Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.     

Aurothioglucose inhibits murine thioredoxin reductase activity in vivo

Gold (I)-containing compounds, including aurothioglucose (ATG), are potent in vitro inhibitors of several selenocysteine-containing enzymes. Gold compounds have also been shown to potentiate the virulence of several viruses in mice, including coxsackievirus, implicated as a possible infectious agent in Keshan disease. One possible mechanism by which gold compounds may be increasing the virulence of viral infections in mice is by acting as a selenium antagonist in vivo and inducing oxidative stress. To investigate the possible role of gold compounds in inducing oxidative stress in mice, we assessed the ability of ATG administered in vivo to inhibit the activity of the selenocysteine-containing enzymes thioredoxin reductase (TR) and glutathione peroxidase (GPX1). Doses as low as 0. 025 mg ATG/g body weight caused significant and prolonged inhibition of TR activity in all tissues examined. No such inhibition of GPX1 activity was seen, indicating differential in vivo sensitivity of the enzymes to inhibition by ATG. In liver and heart, some recovery of TR activity was observed after a 7-d period, but no recovery was observed in pancreas or kidney. Because TR is involved in several important cellular redox functions, its inhibition most likely will affect multiple cellular processes. These results indicate that in vivo administration of ATG results in significant and long-lasting inhibition of TR activity. Such inhibition of TR could lead to increased levels of oxidative stress in vivo, thereby increasing the virulence of several viruses including the coxsackievirus.     

A negative regulatory region in the intracellular domain of the human interferon-alpha receptor

Interferon-alpha (IFN-alpha)-mediated intracellular signaling is initiated by ligand-induced receptor dimerization, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. The IFN-alpha receptor consists of at least two distinct subunits. One subunit, IFNAR1, has low affinity binding for interferon yet is required for signal transduction. We introduced mutations in the cytoplasmic domain of human IFNAR1 in order to identify residues involved in the mediation of biological responses. We took advantage of the species specificity of the interferon receptors by analyzing human IFN-alpha-induced major histocompatibility complex class I antigen expression in mouse L929 cells stably transfected with mutant human receptors. The membrane proximal 60-amino acids were insufficient to signal a biological response even though within these residues Tyk2 and Stat2 binding sites have been identified. IFN-alpha-induced receptor tyrosine phosphorylation was not critical for signaling because mutation of Tyr residues to Phe did not prevent the biological response to IFN-alpha. The deletion of a 16-amino acid region highly homologous between species created a receptor which signals an enhanced response. Tyrosine dephosphorylation is a component of this enhanced response as mutation of the Tyr residues within this region to Phe resulted in a receptor with increased sensitivity to IFN. The known signaling molecules that interact with IFNAR1 are positive regulators of IFN-alpha function. The presence of this domain in the COOH-terminal region suggests that the receptor may interact with signaling molecules that negatively regulate interferon responses.     

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.     

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.     

Contact areas in the thumb carpometacarpal joint

The thumb carpometacarpal joint is a common site of osteoarthritis. It has been hypothesized that peaks of localized stress on the dorsoradial or volar-ulnar regions, or both, of the articular surfaces of the trapezium and metacarpal lead to erosion of cartilage and may be responsible for the progression of the disease. The objective of this study was to determine the contact areas in this joint under the functional position of lateral (key) pinch and in the extremes of range of motion of the joint. These contact areas were assessed relative to the observed sites of cartilage thinning. Eight hands from cadavers of women and five from cadavers of men were tested in vitro with the thumb under a 25 N load in the lateral pinch position, and under small muscle loads (0-5 N) with the thumb in flexion, extension, abduction, adduction, and neutral positions. Contact areas of articular surfaces of the thumb carpometacarpal joint were determined for these positions using a stereophotogrammetric technique. The lateral pinch position produced contact areas predominantly on the central, volar, and volar-ulnar regions of the trapezium and the metacarpal. In three specimens, contact areas were distinctly separated between the dorsoradial and volar-ulnar regions, and in one specimen, from a man, contact occurred exclusively on the dorsoradial region of the trapezium. Using stereophotogrammetry, maps of cartilage thickness also were determined for a subset of nine specimens. The volar-ulnar, ulnar, and dorsoradial regions of the trapezium were the most common sites of thin cartilage, and these may be sites of cartilage wear.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of scales measuring attitudes, self-efficacy, and intention related to smoking among middle school students.

Attitudes toward smoking, self-efficacy to avoid smoking, and smoking intention, widely cited correlates of youth smoking prevention, are often measured in large-scale youth tobacco surveys. The psychometric properties of these scales have not been well studied among middle school youth. We examined the factorial, discriminate, and convergent validity of these scales among sixth to eighth graders from a convenience sample of 22 Texas middle schools (51.2% female; 51.21% White, 32.1% Hispanic, 16.9% African American, and 8.8% Other; 67.8% nonsmokers, 21.9% experimental smokers; 3.3% former smokers; and 7.6% current smokers). Confirmatory factor analysis and invariance testing suggest that smoking attitudes, self-efficacy, and intention have evidence of construct validity in this multiethnic sample, and the scales are appropriate to assess these constructs among middle school adolescents. Additional studies are needed to establish additional evidence of validity of these constructs in other middle school samples and other subgroups (e.g. current, experimental, and former smokers). (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A self-consistent two-dimensional model of quantum-wellsemiconductor lasers: optimization of a GRIN-SCH SQW laserstructure

A two-dimensional model for quantum-well lasers that solves, self-consistently, the semiconductor equations together with the complex scalar wave equation is described. It incorporates a position- and wavelength-dependent gain function which is derived from a quantum mechanical calculation. Such a model enables one to predict the characteristics of a quantum-well laser with a minimal number of empirical parameters. The output of the model includes light-current characteristics, the current distribution, and the optical field intensity distribution, obtained simultaneously in the calculation. Examples for modeling GRIN-SCH SQW (graded-index separate confinement heterostructure single quantum well) ridge wave guide lasers are given, and good agreement with experimental results is obtained. The model is used to optimize the geometry of a GRIN-SCH SQW laser for minimum threshold current and maximum efficiency