Initial synthesis and characterization of an alpha7 nicotinic receptor cellular membrane affinity chromatography column: effect of receptor subtype and cell type

In this study, cellular membrane fragments from SH-EP1-pCEP4-halpha7 and alpha7 HEK-293 cell lines were used to synthesize cellular membrane affinity chromatography (CMAC) columns containing functional alpha7 nicotinic acetylcholine receptors, CMAC(alpha7 nAChR) columns. The synthesis of stable columns required the addition of cholesterol to the 2% cholate solubilization/immobilization (s/i) buffer and to the mobile phase. In addition, when membranes from the SH-EP1 cell line were used, l-alpha-phosphatidylserine and l-alpha-phosphatidylethanolamine also had to be added to the s/i buffer. A CMAC(alpha4beta2 nAChR) column was prepared using membrane fragments from a SH-EP1-pCEP4-halpha4beta2 cell line, and this process required the addition of l-alpha-phosphatidylserine and l-alpha-phosphatidylethanolamine to the s/i buffer, but not cholesterol. The s/i buffers from the three columns were compared with the s/i buffer utilized in the preparation of a CMAC(alpha4beta2 nAChR) column prepared using an alpha4beta2 HEK-293 cell line, which required no additions to the 2% cholate s/i buffer. The data demonstrate that both cell type and receptor type affect the protocol required to produce a stable CMAC column and that, at the current time, the development of an optimum immobilization protocol is an empirical process. The results are also consistent with the observation that the alpha7 nAChR is localized in lipid rafts in both of these cell lines and that the cholate detergent removed cholesterol from these microdomains.     

Mechanical properties of reactive powder concretes

Reactive Powder Concretes (RPC) are a set of ultrahigh-strength concretes reinforced with steel fibers. Their compressive strength is between 200 and 800MPa, and their flexural strength can reach 140MPa.RPC200 has been studied with respect to compressive strength and two-point loading strength to define its mechanical behavior.RPC800, which has been mostly studied from the point of view of compressive strength, displays hardening elastic non-linear behavior at low stress. This behavior is similar to that of some natural rocks. The critical stress intensity factorK _Ic , and the average fracture energy, , ofRPC200 andRPC800 have been studied experimentally by applying the theory of linear fracture mechanics (compliance method). The fracture energy, which is a measurement of ductility, can reach 40,000 J/m² forRPC200, as compared to 100 to 150 J/m² for ordinary concretes. Fracture energy depends on the volume of fibers added to the concrete. The optimum content is between 2 and 3% by volume.

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Résumé Les Bétons de Poudres Réactives (BPR) constituent une famille de bétons à ultra hautes performances renforcés de fibres métalliques. Leur résistance en compression se situe entre 200 et 800 MPa et leur résistance en flexion peut atteindre 140 MPa. Le BPR200 a été étudié en compression et en flexion 4-points pour définir son comportement mécanique. Le BPR800, étudié principalement en compression, présente un comportement non linéaire élastique à faible contrainte. Ce comportement s'apparente à celui de certaines roches naturelles. Le facteur d'intensité de contrainte K_Ic et l'énergie moyenne de fracturation du BPR20 et du BPR800 ont été étudiés expérimentalement en appliquant la théorie de la mécanique linéaire de la rupture (méthode de la complaisance). L'énergie de fracturation qui caractérise la ductilité peut atteindre 40 000 J/m² pour le BPR200 à comparer à des valeurs de 100 à 150 J/m² pour les bétons traditionnels. L'énergie de fracturation dépend du volume de fibres ajouté au béton. Le dosage optimal se situe entre 2 et 3 vol-%.

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Editorial note Gérard Bernier is working at the Laboratoire de Mécanique et de Technologie, école Normale Supérieure, Cachan (France). This laboratory is a RILEM Associate Member.     

An accelerated methodology for the evaluation of critical properties in polyphase alloys

A system for more rapidly determining the strength and stiffness of polyphase alloys is presented that is based on a digital representation of the material structure. Working in concert with the representation are a number of digital tools and probes that are analogues of testing equipment and instrumentation of traditional laboratory methods. These are combined with nontraditional mechanical testing methods to complete the system. An example of an Fe-Cu system is used to illustrate the methodology. This article is based on a presentation made in the symposium entitled “Three Dimensional Materials Science” during the 2003 MS&T ’03: Materials Science & Technology Conference 2003 in Chicago, Illinois, on November 11 & 12, 2003, under the auspices of the ASM/MSCTS: Materials Science Critical Technology Sector Committee and the TMS/SMD: Structural Materials Division Committee.     

