Coarser connected metrizable topologies

We show that every metric space, X, with w(X)?c has a coarser connected metrizable topology.     

Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism

Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K(D) approximately 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.     

Parametric ranked set sampling

Ranked set sampling was introduced by McIntyre (1952,Australian Journal of Agricultural Research,3, 385–390) as a cost-effective method of selecting data if observations are much more cheaply ranked than measured. He proposed its use for estimating the population mean when the distribution of the data was unknown. In this paper, we examine the advantage, if any, that this method of sampling has if the distribution is known, for a specific family of distributions. Specifically, we consider estimation of and for the family of random variables with cdf's of the formF(x–/). We find that the ranked set sample does provide more information about both and than a random sample of the same number of observations. We examine both maximum likelihood and best linear unbiased estimation of and , as well as methods for modifying the ranked set sampling procedure to provide even better estimation.This paper has been prepared with partial support from the United States Environmental Protection Agency under Cooperative Agreement Number CR821801-01-0. The contents have not been subjected to Agency review and therefore do not necessarily reflect the views or policies of the Agency and no official endorsement should be inferred.     

Incorporation of poly(2‐acrylamido‐2‐methyl‐N‐propanesulfonic acid) segments into block and brush copolymers by ATRP

2‐Acrylamido‐2‐methyl‐N‐propanesulfonic acid (AMPSA) was successfully polymerized via atom transfer radical polymerization (ATRP) using a copper chloride/2,2′‐bipyridine (bpy) catalyst complex after in situ neutralization of the acidic proton in AMPSA with tri(n‐butyl)amine (TBA). A 5 mol % excess of TBA was required to completely neutralize the acid and prevent protonation of the bpy ligand, as well as to avoid side reactions caused by large excess of TBA. The use of activators generated by electron transfer (AGET) ATRP with ascorbic acid as reducing agent resulted in both increased conversion of the AMPSA monomer during polymerization (up to 50% with a 0.8 [ascorbic acid]/[Cu(II)] ratio) and much shorter polymerization times (<30 min). Block copolymers and molecular brushes containing AMPSA side chains were prepared using this method, and the solution and surface behavior of these materials were investigated. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5386–5396, 2009     

Thermal reactions of 8-methylbicyclo[4.2.0]oct-2-enes: competitive diradical-mediated [1,3] sigmatropic, stereomutation, and fragmentation processes

[reaction: see text] At 275 degrees C, 8-exo-methylbicyclo[4.2.0]oct-2-ene (1a) undergoes a [1,3] sigmatropic rearrangement to 5-methylbicyclo[2.2.2]oct-2-enes, of which the orbital symmetry-allowed si product is only marginally favored over the forbidden sr product; that is, si/sr is 2.4. Accompanying the [1,3] shift are significant amounts of epimerization and fragmentation. The 8-endo epimer 1b, which yields no [1,3] product, experiences primarily direct fragmentation and secondarily epimerization. A diradical intermediate can account for all such observations.     

The solubilities of zinc complexes of 8-quinolinol and some derivatives of 8-quinolinol

The solubilities of bis-8-quinolinolo-zinc^II, bis-2-methyl-8-quinolinolo-zinc^II and bis-5 : 7-dibromo-S-quinolinolo-zinc^II have been measured over the pH range of 4.5 to 9 at ionic strengths of 0.114 and 0.230 and at 25° and 40°. A mathematical treatment of the results gave nearly constant solubility products only when a dissolved but undissociated molecule, in addition to all of the dissociated and partly dissociated species, was considered. The relative abundance of each species of molecule in solution has been calculated.     

Characterization of fibrinogen adsorption onto glass microcapillary surfaces by ELISA

Adsorption of biomolecules onto microchannel surfaces remains a critical issue in microfluidic devices. This paper investigates the adsorption of fibrinogen on glass microcapillaries using an immunoassay method (ELISA) and X-ray photoelectron spectroscopy (XPS). Various adsorption conditions such as protein concentrations and incubation times, buffer pH, buffer ionic strengths and effects of flow are presented. ELISA is successfully demonstrated as a facile and robust technique to examine these phenomena. The highest adsorption level occurs near the isoelectric point of fibrinogen (pH 5.0) and low buffer ionic strengths (0-8 mM). Microchannel surface saturation was achieved at a fibrinogen solution concentration of approximately 50 microg ml(-1). Fibrinogen adsorption under flow was always higher than that seen in static systems. The importance of diffusion phenomena in microchannels on protein adsorption was demonstrated. ELISA experiments using fused silica and PEEK have also confirmed significant adsorption on these mass spectrometer transfer line materials.     

Phosphoesterase activity and phosphate release from tributyl phosphate by aCitrobacter sp.

Tributyl phosphate (TBP) and other alkyl phosphates represent a class of persistent organophosphorus compounds of widespread use. Biodegradation of the phosphotriesters is postulated to occur through sequential hydrolytic cleavages via the phosphodiester and monoester intermediates to alcohol and inorganic phosphate (P_i). Immobilized cells of aCitrobacter sp. liberated P_i upon challenge with TBP but the reaction was short-lived. In vitro studies with purified phosphomonoesterase (phosphatase) used³¹P nuclear magnetic resonance to demonstrate P_i transfer onto ethanol (phosphotransferase activity). This suggested that in vivo the onset of a futile phosphohydrolytic and transphosphorylation cycle would limit the extent of phosphate production. A mutant deficient in the transphosphorylating phosphomonoesterase showed an extended release of P_i under challenge with TBP that was not subject to the complete and premature reaction termination that precluded application of the parent strain to possible industrial processes for alkyl phosphate biodegradation.     

Autoantibodies to thyroglobulin in health and disease

Thyroglobulin (Tg)—a heavily glycosylated, iodinated protein—isa major autoantigen in autoimmune thyroiditis. Tg also induces thyroiditis by immunization of experimental animals. Humans with chronic lymphocytic thyroiditis characteristically produce autoantibodies to thyroglobu lin, but similar autoantibodies are also found in some clinically normal, euthyroid individuals. A comparison of the fine specificity of autoantibodies in humans and in experimentally immunized mice was carried out, based on their ability to inhibit a panel of monoclonal antibodies (MAbs). Patients with autoimmune thyroid disease, as well as normal individuals, produced autoantibodies mainly to the conserved, cross-reactive determinants of thyroglobulin. Patients developed additional autoantibodies to species-restricted epitopes. The determinants recognized by patients with Graves' disease differed in some respects from epitopes recognized by thyroiditis patients or patients with differentiated thyroid carcinoma. Similarly, mice that are genetically susceptible to thyroiditis produced autoantibodies that reacted with the mouse-specific antigenic determinants. Using an autoantibody that reacts with one of the epitopes associated with thyroiditis, a reactive 15-k Da fragment of human Tg—localized at the carboxy end of the molecule—was isolated and sequenced. Iodine plays an important role in the precise specificity of the disease-associated epitope, since T cells from patients with thyroiditis react with iodinated but not noniodinated human thyroglobulin. Addition of iodine to Tg generates new or cryptic epitopes. Use of a selected MAb as a surrogate for the T-cell receptor suggests that a specific iodine-containing epitope is sometimes involved in recognition. Finally, thyroglobulin-reactive autoantibodies exhibit proteolytic activity on thyroglobulin.