Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

DC-SIGN and alpha 2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells

Intravenous immunoglobulin (IVIg) is widely used to treat autoimmune diseases. Several mutually nonexclusive mechanisms are proposed to explain the beneficial effects of IVIg in patients (1, 2). Lately, Ravetch and colleagues (3) demonstrate that anti-inflammatory activity of IVIg is mediated mainly by antibodies that contain terminal _2,6-sialic acid linkages at the Asn297-linked glycan of Fc region.

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a process 5'-3' single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg2+ or Mn2+ and inhibited by Cd2+ suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays, Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interactio between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.

The effects of cholesterol inclusion on the vesicular membranes of cationic lipids

Small unilamellar vesicles formed from four cationic lipids in the absence and the presence of varying amounts of cholesterol were studied using fluorescence polarization and H-1-NMR techniques. The fluorescence polarization data clearly indicate that the packing order in the cationic lipid bilayers are affected by inclusion of cholesterol. importantly, this effect exists also with a cationic lipid that is devoid of any formal linkage region where the interaction of the lipid with cholesterol through hydrogen bonding is not feasible. The interactions of cholesterol with different types of cationic lipids in excess water have also been examined in multilamellar dispersions using proton magnetic resonance spectroscopy. In all the cases, the methylene proton linewidths in the NMR spectra respond to the addition of cholesterol to vesicles. Hydrophobic association of the lipid and cholesterol imposes restriction on the chain (CH2)(n) motions, leaving the terminal CH3 groups relatively mobile. On the basis of energy-minimized structural models, a rationale of the cholesterol-cationic lipid assembly has also been presented.

Identifying and addressing equivocal trouble in understanding within classroom interaction

Maintaining intersubjectivity is crucial for accomplishing coordinated social action. Although conversational repair is a recognised defence of intersubjectivity and routinely used to address ostensible sources of trouble in social interaction, it is less clear how people address more equivocal trouble. This study uses conversation analysis to examine preschool classroom interaction, focusing on practices used to identify and address such trouble. Repair is found to be a recurrent frontline practice for addressing equivocal trouble, occasioning space for further information that might enable identifying a specific trouble source. Where further information is forthcoming, a range of strategies are subsequently employed to address the trouble. Where this is not possible or does not succeed, a secondary option is to progress a broader activity-in-progress. This allows for the possibility of another opportunity to identify and address the trouble. Given misunderstandings can jeopardise interactants’ ability to mutually accomplish courses of action, these practices defend intersubjectivity against the threat of equivocal trouble.

Pt metal-CeO2 interaction: Direct observation of redox coupling between Pt-0/Pt2+/Pt4+ and Ce4+/Ce3+ states in Ce0.98Pt0.02O2-delta catalyst by a combined electrochemical and x-ray photoelectron spectroscopy study

Pt ions-CeO2 interaction in Ce1-xPtxO2-delta (x=0.02) has been studied for the first time by electrochemical method combined with x-ray diffraction and x-ray photoelectron spectroscopy. Working electrodes made of CeO2 and Ce0.98Pt0.02O2-delta mixed with 30% carbon are treated electrochemically between 0.0-1.2 V in potentiostatic (chronoamperometry) and potentiodynamic (cyclic voltametry) mode with reference to saturated calomel electrode. Reversible oxidation of Pt-0 to Pt2+ and Pt4+ state due to the applied positive potential is coupled to simultaneous reversible reduction of Ce4+ to Ce3+ state. CeO2 reduces to CeO2-y (y=0.35) after applying 1.2 V, which is not reversible; Ce0.98Pt0.02O2-delta reaches a steady state with Pt2+:Pt4+ in the ratio of 0.60:0.40 and Ce4+:Ce3+ in the ratio of 0.55:0.45 giving a composition Ce0.98Pt0.02O1.74 at 1.2 V, which is reversible. Composition of Pt ion substituted compound is reversible between Ce0.98Pt0.02O1.95 to Ce0.98Pt0.02O1.74 within the potential range of 0.0-1.2 V. Thus, Ce0.98Pt0.02O2-delta forms a stable electrode for oxidation of H2O to O-2 unlike CeO2. A linear relation between oxidation of Pt2+ to Pt4+ with simultaneous reduction in Ce4+ to Ce3+ is observed demonstrating Pt-CeO2 metal support interaction is due to reversible Pt-0/Pt2+/Pt4+ interaction with Ce4+/Ce3+ redox couple.

Molecule Modeling of Human Pentameric alpha(7) Neuronal Nicotinic Acetylcholine Receptor and Its Interaction with its Agonist and Competitive Antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved i interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Interaction of Co, Mn, Mg and AI with d(GCGTACGC): a spectroscopic study

Spectroscopic study on the interactions of trace elements Co, Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter T-m values. Mn enhanced T-m at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the T-m. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.

A comparative Raman study of the interaction of electron donor and acceptor molecules with graphene prepared by different methods

Interaction of tetrathiafulvalene (TTF) and tetracyanoethylene (TCNE) with few-layer graphene samples prepared by the exfoliation of graphite oxide (EG), conversion of nanodiamond (DG) and arc-evaporation of graphite in hydrogen (HG) has been investigated by Raman spectroscopy to understand the role of the graphene surface. The position and full-width at half maximum of the Raman G-band are affected on interaction with TTF and TCNE and the effect is highest with EG and least with HG. The effect of TTF and TCNE on the 2D-band is also maximum with EG. The magnitude of interaction between the donor/acceptor molecules varies in the same order as the surface areas of the graphenes. (C) 2009 Published by Elsevier B. V.

