RPC. Based on magmatism, deformation, basin development and metamorphism, we propose a scheme for the Precambrian tectonic evolution of Uruguay, which is summarized in the first Palaeoproterozoic tectonic map of the Rio de La Plata craton.

HFD groups in the Solovay model

Hajnal and Juhasz proved that under CH there is a hereditarily separable, hereditarily normal topological group without non-trivial convergent sequences that is countably compact and not Lindelof. The example constructed is a topological subgroup H subset of 2(omega 1) that is an HFD with the following property (P) the projection of H onto every partial product 2(I) for I is an element of vertical bar omega(1)vertical bar(omega) is onto. Any such group has the necessary properties. We prove that if kappa is a cardinal of uncountable cofinality, then in the model obtained by forcing over a model of CH with the measure algebra on 2(kappa), there is an HFD topological group in 2(omega 1) which has property (P). Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Mitochondrial ion transport pathways: Role in metabolic diseases

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H(+) transport (promoted by uncoupling proteins, UCPs). Ca(2+) and K(+) uniporters which may be determinant in metabolic disorders. (C) 2009 Elsevier B.V. All rights reserved.

Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.

EVALUATION OF METAL TOXICITY BY A MODIFIED METHOD BASED ON THE FUNGUS GERRONEMA VIRIDILUCENS BIOLUMINESCENCE IN AGAR MEDIUM

Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+), Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables LIS to propose a metal toxicity series to Gerronenia viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximate to Ni(2+) approximate to Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximate to Na(+) > Ca(2+), and to shed some li-ht on the mechanism of toxic action of metal cations to basidiomycete fungi. Environ. Toxicol. Chem. 2010;29:320-326. (C) 2009 SETAC

A compact and high-resolution version of a capacitively coupled contactless conductivity detector

An all-in-one version of a capacitively coupled contactless conductivity detector is introduced. The absence of moving parts (potentiometers and connectors) makes it compact (6.5 cm(3)) and robust. A local oscillator, working at 1.1 MHz, was optimized to use capillaries of id from 20 to 100 lam. Low noise circuitry and a high-resolution analog-to-digital converter (ADC) (21 bits effective) grant good sensitivities for capillaries and background electrolytes currently used in capillary electrophoresis. The fixed frequency and amplitude of the signal generator is a drawback that is compensated by the steady calibration curves for conductivity. Another advantage is the possibility of determining the inner diameter of a capillary by reading the ADC when air and subsequently water flow through the capillary. The difference of ADC reading may be converted into the inner diameter by a calibration curve. This feature is granted by the 21-bit ADC, which eliminates the necessity of baseline compensation by hardware. In a typical application, the limits of detection based on the 3 sigma criterion (without baseline filtering) were 0.6, 0.4, 0.3, 0.5, 0.6, and 0.8 mu mol/L for K(+), Ba(2+), Ca(2+), Na(+), Mg(2+), and Li(+), respectively, which is comparable to other high-quality implementations of a capacitively coupled contactless conductivity detector.

Removal of metal ions from aqueous solution by chelating polymeric hydrogel

Polysaccharide natural seed coat from the tree Magonia pubescens, in the form of hydrogel was used to remove metals in aqueous solution. Swelling tests indicate that seed coat presents hydrogel behavior, with maximum water absorption of 292 g water/g. Adsorption experiments performed using Na(+), Mg(2+), K(+), Ca(2+), Cr(3+), Fe(3+) and Zn(2+) demonstrated that the polysaccharide structure has a high capacity to extract these ions from the aqueous solution. Scanning electron microscopy revealed significant morphological changes of the material before and after water contact. Differential scanning calorimetry measurements indicate a signal shift of the water evaporation temperature in the material with adsorbed zinc. X-ray photoelectron spectroscopy analysis combined with theoretical studies by the density functional theory and on Hartree-Fock (HF) level evidence that the metallic ions were adsorbed through coordination with hydroxyl groups of polysaccharide. In the case of Zn(2+) the lowest HF energy was observed for the tetracoordination mode, where Zn(2+) is coordinated by two hydroxyl groups and two water molecules.

Simultaneous Differential Pulse Voltammetric Determination of Ascorbic Acid and Caffeine in Pharmaceutical Formulations Using a Boron-Doped Diamond Electrode

A cathodically pretreated boron-doped diamond electrode was used for the simultaneous anodic determination of ascorbic acid (AA) and caffeine (CAF) by differential pulse voltammetry Linear calibration curves (r = 0 999) were obtained from 1 9 x 10(-5) to 2 I x 10(-4) mol L(-1) for AA and from 9 7 x 10(-6) to 1 1 x 10-4 mol L(-1) for CAF. with detection limits of 19 wool L(-1) and 7 0 mu nol L(-1). respectively This method was successfully applied for the determination of AA and CAF in pharmaceutical formulations. with results equal to those obtained using a HPLC reference method