Characterization of AT4 receptor from bovine aortic endothelium with photosensitive analogues of angiotensin IV

Newly developed photosensitive analogues of AngIV were used to characterize the AT4 receptor of bovine aortic endothelial cells. The photoactivatable AngIV analogues [N3-Phe6]AngIV and [Bpa6]AngIV displayed high affinities for AT4 receptor, with IC50's of 3.7 +/- 0.3 and 19.1 +/- 3.5 nM, respectively. The radioiodinated ligands showed a good efficiency of photoaffinity labeling demonstrated by high proportions (60-75%) of acid-resistant binding. Covalently labeled receptor was solubilized under reducing or nonreducing conditions and subjected to SDS-PAGE. Under nonreducing conditions, autoradiographies revealed a major band of Mr 186 +/- 2 kDa and a minor band of Mr 241 +/- 6 kDa. The labeling of these bands was completely abolished in the presence of 10 microM AngIV. Under reducing conditions, only the low Mr 186 kDa band was revealed. After endoglycosidase digestion with an enzyme that cleaves N-linked saccharides, the Mr of the denatured AT4 receptor was decreased by 31% to a value of 129 +/- 10 kDa. Kinetic studies revealed a stepwise process of AT4 receptor deglycosylation by endoglycosidase F, suggesting at least two different sites of N-linked saccharides. Mild trypsin treatment of photolabeled endothelial cell membranes released a large fragment of Mr 177 +/- 3 kDa which accounts for about 95% of the whole receptor molecular mass. These results demonstrate that [N3-Phe6]AngIV and [Bpa6]AngIV are very efficient tools for selective photoaffinity labeling of AT4 receptor. We have shown that AT4 receptor is a 186 kDa integral membrane glycoprotein with a very large extracellular domain. These properties are consistent with those of a growth factor or cytokine receptor.     

Myocardial color scan with 43K in ischemic heart disease

The value of myocardial scanning with 43K was assessed in 64 consecutive patients undergoing coronary arteriography, and in five young volunteers. Myocardial scans at rest detected only 16 of the 35 transmural infarcts documented on electrocardiograms, 11 of 11 anterior infarcts and five of 24 in other sites. Myocardial scans were obtained immediately after a graded exercise test in the five normal volunteers, in nine patients with normal coronary arteriograms and in 25 patients with atherosclerotic narrowing greater than 75% involving the left anterior descending artery, with or without disease of other coronary vessels. All patients with normal coronary arteriograms had normal myocardial scans. A regional perfusion deficit was observed after exercise in all six patients with single vessel disease, but in only 11 of the 19 patients with disease involving two or three vessels. Although the technique was specific, it lacked sensitivity, due mostly to poor resolution and the location of the disease.     

Localization by TEM and EELS of deuterium trapping sites in CFC exposed to plasma irradiation in Tore Supra

Carbon fiber composite (CFC) Sepcarb^® N11 is used in the tokamak Tore Supra as plasma-facing components. To investigate the fuel retention capability of this material, a mobile sample holder was used to expose CFC N11 samples to direct irradiation by the scrape-off layer plasma of Tore Supra at fluences up to 1 × 10²⁵ m⁻². Deuterium (D) elemental mapping using nuclear reaction analysis for the most-exposed CFC sample showed that D retention occurs at depths greater than 8 μm due to the presence of deep (>3.5 μm) local retention sites. In this work, combining transmission electron microscopy (TEM) and electron energy-loss spectroscopy (EELS), we describe at a high spatial resolution where and how D atoms are trapped in these sites. TEM experiments performed on thin cross-sections of the plasma-modified surface show evidence of the presence of a 3.5 μm-thick deuterated amorphous carbon layer deposited on the CFC surface. We show that specific localized retention sites correspond to the filling of relatively large (∼3 μm.) and deep (at least 3 μm below the initial CFC surface) cracks between fibres and matrix by the deuterated amorphous carbon layer.     

Stability of various doping species in trans-polyacetylene

Polyacetylene, a simple conjugated organic polymer, a material of strong interest if doped by various chemical species such as iodine or antimony pentafluoride : its electrical conductivity increases by more than twelve orders of magnitude. By increasing the dopant concentration one obtains a semiconductive or a metallic regime. Such properties allow us to make electronic material and then devices by convenient n or p type doping of polyacetylene. However,it is necessary to determine the thermal stability of the various dopant species. In this paper, we compared the stability of iodine, SbF₅ and CF₃SO₃H dopants by studying the mass loss and the electrical conductivity decrease at various temperatures from 20‡C up to 180‡C. It is shown that only SbF₅ is a relatively stable dopant up to 80‡C, a temperature which may be reached in some active electronic devices. Raman spectroscopy allowed us to measure the ratio of I₋₃ to I₋₅ ions during the desorption process and to propose a possible chemical reaction which produces gaseous iodine from the active dopant species.