Reduction in arterial wall strain with aggressive lipid-lowering therapy in patients with carotid artery disease

Background: Inflammation and biomechanical factors have been associated with the development of vulnerable atherosclerotic plaques. Lipid-lowering therapy has been shown to be effective in stabilizing them by reducing plaque inflammation. Its effect on arterial wall strain, however, remains unknown. The aim of the present study was to investigate the role of high- and low-dose lipid-lowering therapy using an HMG-CoA reductase inhibitor, atorvastatin, on arterial wall strain. Methods and Results: Forty patients with carotid stenosis >40% were successfully followed up during the Atorvastatin Therapy: Effects on Reduction Of Macrophage Activity (ATHEROMA; ISRCTN64894118) Trial. All patients had plaque inflammation as shown by intraplaque accumulation of ultrasmall super paramagnetic particles of iron oxide on magnetic resonance imaging at baseline. Structural analysis was performed and change of strain was compared between high- and low-dose statin at 0 and 12 weeks. There was no significant difference in strain between the 2 groups at baseline (P=0.6). At 12 weeks, the maximum strain was significantly lower in the 80-mg group than in the 10-mg group (0.085±0.033 vs. 0.169±0.084; P=0.001). A significant reduction (26%) of maximum strain was observed in the 80-mg group at 12 weeks (0.018±0.02; P=0.01). Conclusions: Aggressive lipid-lowering therapy is associated with a significant reduction in arterial wall strain. The reduction in biomechanical strain may be associated with reductions in plaque inflammatory burden.

Study of carotid arterial plaque stress for symptomatic and asymptomatic patients

Stroke is one of the leading causes of death in the world, resulting mostly from the sudden ruptures of atherosclerosis carotid plaques. Until now, the exact plaque rupture mechanism has not been fully understood, and also the plaque rupture risk stratification. The advanced multi-spectral magnetic resonance imaging (MRI) has allowed the plaque components to be visualized in-vivo and reconstructed by computational modeling. In the study, plaque stress analysis using fully coupled fluid structure interaction was applied to 20 patients (12 symptomatic and 8 asymptomatic) reconstructed from in-vivo MRI, followed by a detailed biomechanics analysis, and morphological feature study. The locally extreme stress conditions can be found in the fibrous cap region, 85% at the plaque shoulder based on the present study cases. Local maximum stress values predicted in the plaque region were found to be significantly higher in symptomatic patients than that in asymptomatic patients (200±43. kPa vs. 127±37. kPa, p=0.001). Plaque stress level, defined by excluding 5% highest stress nodes in the fibrous cap region based on the accumulative histogram of stress experienced on the computational nodes in the fibrous cap, was also significantly higher in symptomatic patients than that in asymptomatic patients (154±32. kPa vs. 111±23. kPa, p<0.05). Although there was no significant difference in lipid core size between the two patient groups, symptomatic group normally had a larger lipid core and a significantly thinner fibrous cap based on the reconstructed plaques using 3D interpolation from stacks of 2D contours. Plaques with a higher stenosis were more likely to have extreme stress conditions upstream of plaque throat. The combined analyses of plaque MR image and plaque stress will advance our understanding of plaque rupture, and provide a useful tool on assessing plaque rupture risk.

Stress analysis of carotid atheroma in a transient ischaemic attack patient using the MRI-based fluid-structure interaction method

Rupture of atherosclerotic plaque is a major cause of mortality. Plaque stress analysis, based on patient-specific multisequence in vivo MRI, can provide critical information for the understanding of plaque rupture and could eventually lead to plaque rupture prediction. However, the direct link between stress and plaque rupture is not fully understood. In the present study, the plaque from a patient who recently experienced a transient ischaemic attack (TIA) was studied using a fluid-structure interaction method to quantify stress distribution in the plaque region based on in vivo MR images. The results showed that wall shear stress is generally low in the artery with a slight increase at the plaque throat owing to minor luminal narrowing. The oscillatory shear index is much higher in the proximal part of the plaque. Both local wall stress concentrations and the relative stress variation distribution during a cardiac cycle indicate that the actual plaque rupture site is collocated with the highest rupture risk region in the studied patient.

Fluid-structure interaction study on human arterial plaque with patient specific geometry and boundary conditions

Atherosclerotic plaque rupture has been extensively considered as the leading cause of death in western countries. It is believed that high stresses within plaque can be an important factor on triggering the rupture of the plaque. Stress analysis in the coronary and carotid arteries with plaque have been developed by many researchers from 2D to 3-D models, from structure analysis only to the Fluid-Structure Interaction (FSI) models[1].

Carotid arterial plaque stress analysis using fluid-structure interactive simulation based on in-vivo magnetic resonance images of four patients

The rupture of atherosclerotic plaques is known to be associated with the stresses that act on or within the arterial wall. The extreme wall tensile stress (WTS) is usually recognized as a primary trigger for the rupture of vulnerable plaque. The present study used the in-vivo high-resolution multi-spectral magnetic resonance imaging (MRI) for carotid arterial plaque morphology reconstruction. Image segmentation of different plaque components was based on the multi-spectral MRI and co-registered with different sequences for the patient. Stress analysis was performed on totally four subjects with different plaque burden by fluid-structure interaction (FSI) simulations. Wall shear stress distributions are highly related to the degree of stenosis, while the level of its magnitude is much lower than the WTS in the fibrous cap. WTS is higher in the luminal wall and lower at the outer wall, with the lowest stress at the lipid region. Local stress concentrations are well confined in the thinner fibrous cap region, and usually locating in the plaque shoulder; the introduction of relative stress variation during a cycle in the fibrous cap can be a potential indicator for plaque fatigue process in the thin fibrous cap. According to stress analysis of the four subjects, a risk assessment in terms of mechanical factors could be made, which may be helpful in clinical practice. However, more subjects with patient specific analysis are desirable for plaque-stability study.