Simulation of Sawmill Yard Operations Using Software Agents

Bergkvist insjön AB is a sawmill yard which is capable of producing 350,000 cubic meter of timber every year this requires lot of internal resources. Sawmill operations can be classified as unloading, sorting, storage and production of timber. In the company we have trucks arriving at random they have to be unloaded and sent back at the earliest to avoid queuing up of trucks creating a problem for truck owners. The sawmill yard has to operate with two log stackers that does several tasks including transporting the logs from trucks to measurement station where the logs will be sorted into classes and dropped into pockets from pockets to the sorted timber yard where they are stored and finally from there to sawmill for final processing. The main issue that needs to be answered here is the lining up trucks that are waiting to be unload, creating a problem for both sawmill as well as the truck owners and given huge production volume, it is certain that handling of resources is top priority. A key challenge in handling of resources would be unloading of trucks and finding a way to optimize internal resources.To address this problem i have experimented on different ways of using internal resources, i have designed different cases, in case 1 we have both the log stackers working on sawmill and measurement station. The main objective of having this case is to make sawmill and measurement station to work all the time. Then in case 2, i have divided the work between both the log stackers, one log stacker will be working on sawmill and pocket_control and second log stacker will be working on measurement station and truck. Then in case 3 we have only one log stacker working on all the agents, this case was designed to reduce cost of production, as the experiment cannot be done in real-time due to operational cost, for this purpose simulation is used, preliminary investigation into simulation results suggested that case 2 is the best option has it reduced waiting time of trucks considerably when compared with other cases and it showed 50% increase in optimizing internal resources.

Argilominerais e ostracodes da Formação Alagamar (cretáceo inferior), Bacia Potiguar, NE - Brasil : paleoambiente e indicadores térmicos

A Fonnação Alagamar corresponde ao estágio transicional na evolução tectono-sedimentar da bacia Potiguar, representada inicialmente por um ambiente tlúvio-deltaico passando a lagunar restrito com indícios de intluência marinha. Os resultados obtidos nas análises por DRX, a partir das frações> 2 11me < 211m, demonstram variações na composição mineralógica ao longo das perfurações analisadas. A principal característica é a forte contribuição de minerais detríticos na fração >2 I-lme a predominância de argilominerais na fração <2 I-lmrepresentados por esmectita, ilita, caulinita, clorita, sepiolita e interestratificados de ilitalesmectita (IIE) e cloritalesmectita (CIE). As carapaças de ostracodes identificadas correspondem a espécies nãomarinhas de três famílias mais comuns no Cretáceo: Cyprididae, Limnocytheridae e Darwinulidae. A avaliação estatística da composição ontogenética das espécies fósseis, tomou-se útil neste trabalho para estimar os níveis de energia do paleoambiente deposicional. As associações de argilominerais e ostracodes, caracterizadas ao longo dos cilindros de sondagens estudados na Fonnação Alagamar, sugerem a variação dos ambientes deposicionais a partir de um ambiente lacustre e de clima árido. Ainda a passagem por uma fase transgressiva com o aumento da salinidade, condições de fundo redutoras e margens subaquosas, ocasionam exposições subaéreas intennitentes, culminandono topo com um período mais úmido e condições de águas salinas O rico conteúdo em esmectita assemelha os depósitos da Fonnação Alagamar aos depósitos neoaptianos, os quais estão associados a condições climáticas com tendência à aridez. Sugere, ainda, morfologia pouco acidentada e drenagem reduzida ao redor de um paleolago restrito, o que favorece o desenvolvimento de solos do tipo vertissolo, fonte principal da esmectita da área. Os teores baixos de ilita e caulinita são, em grande parte, do resultado de erosão reduzida, atribuído ao relevo pouco acidentado da área. A associação dos argilominerais ilita, esmectita e interestratificados não ordenados indica condições rasas de soterramento e baixas temperaturas de diagênese nas amostras da base da perfuração RN6, alto de Macau. Ainda, a coloração apresentada pelas carapaças de ostracodes, entre o amarelo-laranja muito claro (2,5 Y 8/4) e amarelo amarronado(10 YR 6/6), sugerem níveis imaturos quanto à maturação da matéria orgânica. Na perfuração RN9, próxima a falha de Ubarana, condições de maturação da matéria orgânica são indicadas pelas cores cinza muito preto (5 Y 3/1) a preto (5 Y 2,5/1) apresentadas pelas carapaças de Candona sp.1, originalmente de cor branca (5 YR S/1 aiO YR 8/1). As intensidades e a fonna dos picos da ilita e da caulinita, a ausência de interestratificados e de argilominerais expansivos indicam condições de diagênese mais intensas que na perfuraçãoRN6. Associações de argilominerais identificadas em análises de difração de raios-X e mudanças na coloração das carapaças de ostracodes sugerem a utilização destes constituintes como indicadores ténnicos (geotennômetros) da maturação da matéria orgânica presente em rochas geradoras de hidrocarbonetos. Palavras chaves: Fonnação Alagamar, argilominerais, ostracodes, geotermômetros, bacia Potiguar.